TLR3 preconditioning induces anti-inflammatory and anti-ictogenic effects in mice mediated by the IRF3/IFN-ß axis.

Activation of Toll-like receptor 3 (TLR3) was previously shown to contribute to the generation of epileptic seizures in rodents by evoking a proinflammatory response in the forebrain. This suggests that TLR3 blockade may provide therapeutic effects in epilepsy. We report that brain activation of TLR3 using the synthetic receptor ligand Poly I:C may also result in remarkable dose- and time-dependent inhibitory effects on acute seizures in mice without inducing inflammation. These inhibitory effects are associated with reduced neuronal excitability in the hippocampus as shown by a decrease in the population spike amplitude of CA1 pyramidal neurons following Schaffer collaterals stimulation. TLR3 activation which results in seizure inhibition does not evoke NF-kB-dependent inflammatory molecules or morphological activation of glia, however, it induces the alternative interferon (IFN) regulatory factor (IRF)-3/IFN-ß signaling pathway. IFN-ß reproduced the inhibitory effects of Poly I:C on neuronal excitability in hippocampal slices. Seizure inhibition attained with activation the TLR3-IRF3/IFN-ß axis should be carefully considered when TLR3 are targeted for therapeutic purposes.

Review: Neuroinflammatory pathways as treatment targets and biomarker candidates in epilepsy: emerging evidence from preclinical and clinical studies.

Accumulating evidence indicates an important pathophysiological role of brain inflammation in epilepsy. In this review, we will provide an update of specific inflammatory pathways that have been proposed to be crucial in the underlying molecular mechanisms of epilepsy, including the interleukin-1 receptor/toll-like receptor signalling, cyclooxygenase-2, tumour necrosis factor-alpha, complement signalling and chemokines. Furthermore, by drawing on evidence from preclinical and clinical studies we will discuss the potential of these signalling pathways targets for novel therapeutic interventions that control drug-resistant seizures or have disease-modifying effects. Finally, we will assess the use of these inflammatory pathways as potential biomarkers for the development of epilepsy or to measure the effectiveness of therapeutic interventions.

Pharmacological blockade of IL-1ß/IL-1 receptor type 1 axis during epileptogenesis provides neuroprotection in two rat models of temporal lobe epilepsy.

We studied whether pharmacological blockade of the IL-1ß-mediated signaling, rapidly activated in forebrain by epileptogenic injuries, affords neuroprotection in two different rat models of status epilepticus (SE). As secondary outcome, we measured treatment's effect on SE-induced epileptogenesis. IL-1ß signaling was blocked by systemic administration of two antiinflammatory drugs, namely human recombinant IL-1 receptor antagonist (anakinra), the naturally occurring and clinically used competitive IL-1 receptor type 1 antagonist, and VX-765 a specific non-peptide inhibitor of IL-1ß cleavage and release. Antiinflammatory drugs were given 60min after antiepileptic (AED) drug-controlled SE induced by pilocarpine, or 180min after unrestrained electrical SE, for 7days using a protocol yielding therapeutic drug levels in brain. This drug combination significantly decreased both IL-1ß expression in astrocytes and cell loss in rat forebrain. Neuroprotection and the antiinflammatory effect were more pronounced in the electrical SE model. Onset of epilepsy, and frequency and duration of seizures 3months after electrical SE were not significantly modified. Transcriptomic analysis in the hippocampus showed that the combined treatment did not affect the broad inflammatory response induced by SE during epileptogenesis. In particular, the treatment did not prevent the induction of the complement system and Toll-like receptors, both contributing to cell loss and seizure generation. We conclude that the IL-1ß signaling represents an important target for reducing cell loss after SE. The data highlight a new class of clinically tested agents affording neuroprotection after a delayed post-injury intervention. Earlier blockade of this rapid onset inflammatory pathway during SE, or concomitant treatment with antiinflammatory drugs targeting additional components of the broad inflammatory response to SE, or co-treatment with AEDs, is likely to be required for optimizing beneficial outcomes.

