Lack of Direct Effects of Neurotrophic Factors in an In Vitro Model of Neuroinflammation.

Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.

Viral Vector-Based Gene Therapy for Epilepsy: What Does the Future Hold?

In recent years, many pre-clinical studies have tested gene therapy approaches as possible treatments for epilepsy, following the idea that they may provide an alternative to conventional pharmacological and surgical options. Multiple gene therapy approaches have been developed, including those based on anti-sense oligonucleotides, RNA interference, and viral vectors. In this opinion article, we focus on translational issues related to viral vector-mediated gene therapy for epilepsy. Research has advanced dramatically in addressing issues like viral vector optimization, target identification, strategies of gene expression, editing or regulation, and safety. Some of these pre-clinically validated potential gene therapies are now being tested in clinical trials, in patients with genetic or focal forms of drug-resistant epilepsy. Here, we discuss the ongoing translational research and the advancements that are needed and expected in the near future. We then describe the clinical trials in the pipeline and the further challenges that will need to be addressed at the clinical and economic levels. Our optimistic view is that all these issues and challenges can be overcome, and that gene therapy approaches for epilepsy will soon become a clinical reality.

The NLRP3 Inflammasome in Neurodegenerative Disorders: Insights from Epileptic Models.

Neuroinflammation represents a dynamic process of defense and protection against the harmful action of infectious agents or other detrimental stimuli in the central nervous system (CNS). However, the uncontrolled regulation of this physiological process is strongly associated with serious dysfunctional neuronal issues linked to the progression of CNS disorders. Moreover, it has been widely demonstrated that neuroinflammation is linked to epilepsy, one of the most prevalent and serious brain disorders worldwide. Indeed, NLRP3, one of the most well-studied inflammasomes, is involved in the generation of epileptic seizures, events that characterize this pathological condition. In this context, several pieces of evidence have shown that the NLRP3 inflammasome plays a central role in the pathophysiology of mesial temporal lobe epilepsy (mTLE). Based on an extensive review of the literature on the role of NLRP3-dependent inflammation in epilepsy, in this review we discuss our current understanding of the connection between NLRP3 inflammasome activation and progressive neurodegeneration in epilepsy. The goal of the review is to cover as many of the various known epilepsy models as possible, providing a broad overview of the current literature. Lastly, we also propose some of the present therapeutic strategies targeting NLRP3, aiming to provide potential insights for future studies.

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats during EEG Recordings.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Low-dose 7,8-Dihydroxyflavone Administration After Status Epilepticus Prevents Epilepsy Development.

Temporal lobe epilepsy often manifests months or even years after an initial epileptogenic insult (e.g., stroke, trauma, status epilepticus) and, therefore, may be preventable. However, no such preventive treatment is currently available. Aim of this study was to test an antioxidant agent, 7,8-dihydroxyflavone (7,8-DHF), that is well tolerated and effective in preclinical models of many neurological disorders, as an anti-epileptogenic drug. However, 7,8-DHF also acts as a TrkB receptor agonist and, based on the literature, this effect may imply an anti- or a pro-epileptogenic effect. We found that low- (5 mg/kg), but not high-dose 7,8-DHF (10 mg/kg) can exert strong anti-epileptogenic effects in the lithium-pilocarpine model (i.e., highly significant reduction in the frequency of spontaneous seizures and in the time to first seizure after status epilepticus). The mechanism of these different dose-related effects remains to be elucidated. Nonetheless, considering its excellent safety profile and antioxidant properties, as well as its putative effects on TrkB receptors, 7,8-DHF represents an interesting template for the development of effective and well-tolerated anti-epileptogenic drugs.

Antipsychotic drugs counteract autophagy and mitophagy in multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.

Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells.

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

Implication of sestrin3 in epilepsy and its comorbidities.

Epilepsy is a serious neurological disorder affecting about 1% of the population worldwide. Epilepsy may arise as a result of acquired brain injury, or as a consequence of genetic predisposition. To date, genome-wide association studies and exome sequencing approaches have provided limited insights into the mechanisms of acquired brain injury. We have previously reported a pro-epileptic gene network, which is conserved across species, encoding inflammatory processes and positively regulated by sestrin3 (SESN3). In this study, we investigated the phenotype of SESN3 knock-out rats in terms of susceptibility to seizures and observed a significant delay in status epilepticus onset in SESN3 knock-out compared to control rats. This finding confirms previous in vitro and in vivo evidence indicating that SESN3 may favour occurrence and/or severity of seizures. We also analysed the phenotype of SESN3 knock-out rats for common comorbidities of epilepsy, i.e., anxiety, depression and cognitive impairment. SESN3 knock-out rats proved less anxious compared to control rats in a selection of behavioural tests. Taken together, the present results suggest that SESN3 may regulate mechanisms involved in the pathogenesis of epilepsy and its comorbidities.

Gene Therapy Tools for Brain Diseases.

