Search

Summary: Purpose: Overexpression of multidrug transporters such as P-glycoprotein (P-gp) may play a significant role in pharmacoresistance, by preventing antiepileptic drugs (AEDs) from reaching their targets in the brain. Until now, many studies have described increased P-gp expression in epileptic tissue or have shown that several AEDs act as substrates for P-gp. However, definitive proof showing the functional involvement of P-gp in pharmacoresistance is still lacking. Here we tested whether P-gp contributes to pharmacoresistance to phenytoin (PHT) by using a specific P-gp inhibitor in a model of spontaneous seizures in rats. Methods: The effects of PHT on spontaneous seizure activity were investigated in the electrical post–status epilepticus rat model for temporal lobe epilepsy, before and after administration of tariquidar (TQD), a selective inhibitor of P-gp. Results: A 7-day treatment with therapeutic doses of PHT suppressed spontaneous seizure activity in rats, but only partially. However, an almost complete control of seizures by PHT (93 ± 7%) was obtained in all rats when PHT was coadministered with TQD. This specific P-gp inhibitor was effective in improving the anticonvulsive action of PHT during the first 3–4 days of the treatment. Western blot analysis confirmed P-gp upregulation in epileptic brains (140–200% of control levels), along with ∼20% reduced PHT brain levels. Inhibition of P-gp by TQD significantly increased PHT brain levels in chronic epileptic rats. Conclusions: These findings show that TQD significantly improves the anticonvulsive action of PHT, thus establishing a proof-of-concept that the administration of AEDs in combination with P-gp inhibitors may be a promising therapeutic strategy in pharmacoresistant patients.

Interleukin-1beta is rapidly synthesized by glia after the induction of seizures. Recent evidence shows that endogenous IL-1beta has proconvulsant actions mediated by interleukin-1 receptor type I. This receptor also mediates interleukin-1beta effects on neuronal susceptibility to neurotoxic insults. In this study, we investigated the basal and seizure-induced expression of interleukin-1 receptor type I in rat forebrain to identify the cells targeted by interleukin-1beta during epileptic activity. Self-sustained limbic status epilepticus was induced in rats by electrical stimulation of the ventral hippocampus. Interleukin-1 receptor type I immunoreactivity was barely detectable in neurons in control brain tissue. During status epilepticus, interleukin-1 receptor type I was induced in the hippocampal neurons firstly, and several hours later in astrocytes localized in limbic and extralimbic areas. Neuronal interleukin-1 receptor type I expression in the hippocampus outlasted the duration of spontaneous electroencephalographic seizure and was not observed in degenerating neurons. Astrocytic expression occurred transiently, between six and 18 h after the induction of status epilepticus and was invariably found in regions of neuronal damage. These time-dependent, cell- and region-specific changes in interleukin-1 receptor type I expression during status epilepticus suggest that interleukin-1 receptor type I in neurons mediates interleukin-1beta-induced fast changes in hippocampal excitability while interleukin-1 receptor type I receptors in astrocytes may mediate interleukin-1beta effects on neuronal survival in hostile conditions.

Summary: Purpose: We measured the brain-to-plasma partition of 10,11-dihydro-10-hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide (10-OHCBZ) in epilepsy patients undergoing surgery to alleviate drug-resistant seizures and administered with different oral doses of oxcarbazepine (OXC). We addressed the possible contribution of the multidrug transporter P-glycoprotein (P-gp or MDR1) in determining 10-OHCBZ brain levels by measuring whether this active metabolite is a substrate of P-gp and the relation between the level of expression of MDR1 and the drug concentration in the same brain tissue specimens. Methods: Steady-state plasma and brain concentrations (Css) of 10-OHCBZ were determined intraoperatively in 11 patients by high-performance liquid chromatography (HPLC) with UV detection. The level of expression of MDR1 mRNA was measured in surgically resected brain tissue by reverse transcriptase polymerase chain reaction (RT-PCR). The ability of 10-OHCBZ to act as substate of P-gp was evaluated by measuring its uptake in cell lines expressing different levels of P-gp, in the presence or absence of a selective P-gp inhibitor. Results: OXC was converted to 10-OHCBZ and to Di-OHCBZ, the two main metabolites measured in plasma. The brain concentrations of the active metabolite 10-OHCBZ did not reflect plasma Css. A significant inverse linear correlation was found between 10-OHCBZ brain-to-plasma concentration ratio and the level of brain expression of MDR1 mRNA. In vitro uptake studies demonstrated lower intracellular 10-OHCBZ levels in cells with higher P-gp expression. Intracellular drug concentration was increased by XR9576, a specific P-gp blocker. Conclusions: Pharmacologic failure of OXC in pharmacoresistant epilepsy is unlikely to be due to alterations in drug metabolism. 10-OHCBZ does not appear to cross the blood–brain barrier by simple diffusion, and it acts as a substrate of P-gp. The level of expression of MDR1 is inversely correlated with 10-OHCBZ concentration in the epileptic tissue. P-gp may play a role in the pharmacoresistance to OXC by determining the attainment of insufficient concentrations of its active metabolite at neuronal targets.

Neuropeptide Y (NPY) potently inhibits glutamate release and seizure activity in rodent hippocampus in vitro and in vivo, but the nature of the receptor(s) mediating this action is controversial. In hippocampal slices from rats and several wild-type mice, a Y2-preferring agonist mimicked, and the Y2-specific antagonist BIIE0246 blocked, the NPY-mediated inhibition both of glutamatergic transmission and of epileptiform discharges in two different slice models of temporal lobe epilepsy, stimulus train-induced bursting (STIB) and 0-Mg2+ bursting. Whereas Y5 receptor-preferring agonists had small but significant effects in vitro, they were blocked by BIIE0246, and a Y5 receptor-specific antagonist did not affect responses to any agonist tested in any preparation. In slices from mice, NPY was without effect on evoked potentials or in either of the two slice seizure models. In vivo, intrahippocampal injections of Y2- or Y5-preferring agonists inhibited seizures caused by intrahippocampal kainate, but again the Y5 agonist effects were insensitive to a Y5 antagonist. Neither Y2- nor Y5-preferring agonists affected kainate seizures in mice. A Y5-specific antagonist did not displace the binding of two different NPY ligands in WT or mice, whereas all NPY binding was eliminated in the mouse. Thus, we show that Y2 receptors alone mediate all the anti-excitatory actions of NPY seen in the hippocampus, whereas our findings do not support a role for Y5 receptors either in vitro or in vivo. The results suggest that agonists targeting the Y2 receptor may be useful anticonvulsants.