Interleukin-1 type 1 receptor/Toll-like receptor signalling in epilepsy: the importance of IL-1beta and high-mobility group box 1.

Inflammatory processes in brain tissue have been described in human epilepsy of various aetiologies and in experimental models of seizures. This, together with the anticonvulsant properties of anti-inflammatory therapies both in clinical and in experimental settings, highlights the important role of brain inflammation in the aetiopathogenesis of seizures. Preclinical investigations in experimental models using pharmacological and genetic tools have identified a significant contribution of interleukin-1 (IL-1) type 1 receptor/Toll-like receptor (IL-1R/TLR) signalling to seizure activity. This signalling can be activated by ligands associated with infections (pathogen-associated molecular patterns) or by endogenous molecules, such as proinflammatory cytokines (e.g. IL-1beta) or danger signals [damage-associated molecular patterns, e.g. high-mobility group box 1 (HMGB1)]. IL-1beta and HMGB1 are synthesized and released by astrocytes and microglia in the rodent brain during seizures. Notably, a rapid release of HMGB1 from neurons appears to be triggered by proconvulsant drugs even before seizure occurrence and is involved in their precipitation of seizures. The activation of IL-1R/TLR signalling mediates rapid post-translational changes in N-methyl-d-aspartate-gated ion channels in neurons. A long-term decrease in seizure threshold has also been observed, possibly mediated by transcriptional activation of genes contributing to molecular and cellular plasticity. This emerging evidence identifies specific targets with potential anticonvulsant effects in drug-resistant forms of epilepsy.

Molecular and functional interactions between tumor necrosis factor-alpha receptors and the glutamatergic system in the mouse hippocampus: implications for seizure susceptibility.

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine acting on two distinct receptor subtypes, namely p55 and p75 receptors. TNF-alpha p55 and p75 receptor knockout mice were previously shown to display a decreased or enhanced susceptibility to seizures, respectively, suggesting intrinsic modifications in neuronal excitability. We investigated whether alterations in glutamate system function occur in these naive knockout mice with perturbed cytokine signaling that could explain their different propensity to develop seizures. Using Western blot analysis of hippocampal homogenates, we found that p55(-/-) mice have decreased levels of membrane GluR3 and NR1 glutamate receptor subunits while GluR1, GluR2, GluR6/7 and NR2A/B were unchanged as compared to wild-type mice. In p75(-/-) mice, GluR2, GluR3, GluR6/7 and NR2A/B glutamate receptor subunits were increased in the hippocampus while GluR1 and NR1 did not change. Extracellular single-cell recordings of the electrical activity of hippocampal neurons were carried out in anesthetized mice by standard electrophysiological techniques. Microiontophoretic application of glutamate increased the basal firing rate of hippocampal neurons in p75(-/-) mice versus wild-type mice, and this effect was blocked by 2-amino-5-phosphopentanoic acid and 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione denoting the involvement of N-methyl-D-aspartic acid and AMPA receptors. In p55(-/-) mice, hippocampal neurons responses to glutamate were similar to wild-type mice. Spontaneous glutamate release measured by in vivo hippocampal microdialysis was significantly decreased only in p55(-/-) mice. No changes were observed in KCl-induced glutamate release in both receptor knockout mice strains versus wild-type mice. These findings highlight specific molecular and functional interactions between p55 and p75 receptor-mediated signaling and the glutamate system. These interactions may be relevant for controlling neuronal excitability in physiological and pathological conditions.

The IL-1beta system in epilepsy-associated malformations of cortical development.

