Optical mapping of neuronal activity during seizures in zebrafish

Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype

Marshall Hall, the reflex arc and epilepsy

Marshall Hall (1790-1857), who graduated from the University of Edinburgh's Medical School in 1812, was considered one of the greatest physiologists of his day. He advanced knowledge in various areas of medicine, in particular elucidating the mechanism of reflex activity in 1833. Hall suggested that convulsive epileptic seizures arose from heightened activity in the afferent limb or the central component of the reflex arc. From 1838 onwards he developed the idea that reflex-mediated neck muscle spasm in seizures obstructed cerebral venous return, congested the brain and thus caused unconsciousness. Associated reflex-mediated laryngeal spasm then caused convulsing. This was the most comprehensive physiologically based explanation of the major features of the convulsive epileptic seizure then available. Hall subsequently advocated and employed tracheotomy to prevent epileptic convulsing. His idea was taken up, modified and made more acceptable by others, and for a generation was the widely acknowledged basis for interpreting epileptogenesis. However, from 1870 onwards it was superseded by John Hughlings Jackson's accumulating evidence that epileptic seizures often arose in the cerebral cortex

Dog electroencephalogram for early safety seizure liability assessments and investigation of species-specific sensitivity for neurological symptoms

Preclinical safety is an important part of drug development in animals and humans. In toxicology studies, seizure liability can be detected at high doses as convulsions. Non-convulsive seizures induce only subtle behavioral changes and their assessment in animals is challenging. Electroencephalography (EEG) is the only method to correlate animal behavior to seizure activity and video-EEG is the current gold-standard for preclinical seizure liability assessments (Authier et al., 2014b). In most cases there are no clear premonitory signs that forewarn of convulsions but epileptiform EEG activity prior to clinical manifestation has been reported during a period potentially sufficient for prophylactic anticonvulsive treatment (Dürmüller et al., 2007). Aim of this thesis was investigation of a study design for assessment of neurological symptoms in dogs. This design should optimize detection of neurological signs while minimizing study duration and animal numbers. Video-EEG was used to increase symptom detection rate and to explore the possibility to refine seizure liability testing by enabling EEG-based anticonvulsive treatment. For establishment of the EEG system in our facility, reference substances were tested first. Then, three in-house drug candidates with different modes of action and known neurological side effects were chosen. Two telemetered beagle dogs were used per experiment. Substance effects on clinical symptoms and on the EEG were investigated. CSF and blood samples for analysis of drug exposure and biomarkers were collected simultaneous to symptoms. Results were compared to previous toxicological studies thereby enabling evaluation of non-rodent species differences in sensitivity for neurological symptoms. Results showed that combination of implants for CSF collection and EEG recording is possible. In this study design, intravenous administration was superior to oral dosing as it led to a reduced variability in exposure levels. Also, experimental time was significantly reduced compared to standard toxicology studies while the same neurological symptoms were induced. This shortened duration enabled continuous clinical observations for a better evaluation of CNS effects and immediate veterinary assistance in the spirit of animal welfare. The EEG was not superior to clinical observations in forewarning of convulsion risk and did not enable convulsion prevention. This was due first to the short latency between onset of abnormal EEG activity and convulsions which was below one minute with in-house compounds. Secondly, accurate interpretation of the unfiltered EEG signal was limited, especially differentiation of artefacts and epileptiform activity. In conclusion, a study design using intravenous infusions is suitable for the characterization of neurological symptoms. All the symptoms, which were already known from studies with a longer duration, were also seen. This allowed better correlation of neurological symptoms to exposure