Summary: Purpose: We investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal damage, 4 and 24 h after kainic acid–induced status epilepticus (SE) in postnatal day (PN) 9, 15, and 21 rats. Methods: Limbic seizures were induced by systemic injection of kainic acid. Glia activation and neuronal cell loss were studied by using immunocytochemistry and Western blot. Cytokine expression was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Southern blot quantification. Results: After SE onset, hippocampal glia activation, cytokine expression, and neuronal damage are all age-dependent phenomena. In the hippocampus, neuronal injury occurs only when cytokines are induced in glia, and cytokine synthesis precedes the appearance of degenerating neurons. Neuronal injury is more pronounced when interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are produced in addition to IL-1β. Conclusions: This study shows that cytokine induction in rat brain after sustained seizures is age dependent, and it is associated with the appearance of cell injury.

Experimental studies suggest that the delivery of antiepileptic agents into the seizure focus might be of potential utility for the treatment of focal-onset epilepsies. Botulinum neurotoxin E (BoNT/E) causes a prolonged inhibition of neurotransmitter release after its specific cleavage of the synaptic protein synaptosomal-associated protein of 25 kDa (SNAP-25). Here, we show that BoNT/E injected into the rat hippocampus inhibits glutamate release and blocks spike activity of pyramidal neurons. BoNT/E effects persist for at least 3 weeks, as determined by immunodetection of cleaved SNAP-25 and loss of intact SNAP-25. The delivery of BoNT/E to the rat hippocampus dramatically reduces both focal and generalized kainic acid-induced seizures as documented by behavioral and electrographic analysis. BoNT/E treatment also prevents neuronal loss and long-term cognitive deficits associated with kainic acid seizures. Moreover, BoNT/E-injected rats require 50% more electrical stimulations to reach stage 5 of kindling, thus indicating a delayed epileptogenesis. We conclude that BoNT/E delivery to the hippocampus is both antiictal and antiepileptogenic in experimental models of epilepsy.

Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y 5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood–brain barrier, and the selective peptide agonist Ala 31 Aib 34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4–5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala 31 Aib 32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y 5 agonist showed stages 4–5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y 5 agonist- or antagonist-treated rats had stages 4–5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y 5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation.

In recent years, increasing evidence has indicated that immune and inflammatory reactions occur in brain in various central nervous system (CNS) diseases. Furthermore, inflammatory processes, such as the production of proinflammatory cytokines and related molecules, have been described in brain after seizures induced in experimental models and in clinical cases of epilepsy. Although little is known about the role of inflammation in epilepsy, it has been hypothesized that activation of the innate immune system and associated inflammatory reactions in brain may mediate some of the molecular and structural changes occurring during and after seizure activity. Whether the innate immune response that takes place in epileptic tissue is beneficial or noxious to the CNS is still an open and intriguing question that should be addressed by further investigations.

Fever can provoke "febrile" seizures (FS). Because complex FS may promote development of temporal lobe epilepsy, understanding their mechanisms is clinically important. Using an immature rodent model and transgenic technology, we examined the role of interleukin-1beta, (IL-1beta), a pyrogenic, proinflammatory cytokine, in FS. IL-1beta receptor-deficient mice were resistant to experimental FS. This resistance appeared independent of genetic background and was attributed to lack of IL-1beta signaling, because exogenous cytokine reduced seizure threshold in wild-type but not receptor-deficient mice independent of strain. In addition, high IL-1beta doses induced seizures only in IL-1beta receptor-expressing mice. These data indicate that IL-1beta signaling contributes critically to fever-induced hyperexcitability underlying FS, constituting a potential target for their prevention.

Recurrent epileptic seizures in the rat enhance the expression of neuropeptide Y (NPY) and its mRNA in various brain areas including the hippocampus, cerebral cortex and the amygdala. In the hippocampus, the most prominent expression of NPY is observed in mossy fibers and in GABAergic interneurons. At the same time, expression of Y2 receptors is also increased whereas Y1 receptors are reduced. Similar changes in Y1 and Y2 receptors were observed in the hippocampus of patients with temporal lobe epilepsy (TLE). In contrast to the rat, NPY expression is not enhanced in mossy fibers in TLE. In the same tissue, surviving NPY interneurons show marked axonal sprouting into areas innervated by mossy fibers (dentate hilus, stratum lucidum, inner molecular layer of the dentate gyrus). Stimulation of presynaptic Y2 receptors inhibits glutamate release, and exert an anticonvulsant action in experimental models. Y1 receptors mediate a weak excitatory component of NPY action. These findings suggest that changes in the NPY system induced by seizures represent an endogenous adaptive mechanism aimed at counteracting hyperexcitability underlying epileptic activity. This concept is strongly supported by evidence that genetically modified rats overexpressing the NPY gene are less susceptible to seizures while deletion of NPY or Y2 receptor genes results in increased susceptibility to seizures.

In adult rats, status epilepticus (SE) induces cytokine production by glia especially when seizures are associated with neuronal injury. This suggests that cytokines may play a role in seizure-induced neuronal damage. As SE-induced injury is age-specific, we used rats of different ages (with distinct susceptibilities to seizure-induced neuronal injury) to elucidate the role of cytokines in this process. Thus, we investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal injury 4 and 24 h following kainic acid-induced SE in postnatal day (PN) 9, 15, and 21 rats. At PN9, there was little activation of microglia and astrocytes at any time point studied. Interleukin-1β (IL), tumor necrosis factor-α (TNF), and IL-6 or the naturally occurring IL-1 receptor antagonist (Ra) mRNA expression did not increase. No evidence of cell injury has been detected. At PN15, immunostaining of microglia and astrocytes was enhanced, but only IL-1β mRNA expression was increased. These changes were observed 4 h after SE. Scattered injured neurons in CA3 and subiculum, but not in any other region, were present 24 h following SE. At PN21, immunostaining of microglia and astrocytes and the mRNA expression of all cytokines studied was significantly increased already 4 h after SE. At 24 h, many injured neurons were present in CA1 and CA3 regions and in 40% of rats in other forebrain areas. These data show that (i) the pattern of glia activation and cytokine gene transcription induced by SE is age-dependent and (ii) neuronal injury in the hippocampus occurs only when cytokines are induced and their synthesis precedes the appearance of neuronal damage. Thus, cytokine expression in immature brain is associated specifically with cell injury rather than with seizures per se, suggesting that proinflammatory cytokines may contribute to the occurence of SE-induced hippocampal damage.

Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7-10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5-10 microM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25-0.5 mM). GABA (10 microM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 microM) and vapreotide (RC 160; 10 microM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 microM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 microM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5-10 microM). Finally, both specific (Rp-8-bromoguanosine 3',5'-monophosphate, 10 microM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 microM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca2+ level.

We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT1A and 5-HT4 receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[3H]hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) to 5-HT1A receptors was selectively increased by 20% on average (p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average (p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase (p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT4 receptors, which colocalize with 5-HT1A receptors on hippocampal neurons, were not modified in kindled tissue. [3H]5-HT uptake and its release as well as the 5-HT1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 microg kg(-1) 8-OH-DPAT or 1,000 microg kg(-1) WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.

We studied the expression and distribution of glutamate receptor subtypes in the spinal cord of mnd mice, a model of motor neuron disorders and neuronal ceroid lipofuscinosis, and control mice using immunocytochemistry and in situ hybridization. The constitutive subunit of the NMDA ionotropic glutamate receptor, NMDAR1, was expressed in all neurons of the grey matter and was not modified in the spinal cord of mnd mice in either its normal or phosphorylated form. The immunoreactivity of GluR2, but not its mRNA, was increased mainly in the substantia gelatinosa both in presymptomatic and in 8-month-old symptomatic mice, suggesting compensatory changes aimed at reducing the Ca2+ permeability of the receptor channel. In spinal cord of mnd mice, mRNA, and protein levels of GluR3 were low only at the symptomatic stage, possibly as a consequence of motor neuron dysfunction. This was not due to motoneuron degeneration, because the number of choline acetyltransferase (ChAT) immunopositive lumbar motor neurons and the ChAT activity in the spinal cord and hind leg muscles of symptomatic mnd mice were no different from control mice. GluR4 mRNA was increased throughout the grey matter, presumably in relation to the marked microglia activation reported in the grey matter of the lumbar spinal cord in mnd mice. These changes in ionotropic glutamate receptors may alter glutamatergic neurotransmission and play some role in the pathology of mnd mice. © 2002 Wiley-Liss, Inc.

We have investigated the role of somatostatin receptor subtypes sst 2 and sst 4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst 2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst 2 knock-out mice. Intrahippocampal injection of three selective sst 2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 μM) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 μM). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst 2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst 2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst 4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst 2 subtypes, suggesting these receptors are functionally coupled.

Purpose: We investigated the changes in the expression of proinflammatory cytokines and related molecules in the rodent hippocampus after the induction of limbic seizures. We then studied the effects of pharmacologic intervention on the interleukin (IL)-1 system on limbic seizures and the susceptibility to seizures of transgenic mice overexpressing the naturally occurring antagonist of IL-1 (IL-1Ra) in astrocytes. Methods: Limbic seizures were induced in rodents by intrahippocampal injection of kainic acid or bicuculline methiodide or by electrical stimulation of the hippocampus causing status epilepticus (SE). Seizure activity was recorded by EEG analysis and behavioral observation according to Racine's scale. Cytokine expression in the hippocampus was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot quantification of the various messenger RNAs (mRNAs) and by immunocytochemistry. Results: We found that limbic seizures rapidly and transiently enhanced IL-1β, IL-6, and tumor necrosis factor (TNF)-α mRNA in the hippocampus with a peak effect at 6 h after SE. Immunoreactivity of the various cytokines was increased in glia. The increase of IL-1 Ra was delayed because the peak effect was observed at 24 h after SE. Moreover, IL-IRa was not produced in large excess, as during peripheral inflammation but in a molar ratio to IL-1β of 1:1. Intrahippocampal injection of IL-1β worsened seizure activity, whereas IL-1Ra was a powerful anticonvulsant in various models of limbic seizures. Transgenic mice overexpressing IL-1Ra in astrocytes were less sensitive to bicuculline-induced seizures. Conclusions: This study shows that limbic seizures in rodents rapidly and reversibly induce proinflammatory cytokines in glia and suggests that changes in the IL-IRa/IL-1β ratio in brain may represent an effective physiopathologic mechanism to control seizures.

Functional studies in epileptic tissue indicate that neuropeptide Y and some of its peptide analogs potently inhibit seizure activity. We investigated seizure susceptibility in transgenic rats overexpressing the rat neuropeptide Y gene under the control of its natural promoter. Seizures were induced in adult transgenic male rats and their wild-type littermates by i.c.v. injection of 0.3 microg kainic acid or by electrical kindling of the dorsal hippocampus. Transgenic rats showed a significant reduction in the number and duration of electroencephalographic seizures induced by kainate by 30% and 55% respectively (P

Experimental and clinical findings have shown in the past decade that specific proinflammatory mediators and their cognate receptors are upregulated in epileptic brain tissue. In particular, the IL-1 receptor (R)/Toll-like receptor (TLR) signaling pathways are activated in experimental models of seizures and in human epileptic tissue from drug-resistant patients. Pharmacological targeting of these proinflammatory pathways using selective receptor antagonists, or the use of transgenic mice with perturbed cell signaling, demostrated that the activation of IL-1R type 1 and TLR4 by their respective endogenous ligands, i.e., interleukin (IL)-1b and High Mobility Group Box 1, is implicated in the precipitation and recurrence of experimentally induced seizures in rodents. This evidence highlights a new target system for pharmacological intervention to inhibit seizures by interfering with mechanisms involved in their genesis and recurrence.