Focal cortical dysplasia (FCD) and glioneuronal tumors (GNT) are recognized causes of chronic intractable epilepsy. The cellular mechanism(s) underlying their epileptogenicity remain largely unknown. Compelling evidence in experimental models of seizures indicates an important role of interleukin (IL)-1beta in the mechanisms of hyperexcitability leading to the occurrence of seizures. We immunocytochemically investigated the brain expression and cellular distribution pattern of IL-1beta, IL-1 receptor (IL-1R) types I and II and IL-1R antagonist (IL-1Ra) in FCD and GNT specimens, and we correlate these parameters with the clinical history of epilepsy in patients with medically intractable seizures. In normal control cortex, and in perilesional regions with histologically normal cortex, IL-1beta, IL-1Rs and IL-1Ra expression was undetectable. In all FCD and GNT specimens, IL-1beta and its signalling receptor IL-1RI were highly expressed by more than 30% of neurons and glia whereas the decoy receptor IL-RII and IL-Ra were expressed to a lesser extent by approximately 10% and 20% of cells, respectively. These findings show a high expression of IL-1beta and its functional receptor (IL-1RI) in FCD and GNT specimens together with a relative paucity of mechanisms (IL-1RII and IL-1Ra) apt to inactivate IL-1beta actions. Moreover, the number of IL-1beta- and IL-1RI-positive neurons was positively correlated with the frequency of seizures, whereas the number of IL-1Ra-positive neurons and astroglial cells was negatively correlated with the duration of epilepsy prior to surgery. The expression of IL-1beta family members in these developmental lesions may contribute to their intrinsic and high epileptogenicity, thus possibly representing a novel target for antiepileptic strategies.

Status epilepticus induces time-dependent neuronal and astrocytic expression of interleukin-1 receptor type I in the rat limbic system.

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.

Testosterone regulates androgen and estrogen receptor immunoreactivity in rat substantia nigra pars reticulata.

At postnatal day (PN)1, there are sex differences in gonadal receptor expression in the rat substantia nigra pars reticulata (SNR). Male pups have lower levels of androgen receptor (AR) and estrogen receptor (ER)beta immunoreactivity (IR) compared to female pups, while ERalpha IR is equally expressed in the two sexes. To test whether these differences are due to sex differences in testosterone exposure, we injected female pups with testosterone propionate (TP) on the day of birth and analyzed the levels of AR and ER IR at PN1. TP-treated females have lower levels of AR and ERbeta IR than control, while there are no differences in the levels of ERalpha IR. TP treatment did not affect the number of AR and ER expressing cells. The regulation of SNR AR and ERbeta IR by testosterone may be important for the development of sex-specific functional systems involved in motor control.

Sex differences in androgen and estrogen receptor expression in rat substantia nigra during development: an immunohistochemical study.

Gonadal hormones are important regulators of sexual differentiation of the CNS. Exposure to testosterone and estrogen during development causes permanent organizational differences between males and females. We previously described functional sex-related differences of the GABA(A)ergic circuits of the rat substantia nigra pars reticulata (SNR) involved in the control of flurothyl seizures. This sexual differentiation of the SNR is regulated by postnatal testosterone. To assess whether the organizing effects of testosterone in the SNR are mediated via the androgen receptor (AR) and/or estrogen receptors (ER), we used immunohistochemistry to study the ontogeny of AR, ERalpha and ERbeta expression in SNR and substantia nigra pars compacta (SNC) of male and female rats. Rats on the day of birth [postnatal day (PN) 0] and at PN1, PN5, PN15 and PN30 were used. AR- and ERbeta-immunopositive cells were present in SNR and SNC in both sexes and at all ages. ERalpha was not detected in male and female SNC at PN0-PN1. In both substantia nigra (SN) regions, there were developmentally regulated sex differences in AR, ERalpha and ERbeta immunoreactivity. In the SN, each receptor showed specific intracellular localization: AR was present in the nucleus, ERalpha and ERbeta were present both in nuclear and extranuclear compartments. ERalpha was detected also in processes. At PN0-PN1, quantitative analysis revealed sex and regional differences in the distribution of SN cells expressing AR and ERalpha, while ERbeta were equally present in both sexes. The presence of gonadal steroid receptors in the SN suggests that the biological effects of gonadal hormones in the CNS extend beyond reproduction-related functions and may affect and modify motor behaviors (including seizures) in a sex-specific manner. Based on the ontogeny of SNR ERbeta, we hypothesize that postnatal injections of testosterone may regulate the nigral GABA(A) system through the aromatization pathway and activation of ERbeta.