and immediate veterinarian treatments. For substances with a high risk to induce severe neurological symptoms, such studies can guide dose selection for longer regulatory toxicological studies to prevent occurrence of severe neurological symptoms.Im Rahmen der Entwicklung von Human- und Veterinärarzneimitteln wird die Anwendersicherheit neuer Medikamente in präklinischen Sicherheitsstudien erforscht. Zentralnervöse Nebenwirkungen werden häufig erst in toxikologischen Prüfungen erkannt, wenn bei hohen Dosierungen Krampfanfälle bei den Versuchstieren auftreten. Epileptische Anfälle können allerdings auch subtilere Symptome, deren Erkennen in Tieren schwierig ist, verursachen. Die Elektroenzephalographie (EEG) bietet in Tierstudien die einzige Möglichkeit, nicht-konvulsive Anfälle zu diagnostizieren. Daher ist die Kombination von Videoüberwachung und EEG in der präklinischen Arzneimittelentwicklung gegenwärtig der Goldstandard für die Sicherheitsbewertung einer Substanz im Hinblick auf ihr Risiko, Anfälle auszulösen (Authier et al., 2014b). Meist gibt es keine klinischen Warnzeichen vor dem Auftreten von Krampfanfällen. Allerdings wurde das Auftreten epileptiformer EEG-Aktivität vor klinischen Symptomen beobachtet. Das beschriebene Zeitfenster ist potentiell ausreichend für prophylaktische antikonvulsive Behandlung (Dürmüller et al., 2007). Ziel dieser Arbeit war es, in Pilotstudien ein neues Studiendesign für die Charakterisierung neurologischer Nebenwirkungen zu evaluieren. Dieses Studiendesign sollte die Erkennungsrate neurologischer Nebenwirkungen optimieren und dabei gleichzeitig eine Reduktion der dazu nötigen Tiere und der Studiendauer ermöglichen. Der Einsatz von EEG und Videoüberwachung sollte es ermöglichen, Substanz-induzierte Anfälle im Frühstadium zu erkennen und ihr klinisches Auftreten zu verhindern. Um das EEG-System in der Forschungseinrichtung neu zu etablieren und um zu evaluieren, ob Implantate für Liquor-Entnahme und EEG-Aufzeichnung kompatibel sind, wurden zuerst Referenzsubstanzen getestet. Zur Beantwortung der eigentlichen Fragestellung wurden drei Arzneimittelkandidaten mit unterschiedlichen Wirkmechanismen ausgewählt, von denen bekannt war, dass sie neurologische Symptome verursachen. Je Substanztest wurden zwei Hunde mit implantierten EEG-Sendern verwendet. Zwei der Substanzen wurden in eskalierenden intravenösen Dosen verabreicht, die dritte wurde als einzelne orale Dosis gegeben. Effekte der Substanzen auf klinische Symptome und auf das EEG wurden evaluiert. Parallel wurden Blut- und Liquor-Proben zur Bestimmung der Substanzspiegel und potentieller Biomarker genommen. Die Auswahl der Substanzen bot zusätzlich die Möglichkeit, die Empfindlichkeit der beiden regelmäßig in Arzneimittelprüfungen verwendeten Nicht-Nager Spezies Hund und Affe für neurologische Symptome vergleichend zu bewerten. Die Ergebnisse zeigen, dass die Kombination von Implantaten für EEG-Aufzeichnung und CSF-Probennahme möglich ist. Die intravenöse Applikation war der oralen Substanzgabe vorzuziehen, da die Variabilität der Substanz-Plasmaspiegel geringer war. Alle Symptome, die aus früheren toxikologischen Studien mit längerer Dauer bekannt waren, wurden ebenso beobachtet. Durch das Dosierungsschema war ihr Auftreten allerdings auf eine verkürzte Zeitspanne reduziert. Die kurze Studiendauer ermöglichte durchgehende klinische Beobachtung, somit die Erkennung aller Symptome und zeitnahe veterinärmedizinische Behandlungen, was im Sinne des Tierschutzes einen Vorteil darstellt. Für eine frühzeitige Erkennung von Krampfanfällen war das EEG nicht besser geeignet als klinische Beobachtung, da die Interpretation des ungefilterten EEG Signals durch das Auftreten von Artefakten erschwert war. Das Studiendesign, in dem das EEG angewendet wurde, ist zur Charakterisierung neurologischer Nebenwirkungen geeignet, da alle Symptome, die aus Studien mit längerer Dauer bekannt waren, ebenso beobachtet wurden. Durch die verkürzte Dauer wurde ermöglicht, Symptome und Substanzplasmaspiegel zu korrelieren und zeitnahe tierärztliche Behandlungen durchzuführen. Bei Substanzen, die ein hohes Risiko für neurologische Nebenwirkungen haben, kann dieses Studiendesign genutzt werden um im Vorfeld von behördlich geforderten toxikologischen Studien Dosierungen zu bestimmen, bei denen keine schweren neurologischen Nebenwirkungen zu erwarten sind

Epilepsy as a Pyridoxine-Dependent Condition: Quantitative Urinary Biomarkers of Epilepsy. Family Disorders of Pyridoxine Metabolism