Increasing evidence underlines that prototypical inflammatory cytokines (IL-1β, TNF-α and IL-6) either synthesized in the central (CNS) or peripheral nervous system (PNS) by resident cells, or imported by immune blood cells, are involved in several pathophysiological functions, including an unexpected impact on synaptic transmission and neuronal excitability. This review describes these unconventional neuromodulatory properties of cytokines, that are distinct from their classical action as effector molecules of the immune system. In addition to the role of cytokines in brain physiology, we report evidence that dysregulation of their biosynthesis and cellular release, or alterations in receptor-mediated intracellular pathways in target cells, leads to neuronal cell dysfunction and modifications in neuronal network excitability. As a consequence, targeting of these cytokines, and related signalling molecules, is considered a novel option for the development of therapies in various CNS or PNS disorders associated with an inflammatory component. This article is part of a Special Issue entitled 'Neuroimmunology and Synaptic Function'.

Around 50 million people in the world now have epilepsy, and it is estimated that 40–70 new cases per 100,000 individuals in the general population will acquire the disease every year in developed countries, with the risk being twice as high in developing countries [1]. An “epilepsy” diagnosis comes with a host of “comorbidities” ranging from significant developmental and cognitive complications, mood and anxiety disorders, and social stigma, to seizure-related injuries. This leads to the staggering annual health care cost of $15.5 billion in the USA alone [2], and over €13.8 billion in Europe [3]. Mortality risk triples for all patients with epilepsy, and the rate of sudden death is increased at least 20 times in those with uncontrolled seizures [4]. The burden of the disease is huge, but there remains an unmet need to better treat it and, more importantly, prevent it. Despite booming rates in new drug development, and new surgical and diagnostic techniques, seizures remain uncontrolled in up to 40 % of patients treated with medications, and in a similar proportion of patients who undergo resective epilepsy surgery [1]. Given this overwhelming disease burden and the limited success of available treatments, the efforts to reduce the development of epilepsy from the outset—preventing “epileptogenesis”—become imperative. Extensive work has been done over the last few decades to mature the concepts of epileptogenesis to where we stand today. The definition of the term “epileptogenesis” itself is constantly evolving, and the venues of research investigating it range from targeted, hypothesis-driven, animal model- or cell-based experimentation, to hypothesis-generating high-throughput “omics” approaches, including metabolomics and connectomics. This issue of Neurotherapeutics will review the most current understanding and research in this field, and provide a window to the future. Throughout the issue, prominent clinical researchers paired with established basic scientists provide a balanced and comprehensive discussion of each topic, illustrating both the mechanistic understanding and the clinical translation. The issue will start with a historical perspective and a discussion of the evolution in our concepts and approaches to understanding epileptogenesis and its biomarkers. The first article, by Pitkanen and Engel [5] is designed to set the stage for the rest of the issue by laying out the main current accepted general principles and the challenges lying ahead. Next, a series of papers dissect the mechanisms of epileptogenesis in each of the major epilepsy etiologies. This section on “mechanisms” is designed to take the “epileptogenesis” discussion to a deeper and more specific level, focusing it on the most frequent and/or challenging epilepsy syndromes, including hippocampal sclerosis, tackled by Patterson et al. [6]; the development of epilepsy after head trauma, addressed by Pitkanen and Immonem [7]; and the defiant issue of epileptogenesis in “non-lesional” epilepsies, undertaken by Guerrini and Marini [8]. A separate article by Aronica and Crino [9] brings to the forefront the state-of-the-art understanding of epileptogenesis in developmental brain tumors and malformations of cortical development. Special pediatric considerations related to pediatric epilepsy syndromes are highlighted in a dedicated paper by Pardo et al. [10] This section is concluded by reviewing mechanisms of epileptogenesis in autoimmune epilepsies, as reviewed by Bien and Bauer [11]. The next major section in this issue focuses on an evaluation of the most promising potential epileptogenesis biomarkers currently available or under investigation. The wide range of molecular, genetic, electrophysiological, and neuroimaging biomarkers are each reviewed in detail. Each paper reviews the evidence and processes underlying specific proposed biomarkers, and discusses their clinical implications. More than anything, this section highlights the wide gaps between the current correlations of these “proposed biomarkers” with specific epilepsy syndromes/seizures and the still elusive goal of predicting future epileptogenesis for most epilepsies. The wealth of research linking epilepsy “comorbidities”, such as depression and emotional challenges, to the processes of epileptogenesis will be reviewed at the end of this section by Kanner et al. [12]. The final section of this issue is devoted to therapeutic approaches, thus reflecting the culmination of the work that has been so far devoted to understanding how epileptogenesis develops (mechanisms) and how to assess it (biomarkers) into how this knowledge can ultimately improve patient care and outcomes. This section includes a set of articles dedicated to the experimental models used to search for effective antiepileptogenic agents by White and Loscher [13], followed by an overview of the most promising such drugs for clinical translation in humans by Kaminski et al. [14]. The particular issues and challenges of designing clinical trials to test potential antiepileptogenic agents are aptly discussed in a dedicated paper by Schmidt et al. [15]. We hope that this issue will provide a comprehensive overview of where the field of epilepsy currently stands in relation to understanding the process of epileptogenesis. More importantly, we hope that the body of work presented here begins to ask the right questions necessary to advance the field.

Inflammation in the central nervous system (CNS) is associated with epilepsy and is characterized by the increased levels of a complex set of soluble molecules and their receptors in epileptogenic foci with profound neuromodulatory effects. These molecules activate receptor-mediated pathways in glia and neurons that contribute to hyperexcitability in neural networks that underlie seizure generation. As a consequence, exciting new opportunities now exist for novel therapies targeting the various components of the immune system and the associated inflammatory mediators, especially the IL-1β system. This review summarizes recent findings that increased our understanding of the role of inflammation in reducing seizure threshold, contributing to seizure generation, and participating in epileptogenesis. We will discuss preclinical studies supporting the hypothesis that pharmacological inhibition of specific proinflammatory signalings may be useful to treat drug-resistant seizures in human epilepsy, and possibly delay or arrest epileptogenesis.