Seizure susceptibility and epileptogenesis are decreased in transgenic rats overexpressing neuropeptide Y.

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<0.05 and 0.01). Transgenic rats were also less susceptible to epileptogenesis than wild-type littermates as demonstrated by a 65% increase in the number of electrical stimuli required to induce stage 5 seizures (P<0.01). This phenotype was associated with a strong and specific expression of neuropeptide Y mRNA in area CA1, a brain area involved in the seizure network. We conclude that endogenous neuropeptide Y overexpression in the rat hippocampus is associated with inhibition of seizures and epileptogenesis suggesting that this system may be a valuable target for developing novel antiepileptic treatments.

Nitric oxide produced by non-motoneuron cells enhances rat embryonic motoneuron sensitivity to excitotoxins: comparison in mixed neuron/glia or purified cultures.

The present study compares the sensitivity to chronic exposure to glutamate agonists of SMI-32-positive rat-derived embryonic motoneurons under both mixed neuron/glia and purified cultures. We found that in spite of a trophic role of glia on cultured motoneurons, SMI-32-positive cells are more sensitive to excitotoxicity in the presence of glia than in purified culture, very likely through nitric oxide released by non-neuronal cells. The rank order of potency for inducing toxicity after 48 h incubation was AMPA>kainate>NMDA, with EC(50): 0.43, 4.9 and 49 microM, respectively, in mixed neuron/glia culture and 14, 32 and 135 microM in purified cultures. The effect of NMDA was dose-dependently potentiated by glycine, with similar potency in the two culture conditions. The effect of agonists was completely antagonized by the specific antagonists CNQX, BNQX and MK801 in both culture conditions. Motoneurons were similarly immunoreactive to NR1 and GluR2 antibodies under both mixed neuron/glia and purified cultures, thus confirming the presence of the calcium-impermeant AMPA receptor subtypes and of the obligatory subunit for NMDA receptors. The effect of kainate in mixed neuron/glia culture was reduced by the addition of 40 microM N-nitro-L-arginine or L-NAME, which shifted the EC(50) to 9 microM. By contrast, L-NAME did not modify the effect of kainic acid in purified cultures. These results suggest that the release of nitric oxide by non-neuronal cells in culture enhances glutamate excitotoxicity in SMI-32-positive cells, and that direct activation of ionotropic glutamate receptors is not enough to explain the mechanism of chronic motoneuron degeneration occurring in vivo in amyotrophic lateral sclerosis (ALS).

Dynamic induction of the long pentraxin PTX3 in the CNS after limbic seizures: evidence for a protective role in seizure-induced neurodegeneration.

Pentraxin 3, a prototypic long pentraxin, is induced by proinflammatory signals in the brain. Inflammatory cytokines are rapidly induced in glia by epileptic activity. We show that pentraxin 3 immunoreactivity and mRNA are enhanced in the rat forebrain above undetectable control levels by limbic seizures with a dual pattern of induction. Within 6 h from seizure onset, pentraxin 3 immunoreactivity was increased in astrocytes. Eighteen to 48 h later, specific neuronal populations and leucocytes were strongly immunoreactive only in areas of neurodegeneration. This staining was abolished when neuronal cell loss, but not seizures, was prevented by blocking N-methyl-D-aspartate receptors. Pentraxin 3 -/- mice had a more widespread seizure-related neuronal damage in the forebrain than their wild-type littermates although both groups had similar epileptic activity. Our results provide evidence that pentraxin 3 is synthesized in brain after seizures and may exert a protective role in seizure-induced neurodegeneration.

Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice.

