Stereoselective Analysis of the Antiseizure Activity of Fenfluramine and Norfenfluramine in Mice: Is l-Norfenfluramine a Better Follow-Up Compound to Racemic-Fenfluramine?

The aim of this study was to investigate the comparative antiseizure activity of the l-enantiomers of d,l-fenfluramine and d,l-norfenfluramine and to evaluate the relationship between their concentration in plasma and brain and anticonvulsant activity. d,l-Fenfluramine, d,l-norfenfluramine and their individual enantiomers were evaluated in the mouse maximal electroshock seizure (MES) test. d,l-Fenfluramine, d,l-norfenfluramine and their individual l-enantiomers were also assessed in the DBA/2 mouse audiogenic seizure model. All compounds were administered intraperitoneally. Brain and plasma concentrations of the test compounds in DBA/2 mice were quantified and correlated with anticonvulsant activity. In the MES test, fenfluramine, norfenfluramine and their enantiomers showed comparable anticonvulsant activity, with ED50 values between 5.1 and 14.8 mg/kg. In the audiogenic seizure model, l-norfenfluramine was 9 times more potent than d,l-fenfluramine and 15 times more potent than l-fenfluramine based on ED50 (1.2 vs. 10.2 and 17.7 mg/kg, respectively). Brain concentrations of all compounds were about 20-fold higher than in plasma. Based on brain EC50 values, l-norfenfluramine was 7 times more potent than d,l-fenfluramine and 13 times more potent than l-fenfluramine (1940 vs. 13,200 and 25,400 ng/g, respectively). EC50 values for metabolically formed d,l-norfenfluramine and l-norfenfluramine were similar to brain EC50 values of the same compounds administered as such, suggesting that, in the audiogenic seizure model, the metabolites were responsible for the antiseizure activity of the parent compounds. Because of the evidence linking d-norfenfluramine to d,l-fenfluramine to cardiovascular and metabolic adverse effects, their l-enantiomers could potentially be safer follow-up compounds to d,l-fenfluramine. We found that, in the models tested, the activity of l-fenfluramine and l-norfenfluramine was comparable to that of the corresponding racemates. Based on the results in DBA/2 mice and other considerations, l-norfenfluramine appears to be a particularly attractive candidate for further evaluation as a novel, enantiomerically pure antiseizure medication.

Epilepsy-predictive magnetic resonance imaging changes following experimental febrile status epilepticus: Are they translatable to the clinic?

OBJECTIVE: A subset of children with febrile status epilepticus (FSE) are at risk for development of temporal lobe epilepsy later in life. We sought a noninvasive predictive marker of those at risk that can be identified soon after FSE, within a clinically realistic timeframe. METHODS: Longitudinal T2 -weighted magnetic resonance imaging (T2 WI MRI) of rat pups at several time points after experimental FSE (eFSE) was performed on a high-field scanner followed by long-term continuous electroencephalography. In parallel, T2 WI MRI scans were performed on a 3.0-T clinical scanner. Finally, chronic T2 WI MRI signal changes were examined in rats that experienced eFSE and were imaged months later in adulthood. RESULTS: Epilepsy-predicting T2 changes, previously observed at 2 hours after eFSE, persisted for at least 6 hours, enabling translation to the clinic. Repeated scans, creating MRI trajectories of T2 relaxation times following eFSE, provided improved prediction of epileptogenesis compared with a single MRI scan. Predictive signal changes centered on limbic structures, such as the basolateral and medial amygdala. T2 WI MRI changes, originally described on high-field scanners, can also be measured on clinical MRI scanners. Chronically elevated T2 relaxation times in hippocampus were observed months after eFSE in rats, as noted for post-FSE changes in children. SIGNIFICANCE: Early T2 WI MRI changes after eFSE provide a strong predictive measure of epileptogenesis following eFSE, on both high-field and clinical MRI scanners. Importantly, the extension of the acute signal changes to at least 6 hours after the FSE enables its inclusion in clinical studies. Chronic elevations of T2 relaxation times within the hippocampal formation and related structures are common to human and rodent FSE, suggesting that similar processes are involved across species.

A novel, noninvasive, predictive epilepsy biomarker with clinical potential.