Neurological disorders affecting the central nervous system (CNS) are still incompletely understood. Many of these disorders lack a cure and are seeking more specific and effective treatments. In fact, in spite of advancements in knowledge of the CNS function, the treatment of neurological disorders with modern medical and surgical approaches remains difficult for many reasons, such as the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ due to the blood-brain barrier. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. Gene therapy can result in a stable or inducible expression of transgene(s), and can allow a nearly specific expression in target cells. In this review, we will discuss the most commonly used tools for the delivery of genetic material in the CNS, including viral and non-viral vectors; their main applications; their advantages and disadvantages. We will discuss mechanisms of genetic regulation through cell-specific and inducible promoters, which allow to express gene products only in specific cells and to control their transcriptional activation. In addition, we will describe the applications to CNS diseases of post-transcriptional regulation systems (RNA interference); of systems allowing spatial or temporal control of expression [optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)]; and of gene editing technologies (CRISPR/Cas9, Zinc finger proteins). Particular attention will be reserved to viral vectors derived from herpes simplex type 1, a potential tool for the delivery and expression of multiple transgene cassettes simultaneously.

A Matter of Genes: The Hurdles of Gene Therapy for Epilepsy.

Gene therapy has recently advanced to the level of standard of care for several diseases. However, its application to neurological disorders is still in the experimental phase. In this review, we discuss recent advancements in the field that provide optimism on the possibility to have first-in-human studies for gene therapy of some forms of epilepsy in the not so distant future.

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Personalized Needles for Microinjections in the Rodent Brain.

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

[Incidence and risk factors associated with the development of pressure ulcers in an Italian general intensive care unit]. / Incidenza e fattori di rischio associatiallo sviluppo di lesioni da pressione in una terapia intensiva generale italiana.

. Incidence and risk factors associated with the development of pressure ulcers in an Italian general intensive care unit. INTRODUCTION: Pressure ulcers (PU) represent one of the most frequent adverse event in intensive care units (ICU). Critical patients are at higher risk of developing a PU, with an incidence between 3.3-39.3%. AIM: To assess the incidence and risk factors for developing PUs in a general ICU of an Italian University hospital. METHODS: Retrospective observational study on a sample of 122 patients admitted to a general intensive care unit, from January to December 2015, with a length of ICU stay > 5 days. PUs were stadied according to the National Pressure Ulcer Advisory Panel. RESULTS: One hundred and twenty two patients were enrolled. The incidence of PUs was 33%, and the incidence rate was 23 PUs for 1.000 days of ICU stay; 29% of PUs were of stage I, 56% of stage II, 8% stage III and 6% stage IV. PUs mainly affected the sacrum (21%) with a very low incidence in the heels (1.6%). Despite several factors were associated to the development of PUs (being female, Sequential Organ Failure Assessment, length of stay, mechanical ventilation > 72 hours, administration of dobutamine, Admission pO 2/FiO2 ratio, mean Braden during ICU stay and admission diagnosis) only ICU lenght of day remained statistically significant. CONCLUSIONS: The incidence of PUs is comparable to other recent studies. Many factors are potentially involved in the onset of PUs. Identifying these factors can help nurses to plan interventions to prevent their development.

Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes.

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

Effects of [Nphe1, Arg14, Lys15] N/OFQ-NH2 (UFP-101), a potent NOP receptor antagonist, on molecular, cellular and behavioural alterations associated with chronic mild stress.

The present study investigated the effect of [Nphe1] Arg14, Lys15-N/OFQ-NH2 (UFP-101), a selective NOP receptor antagonist, in chronic mild stress (CMS) in male Wistar rats. NOP receptor antagonists were reported to elicit antidepressant-like effects in rodents. Our aim was to investigate UFP-101 effects on CMS-induced anhedonia and impairment of hippocampal neurogenesis. UFP-101 (10 nmol/rat intracerebroventricularly) did not influence sucrose intake in non-stressed animals, but reinstated basal sucrose consumption in stressed animals from the second week of treatment. UFP-101 also reversed stress effects in forced swimming test and in open field. Fluoxetine (10 mg/kg intraperitoneally) produced similar effects. Moreover, we investigated whether UFP-101 could affect CMS-induced impairment in hippocampal cell proliferation and neurogenesis, and in fibroblast growth factor (FGF-2) expression. Our data confirm that CMS reduced neural stem cell proliferation and neurogenesis in adult rat hippocampus. Chronic UFP-101 treatment did not affect the reduced proliferation (bromodeoxyuridine-positive cells) observed in stressed animals. However, UFP-101 increased the number of doublecortin-positive cells, restoring neurogenesis. Finally, UFP-101 significantly increased FGF-2 expression, reduced by CMS. These findings support the view that blockade of NOP receptors produces antidepressant-like effects in CMS associated with positive effects on neurogenesis and FGF-2 expression. Therefore, NOP receptors may represent a target for innovative antidepressant drugs.

Is autopsy tissue a valid control for epilepsy surgery tissue in microRNA studies?