Limbic status epilepticus was induced in rats by unilateral 60-min electrical stimulation of the CA3 region of the ventral hippocampus. As assessed by RT-PCR followed by Southern blot analysis, transcripts of interleukin-1β, interleukin-6, interleukin-1 receptor antagonist and inducible nitric oxide synthase were significantly increased 2 h after status epilepticus in the stimulated hippocampus. Induction was maximal at 6 h for interleukin-1β (445%), interleukin-6 (405%) and tumour necrosis factor-α (264%) and at 24 h for interleukin-1 receptor antagonist (494%) and inducible nitric oxide synthase (432%). In rats with spontaneous seizures (60 days after status epilepticus), interleukin-1β mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1β, interleukin-6 and tumour necrosis factor-α was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1β immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 μg/3 μL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-α content in the hippocampus measured 18 h after status epilepticus. Thus, the induction of spontaneously recurring seizures in rats involves the activation of inflammatory cytokines and related pro- and anti-inflammatory genes in the hippocampus. These changes may play an active role in hyperexcitability of the epileptic tissue.

Antagonism of Peripheral Inflammation Reduces the Severity of Status Epilepticus. Marchi N, Fan Q, Ghosh C, Fazio V, Bertolini F, Betto G, Batra A, Carlton E, Najm I, Granata T, Janigro D. Neurobiol Dis 2009;33(2):171–181. Status epilepticus (SE) is one of the most serious manifestations of epilepsy. Systemic inflammation and damage of blood-brain barrier (BBB) are etiologic cofactors in the pathogenesis of pilocarpine SE while acute osmotic disruption of the BBB is sufficient to elicit seizures. Whether an inflammatory-vascular-BBB mechanism could apply to the lithium–pilocarpine model is unknown. LiCl facilitated seizures induced by low-dose pilocarpine by activation of circulating T-lymphocytes and mononuclear cells. Serum IL-1β levels increased and BBB damage occurred concurrently to increased theta EEG activity. These events occurred prior to SE induced by cholinergic exposure. SE was elicited by lithium and pilocarpine irrespective of their sequence of administration supporting a common pathogenetic mechanism. Since IL-1 β is an etiologic trigger for BBB breakdown and its serum elevation occurs before onset of SE early after LiCl and pilocarpine injections, we tested the hypothesis that intravenous administration of IL-1 receptor antagonists (IL-1ra) may prevent pilocarpine-induced seizures. Animals pre-treated with IL-1ra exhibited significant reduction of SE onset and of BBB damage. Our data support the concept of targeting systemic inflammation and BBB for the prevention of status epilepticus.

Recent evidence shows that neuropeptide expression in the CNS is markedly affected by seizure activity, particularly in the limbic system. Changes in neuropeptides in specific neuronal populations depend on the type and intensity of seizures and on their chronic sequelae (i.e. neurodegeneration and spontaneous convulsions). This paper reviews the effects of seizures on somatostatin-containing neurons, somatostatin mRNA and immunoreactivity, the release of this peptide and its receptor subtypes in the CNS. Differences between kindling and status epilepticus in rats are emphasized and discussed in the light of an inhibitory role of somatostatin on hippocampal excitability. Pharmacological studies show that somatostatin affects electrophysiological properties of neurons, modulates classical neurotransmission and has anticonvulsant properties in experimental models of seizures. This peptidergic system may be an interesting target for pharmacological attempts to control pathological hyperactivity in neurons, thus providing new directions for the development of novel anticonvulsant treatments.

Using immunocytochemistry and ELISA, we investigated the production of interleukin (IL)-1β in the rat hippocampus after focal application of kainic acid inducing electroencephalographic (EEG) seizures and CA3 neuronal cell loss. Next, we studied whether EEG seizures per se induced IL-1β and microglia changes in the hippocampus using bicuculline as a nonexcitotoxic convulsant agent. Finally, to address the functional role of this cytokine, we measured the effect of human recombinant (hr)IL-1β on seizure activity as one marker of the response to kainate.Three and 24 hr after unilateral intrahippocampal application of 0.19 nmol of kainate, IL-1β immunoreactivity was enhanced in glia in the injected and the contralateral hippocampi. At 24 hr, IL-1β concentration increased by 16-fold (p< 0.01) in the injected hippocampus. Reactive microglia was enhanced with a pattern similar to IL-1β immunoreactivity. Intrahippocampal application of 0.77 nmol of bicuculline methiodide, which induces EEG seizures but not cell loss, enhanced IL-1β immunoreactivity and microglia, although to a less extent and for a shorter time compared with kainate. One nanogram of (hr)IL-1β intrahippocampally injected 10 min before kainate enhanced by 226% the time spent in seizures (p< 0.01). This effect was blocked by coinjection of 1 μg (hr)IL-1β receptor antagonist or 0.1 ng of 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate, selective antagonists of IL-1β and NMDA receptors, respectively.Thus, convulsant and/or excitotoxic stimuli increase the production of IL-1β in microglia-like cells in the hippocampus. In addition, exogenous application of IL-1β prolongs kainate-induced hippocampal EEG seizures by enhancing glutamatergic neurotransmission.

Seizures increase the synthesis of brain-derived neurotrophic factor in forebrain areas, suggesting this neurotrophin has biological actions in epileptic tissue. The understanding of these actions requires information on the sites and extent of brain-derived neurotrophic factor production in areas involved in seizures onset and their spread. In this study, we investigated by immunocytochemistry the changes in brain-derived neurotrophic factor in the hippocampus, entorhinal and perirhinal cortices of rats at increasing times after acute seizures eventually leading to spontaneous convulsions. We also tested the hypothesis that seizure-induced changes in brain-derived neurotrophic factor induce later modifications in neuropeptide Y expression by comparing, in each instance, their immunoreactive patterns. As early as 100 min after seizure induction, brain-derived neurotrophic factor immunoreactivity increased in CA1 pyramidal and granule neurons and in cells of layers II-III of the entorhinal cortex. At later times, immunoreactivity progressively decreased in somata while increasing in fibres in the hippocampus, the subicular complex and in specific layers of the entorhinal and perirhinal cortices. Changes in neuropeptide Y immunoreactivity were superimposed upon and closely followed those of brain-derived neurotrophic factor. One week after seizure induction, brain-derived neurotrophic factor and neuropeptide Y immunoreactivities were similar to controls in 50% of rats. In rats experiencing spontaneous convulsions, brain-derived neurotrophic factor and neuropeptide Y immunoreactivity was strongly enhanced in fibres in the hippocampus/parahippocampal gyrus and in the temporal cortex. In the dentate gyrus, changes in immunoreactivity depended on sprouting of mossy fibres as assessed by growth-associated protein-43-immunoreactivity. These modifications were inhibited by repeated anticonvulsant treatment with phenobarbital. The dynamic and temporally-linked alterations in brain-derived neurotrophic factor and neuropeptide Y in brain regions critically involved in epileptogenesis suggest a functional link between these two substances in the regulation of network excitability.