A significant proportion of temporal lobe epilepsy (TLE), a common, intractable brain disorder, arises in children with febrile status epilepticus (FSE). Preventative therapy development is hampered by our inability to identify early the FSE individuals who will develop TLE. In a naturalistic rat model of FSE, we used high-magnetic-field MRI and long-term video EEG to seek clinically relevant noninvasive markers of epileptogenesis and found that reduced amygdala T2 relaxation times in high-magnetic-field MRI hours after FSE predicted experimental TLE. Reduced T2 values likely represented paramagnetic susceptibility effects derived from increased unsaturated venous hemoglobin, suggesting augmented oxygen utilization after FSE termination. Indeed, T2 correlated with energy-demanding intracellular translocation of the injury-sensor high-mobility group box 1 (HMGB1), a trigger of inflammatory cascades implicated in epileptogenesis. Use of deoxyhemoglobin-sensitive MRI sequences enabled visualization of the predictive changes on lower-field, clinically relevant scanners. This novel MRI signature delineates the onset and suggests mechanisms of epileptogenesis that follow experimental FSE.

The contribution of NaV1.6 to the efficacy of voltage-gated sodium channel inhibitors in wild type and NaV1.6 gain-of-function (GOF) mouse seizure control.

BACKGROUND AND PURPOSE: Inhibitors of voltage-gated sodium channels (NaVs) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Nav channels as a means of informing the development of improved antiseizure drugs. EXPERIMENTAL APPROACH: We created a series of compounds with diverse selectivity profiles enabling block of NaV1.6 alone or together with NaV1.2. These novel NaV inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding NaV1.6) and in wild-type mice. KEY RESULTS: Pharmacologic inhibition of NaV1.6 in Scn8aN1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. NaV1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS NaV isoforms. CONCLUSIONS AND IMPLICATIONS: Our data suggest NaV1.6 inhibition is a driver of efficacy for NaV inhibitor anti-seizure medicines. Sparing the NaV1.1 channels of inhibitory interneurons did not compromise efficacy. Selective NaV1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies.

Correlated memory defects and hippocampal dendritic spine loss after acute stress involve corticotropin-releasing hormone signaling.

Stress affects the hippocampus, a brain region crucial for memory. In rodents, acute stress may reduce density of dendritic spines, the location of postsynaptic elements of excitatory synapses, and impair long-term potentiation and memory. Steroid stress hormones and neurotransmitters have been implicated in the underlying mechanisms, but the role of corticotropin-releasing hormone (CRH), a hypothalamic hormone also released during stress within hippocampus, has not been elucidated. In addition, the causal relationship of spine loss and memory defects after acute stress is unclear. We used transgenic mice that expressed YFP in hippocampal neurons and found that a 5-h stress resulted in profound loss of learning and memory. This deficit was associated with selective disruption of long-term potentiation and of dendritic spine integrity in commissural/associational pathways of hippocampal area CA3. The degree of memory deficit in individual mice correlated significantly with the reduced density of area CA3 apical dendritic spines in the same mice. Moreover, administration of the CRH receptor type 1 (CRFR(1)) blocker NBI 30775 directly into the brain prevented the stress-induced spine loss and restored the stress-impaired cognitive functions. We conclude that acute, hours-long stress impairs learning and memory via mechanisms that disrupt the integrity of hippocampal dendritic spines. In addition, establishing the contribution of hippocampal CRH-CRFR(1) signaling to these processes highlights the complexity of the orchestrated mechanisms by which stress impacts hippocampal structure and function.

Neuron-restrictive silencer factor-mediated hyperpolarization-activated cyclic nucleotide gated channelopathy in experimental temporal lobe epilepsy.

OBJECTIVE: Enduring, abnormal expression and function of the ion channel hyperpolarization-activated cyclic adenosine monophosphate gated channel type 1 (HCN1) occurs in temporal lobe epilepsy (TLE). We examined the underlying mechanisms, and investigated whether interfering with these mechanisms could modify disease course. METHODS: Experimental TLE was provoked by kainic acid-induced status epilepticus (SE). HCN1 channel repression was examined at mRNA, protein, and functional levels. Chromatin immunoprecipitation was employed to identify the transcriptional mechanism of repressed HCN1 expression, and the basis for their endurance. Physical interaction of the repressor, NRSF, was abolished using decoy oligodeoxynucleotides (ODNs). Video/electroencephalographic recordings were performed to assess the onset and initial pattern of spontaneous seizures. RESULTS: Levels of NRSF and its physical binding to the Hcn1 gene were augmented after SE, resulting in repression of HCN1 expression and HCN1-mediated currents (I(h) ), and reduced I(h) -dependent resonance in hippocampal CA1 pyramidal cell dendrites. Chromatin changes typical of enduring, epigenetic gene repression were apparent at the Hcn1 gene within a week after SE. Administration of decoy ODNs comprising the NRSF DNA-binding sequence (neuron restrictive silencer element [NRSE]), in vitro and in vivo, reduced NRSF binding to Hcn1, prevented its repression, and restored I(h) function. In vivo, decoy NRSE ODN treatment restored theta rhythm and altered the initial pattern of spontaneous seizures. INTERPRETATION: Acquired HCN1 channelopathy derives from NRSF-mediated transcriptional repression that endures via chromatin modification and may provide insight into the mechanisms of a number of channelopathies that coexist with, and may contribute to, the conversion of a normal brain into an epileptic one.