MicroRNAs (miRNAs) are differentially expressed in the brain under pathologic conditions and may therefore represent both therapeutic targets and diagnostic or prognostic biomarkers for neurologic diseases, including epilepsy. In fact, miRNA expression profiles have been investigated in the hippocampi of patients with epilepsy in comparison with control, nonepileptic cases. Unfortunately, the interpretation of these data is difficult because surgically resected epileptic tissue is generally compared with control tissue obtained from autopsies. To challenge the validity of this approach, we performed an miRNA microarray on the laser microdissected granule cell layer of the human hippocampus obtained from surgical samples of patients with epilepsy, autoptic nonepileptic controls, and patients with autoptic epilepsy, using the latter as internal control. Unfortunately, it is extremely difficult to collect autopsy material from documented epilepsy individuals who died of non-epilepsy-related causes-we found only two such cases. However, hierarchical clustering of all samples showed that those obtained from autopsies of patients with epilepsy segregated with the other autoptic samples (controls) and not with the bioptic tissues from the surgery patients, suggesting that the origin of the tissue (surgery or autopsy) may be prevalent over the underlying pathology (epilepsy or not epilepsy). Even taking into account the limitations due to the small number of cases, this observation arises concerns on the use of autopsy tissue as control for this kind of studies.

MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy--comparison with human epileptic samples.

The identification of biomarkers of the transformation of normal to epileptic tissue would help to stratify patients at risk of epilepsy following brain injury, and inform new treatment strategies. MicroRNAs (miRNAs) are an attractive option in this direction. In this study, miRNA microarrays were performed on laser-microdissected hippocampal granule cell layer (GCL) and on plasma, at different time points in the development of pilocarpine-induced epilepsy in the rat: latency, first spontaneous seizure and chronic epileptic phase. Sixty-three miRNAs were differentially expressed in the GCL when considering all time points. Three main clusters were identified that separated the control and chronic phase groups from the latency group and from the first spontaneous seizure group. MiRNAs from rats in the chronic phase were compared to those obtained from the laser-microdissected GCL of epileptic patients, identifying several miRNAs (miR-21-5p, miR-23a-5p, miR-146a-5p and miR-181c-5p) that were up-regulated in both human and rat epileptic tissue. Analysis of plasma samples revealed different levels between control and pilocarpine-treated animals for 27 miRNAs. Two main clusters were identified that segregated controls from all other groups. Those miRNAs that are altered in plasma before the first spontaneous seizure, like miR-9a-3p, may be proposed as putative biomarkers of epileptogenesis.

Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy.

The alterations in GABA release have not yet been systematically measured along the natural course of temporal lobe epilepsy. In this work, we analyzed GABA extracellular concentrations (using in vivo microdialysis under basal and high K(+)-evoked conditions) and loss of two GABA interneuron populations (parvalbumin and somatostatin neurons) in the ventral hippocampus at different time-points after pilocarpine-induced status epilepticus in the rat, i.e. during development and progression of epilepsy. We found that (i) during the latent period between the epileptogenic insult, status epilepticus, and the first spontaneous seizure, basal GABA outflow was reduced to about one third of control values while the number of parvalbumin-positive cells was reduced by about 50% and that of somatostatin-positive cells by about 25%; nonetheless, high K(+) stimulation increased extracellular GABA in a proportionally greater manner during latency than under control conditions; (ii) at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made in humans) this increased responsiveness to stimulation disappeared, i.e. there was no longer any compensation for GABA cell loss; (iii) thereafter, this dysfunction remained constant until a late phase of the disease. These data suggest that a GABAergic hyper-responsiveness can compensate for GABA cell loss and protect from occurrence of seizures during latency, whereas impaired extracellular GABA levels can favor the occurrence of spontaneous recurrent seizures and the maintenance of an epileptic state.

Implication of fibroblast growth factors in epileptogenesis-associated circuit rearrangements.

The transformation of a normal brain in epileptic (epileptogenesis) is associated with extensive morpho-functional alterations, including cell death, axonal and dendritic plasticity, neurogenesis, and others. Neurotrophic factors (NTFs) appear to be very strongly implicated in these phenomena. In this review, we focus on the involvement of fibroblast growth factor (FGF) family members. Available data demonstrate that the FGFs are highly involved in the generation of the morpho-functional alterations in brain circuitries associated with epileptogenesis. For example, data on FGF2, the most studied member, suggest that it may be implicated both in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring acute seizures but reducing seizure-induced cell death. Even if many FGF members are still unexplored and very limited information is available on the FGF receptors, a complex and fascinating picture is emerging: multiple FGFs producing synergic or antagonistic effects one with another (and/or with other NTFs) on biological parameters that, in turn, facilitate or oppose transformation of the normal tissue in epileptic. In principle, identifying key elements in these phenomena may lead to effective therapies, but reaching this goal will require confronting a huge complexity. One first step could be to generate a "neurotrophicome" listing the FGFs (and all other NTFs) that are active during epileptogenesis. This should include identification of the extent to which each NTF is active (concentrations at the site of action); how it is active (local representation of receptor subtypes); when in the natural history of disease this occurs; how the NTF at hand will possibly interact with other NTFs. This is extraordinarily challenging, but holds the promise of a better understanding of epileptogenesis and, at large, of brain function.