The affected pyridoxine metabolism is discussed as an inborn genetic sign of epilepsy. In children with different forms of epilepsy and matched healthy controls, the urinary parameters of pyridoxal phosphate–dependent tryptophan degradation were measured by high-performance liquid chromatography (HPLC) method with simultaneous ultraviolet and fluorimetric detection. Concentrations of compounds, which are formed in the course of tryptophan degradation, and correlations between them turned out to be quantitative biomarkers useful for evaluation of patient’s condition and monitoring individualized antiepileptic treatment. Accumulation of tryptophan, kynurenine, and neurotoxic 3-hydroxykynurenine, along with reduced kynureninase activity, is characteristic of epileptic patients. Growing progressively worse, epilepsy is accompanied by aggravation of pyridoxal phosphate–dependent disturbances of tryptophan metabolism and further inhibition of kynureninase

Molecular Mechanism Involved in the Pathogenesis of Early-Onset Epileptic Encephalopathy

Recent studies have shown that neurologic inflammation may both precipitate and sustain seizures, suggesting that inflammation may be involved not only in epileptogenesis but also in determining the drug-resistant profile. Extensive literature data during these last years have identified a number of inflammatory markers involved in these processes of "neuroimmunoinflammation" in epilepsy, with key roles for pro-inflammatory cytokines such as: IL-6, IL-17 and IL-17 Receptor (IL-17R) axis, Tumor-Necrosis-Factor Alpha (TNF-α) and Transforming-Growth-Factor Beta (TGF-β), all responsible for the induction of processes of blood-brain barrier (BBB) disruption and inflammation of the Central Nervous System (CNS) itself. Nevertheless, many of these inflammatory biomarkers have also been implicated in the pathophysiologic process of other neurological diseases. Future studies will be needed to identify the disease-specific biomarkers in order to distinguish epilepsies from other neurological diseases, as well as recognize different epileptic semiology. In this context, biological markers of BBB disruption, as well as those reflecting its integrity, can be useful tools to determine the pathological process of a variety of neurological diseases. However; how these molecules may help in the diagnosis and prognostication of epileptic disorders remains yet to be determined. Herein, authors present an extensive literature review on the involvement of both, systemic and neuronal immune systems, in the early onset of epileptic encephalopathy

The ILAE classification of seizures and the epilepsies: Modification for seizures in the neonate. Position paper by the ILAE Task Force on Neonatal Seizures

Seizures are the most common neurological emergency in the neonatal period and in contrast to those in infancy and childhood, are often provoked seizures with an acute cause and may be electrographic‐only. Hence, neonatal seizures may not fit easily into classification schemes for seizures and epilepsies primarily developed for older children and adults. A Neonatal Seizures Task Force was established by the International League Against Epilepsy (ILAE) to develop a modification of the 2017 ILAE Classification of Seizures and Epilepsies, relevant to neonates. The neonatal classification framework emphasizes the role of electroencephalography (EEG) in the diagnosis of seizures in the neonate and includes a classification of seizure types relevant to this age group. The seizure type is determined by the predominant clinical feature. Many neonatal seizures are electrographic‐only with no evident clinical features; therefore, these are included in the proposed classification. Clinical events without an EEG correlate are not included. Because seizures in the neonatal period have been shown to have a focal onset, a division into focal and generalized is unnecessary. Seizures can have a motor (automatisms, clonic, epileptic spasms, myoclonic, tonic), non‐motor (autonomic, behavior arrest), or sequential presentation. The classification allows the user to choose the level of detail when classifying seizures in this age group

EVALUATION OF ANTICONVULSANT ACTIVITY OF MIMOSA PUDICA ROOT LINN IN SWISS ALBINO MICE

Objective: To evaluate the anticonvulsant activity of ethanolic extract of Mimosa pudica root (EMPR) in experimental mice models.Methods: Ethanolic extract of root parts of Mimosa pudica (EMPR) was prepared by a continuous method using soxhlet apparatus. EMPR in doses of 1000, 2000 mg/kg body wt along with valproate were administrated to albino mice by oral route and anti-epileptic activity was assessed by maximal electroshock (MES) and pentylenetetrazole (PTZ) induced seizure models. Abolition of tonic hind limb extension phase and an increase in seizure latency period, when compared to control group, were taken as a measure of protection in MES and PTZ induced convulsion models respectively.Results: EMPR in the dose of 1000 and 2000 mg/kg body wt of mice showed significant anti-epileptic property in both MES and PTZ induced seizure models. There was a significant abolition of tonic hind limb extension phase in MES model. There was also a significant increase in seizure latency period in PTZ induced seizure model.Conclusion: Results suggest that ethanolic extract of Mimosa pudica roots possess significant anti-epileptic activity. Further investigations are required to determine its active constituents and also its antiepileptic mechanism of action