Mice lacking doublecortin and doublecortin-like kinase 2 display altered hippocampal neuronal maturation and spontaneous seizures.

Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.

NBI-921352, a first-in-class, NaV1.6 selective, sodium channel inhibitor that prevents seizures in Scn8a gain-of-function mice, and wild-type mice and rats.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

Hippocampal dysfunction and cognitive impairments provoked by chronic early-life stress involve excessive activation of CRH receptors.

Chronic stress impairs learning and memory in humans and rodents and disrupts long-term potentiation (LTP) in animal models. These effects are associated with structural changes in hippocampal neurons, including reduced dendritic arborization. Unlike the generally reversible effects of chronic stress on adult rat hippocampus, we have previously found that the effects of early-life stress endure and worsen during adulthood, yet the mechanisms for these clinically important sequelae are poorly understood. Stress promotes secretion of the neuropeptide corticotropin-releasing hormone (CRH) from hippocampal interneurons, activating receptors (CRF(1)) located on pyramidal cell dendrites. Additionally, chronic CRF(1) occupancy negatively affects dendritic arborization in mouse organotypic slice cultures, similar to the pattern observed in middle-aged, early-stressed (CES) rats. Here we found that CRH expression is augmented in hippocampus of middle-aged CES rats, and then tested whether the morphological defects and poor memory performance in these animals involve excessive activation of CRF(1) receptors. Central or peripheral administration of a CRF(1) blocker following the stress period improved memory performance of CES rats in novel-object recognition tests and in the Morris water maze. Consonant with these effects, the antagonist also prevented dendritic atrophy and LTP attenuation in CA1 Schaffer collateral synapses. Together, these data suggest that persistently elevated hippocampal CRH-CRF(1) interaction contributes importantly to the structural and cognitive impairments associated with early-life stress. Reducing CRF(1) occupancy post hoc normalized hippocampal function during middle age, thus offering potential mechanism-based therapeutic interventions for children affected by chronic stress.

Epileptogenesis provoked by prolonged experimental febrile seizures: mechanisms and biomarkers.

Whether long febrile seizures (FSs) can cause epilepsy in the absence of genetic or acquired predisposing factors is unclear. Having established causality between long FSs and limbic epilepsy in an animal model, we studied here if the duration of the inciting FSs influenced the probability of developing subsequent epilepsy and the severity of the spontaneous seizures. We evaluated if interictal epileptifom activity and/or elevation of hippocampal T2 signal on magnetic resonance image (MRI) provided predictive biomarkers for epileptogenesis, and if the inflammatory mediator interleukin-1beta (IL-1beta), an intrinsic element of FS generation, contributed also to subsequent epileptogenesis. We found that febrile status epilepticus, lasting an average of 64 min, increased the severity and duration of subsequent spontaneous seizures compared with FSs averaging 24 min. Interictal activity in rats sustaining febrile status epilepticus was also significantly longer and more robust, and correlated with the presence of hippocampal T2 changes in individual rats. Neither T2 changes nor interictal activity predicted epileptogenesis. Hippocampal levels of IL-1beta were significantly higher for >24 h after prolonged FSs. Chronically, IL-1beta levels were elevated only in rats developing spontaneous limbic seizures after febrile status epilepticus, consistent with a role for this inflammatory mediator in epileptogenesis. Establishing seizure duration as an important determinant in epileptogenesis and defining the predictive roles of interictal activity, MRI, and inflammatory processes are of paramount importance to the clinical understanding of the outcome of FSs, the most common neurological insult in infants and children.

Altered function of the SCN1A voltage-gated sodium channel leads to gamma-aminobutyric acid-ergic (GABAergic) interneuron abnormalities.

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),(7) severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a(RH/RH) mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a(RH/+) heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a(RH/+) and Scn1a(RH/RH) mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.

Temperature elevation increases GABA(A) -mediated cortical inhibition in a mouse model of genetic epilepsy.