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6-nitro-7-sulphamoyl-benzo(F)quinoxaline-2,3-dione], a selective ( rs)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with ‘normal’ gait (from 50% to 90%, P

Enhancing GABAAReceptor α 1 Subunit Levels in Hippocampal Dentate Gyrus Inhibits Epilepsy Development in an Animal Model of Temporal Lobe Epilepsy Raol YH, Lund IV, Bandyopadhyay S, Zhang G, Roberts DS, Wolfe JH, Russek SJ, Brooks-Kayal AR. J Neurosci 2006;26(44):11342–11346. Differential expression of GABAA receptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the adult rat pilocarpine model of TLE found reduced expression of GABR α1 subunits and increased expression of α4 subunits in dentate gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of spontaneous seizure development, we used adeno-associated virus type 2 containing the α4 subunit gene (GABRA4) promoter to drive transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR α1 subunit mRNA and protein in DG at 1–2 weeks after SE. Enhanced α1 expression in DG resulted in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression can affect the development of epilepsy and suggest that α1 subunit levels are important determinants of inhibitory function in hippocampus.

Ethanol-withdrawal Seizures Are Controlled by Tissue Plasminogen Activator via Modulation of NR2B-containing NMDA ReceptorsPawlak R, Melchor JP, Matys T, Skrzypiec AE, Strickland SProc Natl Acad Sci USA 2005;102:443–448Chronic ethanol abuse causes upregulation of N-methyl-D-aspartate (NMDA) receptors, which underlies seizures and brain damage on ethanol withdrawal (EW). Here we show that tissue plasminogen activator (tPA), a protease implicated in neuronal plasticity and seizures, is induced in the limbic system by chronic ethanol consumption, temporally coinciding with upregulation of NMDA receptors. tPA interacts with NR2B-containing NMDA receptors and is required for upregulation of the NR2B subunit in response to ethanol. As a consequence, tPA-deficient mice have reduced NR2B, extracellular signal-regulated kinase 1/2 phosphorylation, and seizures after EW. tPA-mediated facilitation of EW seizures is abolished by NR2B-specific NMDA antagonist ifenprodil. These results indicate that tPA mediates the development of physical dependence on ethanol by regulating NR2B-containing NMDA receptors.Neuroserpin Portland (Ser52Arg) Is Trapped as an Inactive Intermediate That Rapidly Forms Polymers: Implications for the Epilepsy Seen in the Dementia FENIBBelorgey D, Sharp LK, Crowther DC, Onda M, Johansson J, Lomas DAEur J Biochem 2004;271:3360–3367The dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relation with the age at onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive center loop of one neuroserpin molecule with β-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive center loop of neuroserpin Portland being partially inserted into β-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover, the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.

The search of targets for developing novel drugs that can control seizures resistant to available treatments in children and adults represents a great challenge for basic science. In the past decade, emerging evidence pointed out to the crucial role played by glia, the innate immunity brain-resident cells, in the generation of hyperexcitable neuronal networks underlying seizures. Molecular and pharmacological studies targeting glia, and the inflammatory mediators released by these cells in experimental models of epilepsy, highlighted novel targets for drug intervention aimed at interfering with the disease mechanisms, therefore providing putative disease-modifying treatments. This article will focus on the role of immunity activation in the brain and the concomitant release by glia of inflammatory molecules with neuromodulatory properties, in the pathogenesis of epileptic seizures, cell loss, and comorbidities.

A biomarker is defined as an objectively measured characteristic of a normal or pathological biological process. Identification and proper validation of biomarkers of epileptogenesis, the development of epilepsy, and ictogenesis, the propensity to generate spontaneous seizures, might predict the development of an epilepsy condition; identify the presence and severity of tissue capable of generating spontaneous seizures; measure progression after the condition is established; and determine pharmacoresistance. Such biomarkers could be used to create animal models for more cost-effective screening of potential antiepileptogenic and antiseizure drugs and devices, and to reduce the cost of clinical trials by enriching the trial population, and acting as surrogate markers to shorten trial duration. The objectives of the biomarker subgroup for the London Workshop were to define approaches for identifying possible biomarkers for these purposes. Research to identify reliable biomarkers may also reveal underlying mechanisms that could serve as therapeutic targets for the development of new antiepileptogenic and antiseizure compounds.

Toll-like receptor 4 (TLR4) activation in neuron and astrocytes by High Mobility Group Box 1 (HMGB1) protein is a key mechanism of seizure generation. HMGB1 also activates the Receptor for Advanced Glycation Endproducts (RAGE), but it was unknown whether RAGE activation contributes to seizures or to HMGB1 proictogenic effects. We found that acute EEG seizures induced by 7ng intrahippocampal kainic acid (KA) were significantly reduced in Rage-/- mice relative to wild type (Wt) mice. The proictogenic effect of HMGB1 was decreased in Rage-/- mice, but less so, than in Tlr4-/- mice. In a mouse mesial temporal lobe epilepsy (mTLE) model, status epilepticus induced by 200ng intrahippocampal KA and the onset of the spontaneous epileptic activity were similar in Rage-/-, Tlr4-/- and Wt mice. However, the number of hippocampal paroxysmal episodes and their duration were both decreased in epileptic Rage-/- and Tlr4-/- mice vs Wt mice. All strains of epileptic mice displayed similar cognitive deficits in the novel object recognition test vs the corresponding control mice. CA1 neuronal cell loss was increased in epileptic Rage-/- vs epileptic Wt mice, while granule cell dispersion and doublecortin (DCX)-positive neurons were similarly affected. Notably, DCX neurons were preserved in epileptic Tlr4-/- mice. We did not find compensatory changes in HMGB1-related inflammatory signaling nor in glutamate receptor subunits in Rage-/- and Tlr4-/- naïve mice, except for ~20% NR2B subunit reduction in Rage-/- mice. RAGE was induced in neurons, astrocytes and microvessels in human and experimental mTLE hippocampi. We conclude that RAGE contributes to hyperexcitability underlying acute and chronic seizures, as well as to the proictogenic effects of HMGB1. RAGE and TLR4 play different roles in the neuropathologic sequelae developing after status epilepticus. These findings reveal new molecular mechanisms underlying seizures, cell loss and neurogenesis which involve inflammatory pathways upregulated in human epilepsy.