IL-1beta and its endogenous receptor antagonist (IL-1Ra) are rapidly induced by seizures in the rodent hippocampus. Exogenously applied IL-1beta prolongs seizures in an IL-1R type I-mediated manner. This effect depends on N-methyl-d-aspartate receptor activation. We report here that intrahippocampal application of recombinant IL-1Ra or its selective endogenous overexpression in astrocytes under the control of glial acidic fibrillary protein promoter potently inhibits motor and electroencephalographic seizures induced by bicuculline methiodide in mice. Accordingly, transgenic mice show a reduced seizure-related c-fos mRNA expression in various forebrain areas compared with their wild-type littermates. Recombinant IL-1Ra was ineffective in mice deficient in IL-1R type I, having per se a delayed onset to generalized convulsions. These results demonstrate that IL-1Ra mediates potent anticonvulsant effects acting on IL-1R type I and suggest that the balance between brain IL-1beta and IL-1Ra represents a crucial mechanism to control seizure generalization.

Plastic changes in neuropeptide Y receptor subtypes in experimental models of limbic seizures.

PURPOSE: Neuropetide Y (NPY)-mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. NPY Y2 and Y1 receptors are increased on mossy fiber terminals and decreased on granule cell dendrites after seizures, respectively. We investigated (a) whether seizures modify the NPY Y5 receptors in the hippocampus, and (b) the effect of an agonist at Y2/Y5 receptors and antagonists at Y1 receptors on acute and chronic seizure susceptibility. METHODS: Limbic seizures were induced in rats by electrical stimulation of the dorsal hippocampus, leading to stage 5 kindled seizures, or by intrahippocampal or systemic injections of kainic acid. Pentylentetrazol was administered to epileptic rats to assess their enhanced susceptibility to seizures. NPY Y5 receptor protein was measured in hippocampal homogenates using a specific polyclonal antibody and quantitative Western blotting. RESULTS: Y5 receptors (57-kD band) were transiently decreased (23 to 35%) in all hippocampal subregions 2 and 7 days, but not 2.5 hours, after seizures induced by systemic kainic acid. A minor band of 51 kD was reduced significantly in CA3 and dentate gyrus, although it was increased in CA1, 30 days after seizures, suggesting long-term posttranslational changes in this protein. NPY Y5 receptors were increased by 200% in total homogenate from the stimulated hippocampus 2 days but not 30 days after fully kindled seizures. Intracerebral injections of NPY 13-36 (Y2/Y5 receptor agonist) or BIBP 3225 and BIBO 3304 (selective Y1 receptor antagonists) decreased seizure susceptibility in rats. CONCLUSIONS: These results indicate that NPY Y5 receptors change after limbic seizures and suggest that NPY receptors may provide novel target(s) for the treatment of epilepsy.

Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus.

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-1beta, 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-1beta (445%), interleukin-6 (405%) and tumour necrosis factor-alpha (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-1beta mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1beta, interleukin-6 and tumour necrosis factor-alpha was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1beta immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 microg/3 microL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-alpha 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.

Interleukin-1beta immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures.

Using immunocytochemistry and ELISA, we investigated the production of interleukin (IL)-1beta 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-1beta 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-1beta 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-1beta immunoreactivity was enhanced in glia in the injected and the contralateral hippocampi. At 24 hr, IL-1beta concentration increased by 16-fold (p < 0.01) in the injected hippocampus. Reactive microglia was enhanced with a pattern similar to IL-1beta immunoreactivity. Intrahippocampal application of 0.77 nmol of bicuculline methiodide, which induces EEG seizures but not cell loss, enhanced IL-1beta immunoreactivity and microglia, although to a less extent and for a shorter time compared with kainate. One nanogram of (hr)IL-1beta 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 microgram (hr)IL-1beta receptor antagonist or 0.1 ng of 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate, selective antagonists of IL-1beta and NMDA receptors, respectively. Thus, convulsant and/or excitotoxic stimuli increase the production of IL-1beta in microglia-like cells in the hippocampus. In addition, exogenous application of IL-1beta prolongs kainate-induced hippocampal EEG seizures by enhancing glutamatergic neurotransmission.

Brain-derived neurotrophic factor immunoreactivity in the limbic system of rats after acute seizures and during spontaneous convulsions: temporal evolution of changes as compared to neuropeptide Y.

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.