A missense mutation (R43Q) in the γ2 subunit of the γ-aminobutyric acid (GABA)(A) receptor is associated with generalized (genetic) epilepsy with febrile seizures plus (GEFS+). Heterozygous GABA(A) γ2(R43Q) mice displayed a lower temperature threshold for thermal seizures as compared to wild-type littermates. Temperature-dependent internalization of GABA(A) γ2(R43Q)-containing receptors has been proposed as a mechanism underlying febrile seizure genesis in patients with this mutation. We tested this idea using the GABA(A) γ2(R43Q) knockin mouse model and analyzed GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in acute brain slices after exposure to varying temperatures. Incubation of slices at an elevated temperature increased mIPSC amplitude in neurons from heterozygous mice, with no change seen in wild-type controls. [³H]Flumazenil binding measured in whole-brain homogenates from mutant and control mice following elevation of body temperature showed no temperature-dependent differences in γ2-containing receptor density. Therefore, in vivo mouse data do not support earlier in vitro observations that proposed temperature-dependent internalization of γ2 R43Q containing GABA(A) receptors as the cellular mechanism underlying febrile seizure genesis in patients with the GABA(A) γ2(R43Q) mutation.

Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

Rapid loss of dendritic spines after stress involves derangement of spine dynamics by corticotropin-releasing hormone.

Chronic stress causes dendritic regression and loss of dendritic spines in hippocampal neurons that is accompanied by deficits in synaptic plasticity and memory. However, the responsible mechanisms remain unresolved. Here, we found that within hours of the onset of stress, the density of dendritic spines declined in vulnerable dendritic domains. This rapid, stress-induced spine loss was abolished by blocking the receptor (CRFR(1)) of corticotropin-releasing hormone (CRH), a hippocampal neuropeptide released during stress. Exposure to CRH provoked spine loss and dendritic regression in hippocampal organotypic cultures, and selective blockade of the CRFR(1) receptor had the opposite effect. Live, time-lapse imaging revealed that CRH reduced spine density by altering dendritic spine dynamics: the peptide selectively and reversibly accelerated spine retraction, and this mechanism involved destabilization of spine F-actin. In addition, mice lacking the CRFR(1) receptor had augmented spine density. These findings support a mechanistic role for CRH-CRFR(1) signaling in stress-evoked spine loss and dendritic remodeling.

Fever, febrile seizures and epilepsy.

Seizures induced by fever (febrile seizures) are the most common type of pathological brain activity in infants and children. These febrile seizures and their potential contribution to the mechanisms of limbic (temporal lobe) epilepsy have been a topic of major clinical and scientific interest. Key questions include the mechanisms by which fever generates seizures, the effects of long febrile seizures on neuronal function and the potential contribution of these seizures to epilepsy. This review builds on recent advances derived from animal models and summarizes our current knowledge of the mechanisms underlying febrile seizures and of changes in neuronal gene expression and function that facilitate the enduring effects of prolonged febrile seizures on neuronal and network excitability. The review also discusses the relevance of these findings to the general mechanisms of epileptogenesis during development and points out gaps in our knowledge, including the relationship of animal models to human febrile seizures and epilepsy.

Dexamethasone Attenuates Hyperexcitability Provoked by Experimental Febrile Status Epilepticus.

The role of neuroinflammation in the mechanisms of epilepsy development is important because inflammatory mediators provide tractable targets for intervention. Inflammation is intrinsically involved in the generation of childhood febrile seizures (FSs), and prolonged FS [febrile status epilepticus (FSE)] precedes a large proportion of adult cases of temporal lobe epilepsy (TLE). As TLE is often refractory to therapy and is associated with serious cognitive and emotional problems, we investigated whether its development can be prevented using anti-inflammatory strategies. Using an immature rat model of FSE [experimental FSE (eFSE)], we administered dexamethasone (DEX), a broad anti-inflammatory agent, over 3 d following eFSE. We assessed eFSE-provoked hippocampal network hyperexcitability by quantifying the presence, frequency, and duration of hippocampal spike series, as these precede and herald the development of TLE-like epilepsy. We tested whether eFSE provoked hippocampal microgliosis, astrocytosis, and proinflammatory cytokine production in male and female rats and investigated blood-brain barrier (BBB) breaches as a potential contributor. We then evaluated whether DEX attenuated these eFSE sequelae. Spike series were not observed in control rats given vehicle or DEX, but occurred in 41.6% of eFSE-vehicle rats, associated with BBB leakage and elevated hippocampal cytokines. eFSE did not induce astrocytosis or microgliosis but provoked BBB disruption in 60% of animals. DEX significantly reduced spike series prevalence (to 7.6%) and frequency, and abrogated eFSE-induced cytokine production and BBB leakage (to 20%). These findings suggest that a short, postinsult intervention with a clinically available anti-inflammatory agent potently attenuates epilepsy-predicting hippocampal hyperexcitability, potentially by minimizing BBB disruption and related neuroinflammation.