Using immunocytochemistry and in situ hybridization analysis of mRNA, we investigated the changes in the expression of somatostatin and neuropeptide Y (NPY) in the rat hippocampal principal neurons in kindling or after electrically induced status epilepticus (SE), two models of limbic epilepsy associated with different chronic sequelae of seizures and seizure-related neuropathology. At the preconvulsive stage 2 of kindling and after three consecutive tonic-clonic seizures (stage 5) but not after a single-discharge (AD), somatostatin and NPY immunoreactivity (IR) were markedly increased in interneurons of the deep hilus and the polymorphic cell layer and their presumed projections to the outer molecular layer of the dentate gyrus. Increased mRNA levels were observed in the same neurons. NPY IR and mRNA were highly expressed in pyramidal-shaped basket cells at both stages of kindling. IR was similar two days after stages 2 or 5 of kindling while less pronounced effects were observed one week after kindling completion. Peptide-containing neurons in the hilus appeared well preserved in spite of an average of 24% reduction of Nissl stained cells (p0.01) in the stimulated and contralateral hippocampus at stage 5. No sprouting of mossy fibres in the inner molecular layer was found as assessed by Timm staining. Thirty days after SE, somatostatin IR was slightly reduced or similar to controls in the ventral dentate gyrus and molecular layer in four or six rats (SE-I group) while in the two other post-SE rats (SE-II), somatostatin IR was lost. These changes were associated with a different extent of neurodegeneration as assessed by cell counting of Nissl stained sections. In the granule cells/mossy fibres NPY-IR was transiently expressed at stage 2 and after a single AD. Differently, NPY-IR was persistently enhanced in the mossy fibres of all post-SE rats particularly in the SE-II group. In these rats, NPY immunoreactive fibres were detected in the infrapyramidal region of the stratum oriens CA3 and in the inner molecular layer of the dentate gyrus very likely labeling sprouted mossy fibres. In the hippocampus proper of kindled rats, somatostatin and NPY IR were respectively enhanced in the stratum lacunosum moleculare, the subiculum and in the alveus while no significant changes were observed after SE. Changes in peptide expression were bilateral and involved both the dorsal and the ventral hippocampus. The lasting modifications in peptides IR and mRNA expression in distinct neuronal populations of the hippocampus may reflect functional modifications neurons and play a role in limbic epileptogenesis.

In situ hybridization histochemistry with somatostatin sst1-sst5 receptor messenger RNA-selective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr3-octreotide, [Leu2,D-Trp22,125I-Tyr25]somatostatin-28 and [125I]CGP 23996 ([125I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-1 receptor-selective [125I]Tyr3-octreotide and the non-subtype-selective [Leu3,D-Trp22,125I-Tyr25]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand [125I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, [125I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CA1 subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst1-sst5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-1 receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

1. We studied whether the stimulation of 5-HT1A receptors by 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a specific 5-HT1A receptor agonist, reduced electroencephalographic (EEG) seizures induced by intrahippocampal injection of 0.04 microgram in 0.5 microliter of the glutamate analogue kainic acid in freely-moving rats. 2. Pretreatment with 8-OH-DPAT 15 min earlier at the same site as kainic acid injection, caused a dose-dependent decrease of kainic acid-induced seizure activity. One and 10 micrograms significantly reduced the total time spent in seizures by 72% on average and the total number of seizures by 58% (P < 0.01) and 43% (P < 0.05) respectively. The latency to onset of the first seizure was increased 2.8 times (P < 0.01) only after 1 microgram 8-OH-DPAT; 0.1 microgram was ineffective on all seizure parameters. 3. Systemic administration of 25, 100 and 1000 micrograms kg-1 8-OH-DPAT significantly reduced the total number of seizures and the total time in seizures induced by intrahippocampal kainic acid by 52% and 74% on average. The latency to onset of the first seizure was delayed 1.8 times by 100 and 1000 micrograms kg-1 (P < 0.05). 4. The anticonvulsant action of 8-OH-DPAT given intrahippocampally or systemically was significantly blocked by 5 micrograms, but not 1 microgram WAY 100635, a selective 5-HT1A receptor antagonist, administered in the hippocampus before the agonist. 5. These results indicate that postsynaptic 5-HT1A receptors in the hippocampus mediate the anticonvulsant action of 8-OH-DPAT and that their stimulation has an inhibitory role in the generation of limbic seizures.

The in vitro release of somatostatin and neuropeptide Y, their tissue concentration and immunocytochemical pattern were examined in the entorhinal cortex of chronically epileptic rats. A systemic administration of 12 mg/kg kainic acid causing generalized tonic-clonic seizures for at least 3 h after injection was used to induce, 60 days later, a chronically enhanced susceptibility to seizures in the rats. The release of both peptides under depolarizing conditions was significantly reduced by 15% on average from slices of the entorhinal cortex two days after kainic acid-induced status epilepticus. At 60 days, the spontaneous and 30 mM KCl-induced release of somatostatin was significantly enhanced by 30% on average. The release induced by 100 mM KCl was raised by 70%. The spontaneous, 30 mM and 100 mM KCl-induced release of neuropeptide Y from the same slices was increased, respectively, by 120%, 76% and 36%. The late changes were associated with an increased tissue concentration of neuropeptide Y but not of somatostatin. This was confirmed by immunocytochemical evidence showing that neuropeptide Y-, but not somatostatin-immunoreactive neurons were increased in the entorhinal cortex of kainic acid-treated rats. These results indicate that neurotransmission mediated by somatostatin and neuropeptide Y, two peptides previously shown to play a role in limbic epileptogenesis, is enhanced in the entorhinal cortex of chronically epileptic rats.