Neuropeptide Y: potential role in recurrent developmental seizures.

Seizures induce profound plastic changes in the brain, including altered expression of neuropeptide Y (NPY) and its receptors. Here, I discuss a potential role of NPY plasticity in the developmental brain: in a rat model of febrile seizures (FS), the most common type of seizures in infants and young children, NPY expression was up-regulated in hippocampus after experimentally induced FS. Interestingly, NPY up-regulation was associated with an increased seizure threshold for additional (recurrent) FS, and this effect was abolished when an antagonist against NPY receptor type 2 was applied. These findings suggest that inhibitory actions of NPY, released after seizures, exert a protective effect that reduces the risk of seizure recurrence in the developing brain.

Temporal lobe epilepsy after experimental prolonged febrile seizures: prospective analysis.

Experimental prolonged febrile seizures (FS) lead to structural and molecular changes that promote hippocampal hyperexcitability and reduce seizure threshold to further convulsants. However, whether these seizures provoke later-onset epilepsy, as has been suspected in humans, has remained unclear. Previously, intermittent EEGs with behavioural observations for motor seizures failed to demonstrate spontaneous seizures in adult rats subjected to experimental prolonged FS during infancy. Because limbic seizures may be behaviourally subtle, here we determined the presence of spontaneous limbic seizures using chronic video monitoring with concurrent hippocampal and cortical EEGs, in adult rats (starting around 3 months of age) that had sustained experimental FS on postnatal day 10. These subjects were compared with groups that had undergone hyperthermia but in whom seizures had been prevented (hyperthermic controls), as well as with normothermic controls. Only events that fulfilled both EEG and behavioural criteria, i.e. electro-clinical events, were considered spontaneous seizures. EEGs (over 400 recorded hours) were normal in all normothermic and hyperthermic control rats, and none of these animals developed spontaneous seizures. In contrast, prolonged early-life FS evoked spontaneous electro-clinical seizures in 6 out of 17 experimental rats (35.2%). These seizures consisted of sudden freezing (altered consciousness) and typical limbic automatisms that were coupled with polyspike/sharp-wave trains with increasing amplitude and slowing frequency on EEG. In addition, interictal epileptiform discharges were recorded in 15 (88.2%) of the experimental FS group and in none of the controls. The large majority of hippocampally-recorded seizures were heralded by diminished amplitude of cortical EEG, that commenced half a minute prior to the hippocampal ictus and persisted after seizure termination. This suggests a substantial perturbation of normal cortical neuronal activity by these limbic spontaneous seizures. In summary, prolonged experimental FS lead to later-onset limbic (temporal lobe) epilepsy in a significant proportion of rats, and to interictal epileptifom EEG abnormalities in most others, and thus represent a model that may be useful to study the relationship between FS and human temporal lobe epilepsy.

Dual and Opposing Roles of MicroRNA-124 in Epilepsy Are Mediated through Inflammatory and NRSF-Dependent Gene Networks.

Insult-provoked transformation of neuronal networks into epileptic ones involves multiple mechanisms. Intervention studies have identified both dysregulated inflammatory pathways and NRSF-mediated repression of crucial neuronal genes as contributors to epileptogenesis. However, it remains unclear how epilepsy-provoking insults (e.g., prolonged seizures) induce both inflammation and NRSF and whether common mechanisms exist. We examined miR-124 as a candidate dual regulator of NRSF and inflammatory pathways. Status epilepticus (SE) led to reduced miR-124 expression via SIRT1--and, in turn, miR-124 repression--via C/EBPα upregulated NRSF. We tested whether augmenting miR-124 after SE would abort epileptogenesis by preventing inflammation and NRSF upregulation. SE-sustaining animals developed epilepsy, but supplementing miR-124 did not modify epileptogenesis. Examining this result further, we found that synthetic miR-124 not only effectively blocked NRSF upregulation and rescued NRSF target genes, but also augmented microglia activation and inflammatory cytokines. Thus, miR-124 attenuates epileptogenesis via NRSF while promoting epilepsy via inflammation.

Developmental febrile seizures modulate hippocampal gene expression of hyperpolarization-activated channels in an isoform- and cell-specific manner.

Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (I(H)). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.