Neuropeptide Y (NPY)-mediated neurotransmission undergoes plastic changes during hippocampal or amygdaloid kindling in rats as shown by changes in peptide cellular expression and distribution, its release and receptor subtype plasticity (for review see 37, 51). Electrophysiological and pharmacological evidence in in vitro and in vivo models of seizures indicate that NPY has predominant inhibitory actions on excitatory glutamate-mediated neurotransmission and it displays anticonvulsant properties in acute models of seizures.37, 51 Information about its functional role in epileptogenesis is more limited although recent evidence points to an antiepileptogenic action of this peptide.4, 38 In this chapter, we will review the available evidence concerning the functional role of endogenous NPY in kindling and we will address the contribution of its receptor subtypes in mediating its antiepileptogenic effects providing novel findings on the involvement of Y2 and Y5 receptor subtypes.

Somatostatin-, neuropeptide Y-, neurokinin B- and cholecystokinin-containing neurons were investigated in the rat hippocampus in two chronic models of temporal lobe epilepsy, i.e. 30 days after rapid kindling or electrically induced status epilepticus (post-status epilepticus). After rapid kindling, somatostatin immunoreactivity was strongly increased in interneurons and in the outer and middle molecular layer of the dentate gyrus. In four of six post-status epilepticus rats (status epilepticus I rats), somatostatin immunoreactivity was slightly increased in the dorsal but decreased in the ventral dentate gyrus and molecular layer. Somatostatin immunoreactivity decreased in neurons of the dorsal hilus in the two other post-status epilepticus rats investigated, while a complete loss was found in the respective ventral extension (status epilepticus-II rats). These changes were associated with a different extent of neurodegeneration as assessed by Nissl staining. Similarly, neuropeptide Y immunoreactivity was enhanced in neurons of the hilus and in the middle and outer molecular layer of the dentate gyrus in the dorsal hippocampus of rapidly kindled and status epilepticus-I rats. Neuropeptide Y and neurokinin B immunoreactivity was enhanced in the mossy fibers of all post-status epilepticus rats, but not in the rapidly kindled rats. In status epilepticus-II rats, neuropeptide Y-and neurokinin B-positive fibers were also detected in the infrapyramidal region of the stratum oriens of CA3 and in the inner molecular layer of the dentate gyrus in the dorsal and ventral hippocampus respectively, labeling presumably sprouted mossy fibers. Increased staining of neuropeptide Y and neurokinin B was found in the alveus after rapid kindling. Cholecystokinin immunoreactivity was markedly increased in the cerebral cortex, Ammon's horn and the molecular layer of the dentate gyrus in the ventral hippocampus of rapidly kindled and post-status epilepticus rats. The lasting changes in the immunoreactive pattern of various peptides in the hippocampus may reflect functional modifications in the corresponding peptide-containing neurons. These changes may be involved in chronic epileptogenesis, which evolves in response to limbic seizures.

Using brain microdialysis we studied the effect of high doses of monosodium glutamate (MSG) on the extracellular concentration of glutamate in the hypothalamus and in the hippocampus of freely moving rats. MSG at 4 g/kg (40% solution) given by gavage caused a significant increase in plasma (5.3 +/- 0.4-fold, P < 0.01) and extracellular glutamate in the hippocampus (4.2 +/- 0.6-fold, P < 0.01) and in the hypothalamus (8.9 +/- 1.7-fold, P < 0.01) compared to control rats receiving a 40% sucrose solution (10 ml/kg). The peak increase was found within 40 min after MSG administration then declining to baseline in the next 80 min. No changes were found in glutamate tissue concentrations. Twenty-one days after ad libitum MSG intake with the diet (approximately 4 g/kg) no changes were found, in plasma, in extracellular and tissue concentration of glutamate in the hypothalamus compared to rats fed with a normal diet. Glutamate release induced by 200 mM KCl was not modified as well. Histological analysis of Nissl-stained brain tissue slices did not reveal any obvious cell loss in the hippocampus after acute or chronic MSG administration.

Long-lasting activation of glia occurs in brain during epileptogenesis, which develops after various central nervous system (CNS) injuries. Glia is the cell source of the biosynthesis and release of molecules that play a role in seizure recurrence and may contribute to epileptogenesis, thus representing a putative biomarker of epilepsy development and severity. In this study, we set up an in vivo longitudinal study using (1) H-magnetic resonance spectroscopy (MRS) to measure metabolite content in the rat hippocampus that could reflect the extent and the duration of glia activation. Our aim was to explore if glia activation during epileptogenesis, or in the chronic epileptic phase, can be used as a biomarker of tissue epileptogenicity (i.e., a measure of epilepsy severity). (1) H-MRS measurements were done in the adult rat hippocampus every 24 h for 7 days after status epilepticus (SE) and in chronic epileptic rats, using a 7 T Bruker Biospec MRI (magnetic resonance imaging)/MRS scanner. We studied changes in metabolite levels that reflect astrocytes (myo-inositol, mIns; glutathione, GSH), microglia/macrophage activation and the associated neuronal cell injury/dysfunction (lactate, Lac; N-acetyl-aspartate, NAA). (1) H-MRS results were validated by post hoc immunohistochemistry using cell-specific markers. Data analysis was done to determine whether correlations exist between the metabolite changes and spontaneous seizure frequency or the extent of neuronal cell loss. The analysis of (1) H-MRS spectra showed a progressive increase in mIns and GSH levels after SE, which was maintained in epileptic rats. Lac signal transiently increased during epileptogenesis being undetectable in chronic epileptic tissue. NAA levels were chronically reduced from day 2 post-SE. Immunohistochemistry confirmed the activation of microglia and astrocytes and the progressive neuronal cell loss. GSH levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures, whereas S100β levels in epileptic tissue were positively correlated with this outcome measure. A negative correlation was also found between GSH or mIns levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. (1) H-MRS is a valuable in vivo technique for determining the extent and temporal profile of glia activation after an epileptogenic injury. S100β levels measured in the epileptic tissue may represent a biomarker of seizure frequency, whereas GSH levels during epileptogenesis could serve as a predictive marker of seizure frequency. Both mIns and GSH levels measured before the onset of spontaneous seizures predict the extent of neuronal cell loss in epileptic tissue. These findings highlight the potential of serial (1) H-MRS analysis for searching epilepsy biomarkers for prognostic, diagnostic, or therapeutic purposes.