Author:

Abstract:

Tatiana Afrikanova 14.00 < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";mso-ansi-language:EN-US" lang="EN-US">Validation of an animal model constitutes a large partof successful drug development. In this thesis we describe the development andvalidation of in vivo models of seizures in zebrafish larvae. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";mso-ansi-language:EN-US" lang="EN-US">The process of building an animal model forneurological disorders consists of several steps of development and systematicevaluation of their quality and relevance. Based on a hypothesis aboutbrain-behavior relationships, a model is developed and tested. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";mso-ansi-language:EN-US" lang="EN-US">Validation of a model is a scientific method toimprove the confidence in a model. Validity is defined as the agreement betweena test score and the quality it is believed to measure. Validity is restrictedto a specific use of a model, and must always be open for discussion andre-evaluation. Most animal models are evaluated based on the criteria proposedby Willner: construct, predictive and face validity. Other important factorsare animal welfare, reliability and replicability as well as cross-speciessimilarity. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";mso-ansi-language:EN-US" lang="EN-US">Epilepsy is one of the most common neurologicaldisorders with a high disabling risk; the main symptom of epilepsy is theoccurrence of seizures. An epileptic seizure (according to ILAE) is a transientoccurrence of signs and/or symptoms due to abnormal excessive or synchronousneuronal activity in the brain. Epilepsy is defined as a disorder of the braincharacterized by en enduring predisposition to generate epileptic seizures andby the neurobiologic, cognitive, physiological, and social consequences of thiscondition. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";mso-ansi-language:EN-US" lang="EN-US">There are cases reported where seizures stop occurringspontaneously (self-resolving). Still, in the majority of patients, adequateseizure control or even seizure freedom is achieved with the help ofantiepileptic drugs (AEDs). To date, there are several generations of AEDs onthe market with different or multiple mechanisms of action available,nevertheless, about one third of people diagnosed with epilepsy do not respondto the prescribed treatment. Some of the non-responders can be cured bysurgical intervention removing the brain area involved in seizure generation.Other alternative treatments include ketogenic diet and vagus nervestimulation. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";mso-ansi-language:EN-US" lang="EN-US">To start with, epilepsy involves excessive and/orhighly synchronized neuronal firing, which is otherwise a normal function ofthe brain. Therefore, due to the complexity of disease it’s not possible tomodel it in a biochemical in vitro assay. The use of the existing -predominantly rodent -models of seizures and epilepsy is limited due to theassay costs, timing and labour-intensity despite the value of the data theyprovide. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";color:windowtext;mso-ansi-language:EN-US" lang="EN-US">The constant trade of validity against resourceconsumption leads us to a different approach in model building. It is possibleto use smaller vertebrates to increase the assay’s throughput. On the otherhand, the decrease in construct validity in this case can be partially compensated by the convenience of geneticmanipulation, visual transparency and automation solutions. Assuming that asimpler organism feels less pain, ethical considerations also support zebrafishresearch, as the zebrafish larvae are not even considered laboratory animalsbefore day 6 of life. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";color:windowtext;mso-ansi-language:EN-US" lang="EN-US">Zebrafish video tracking haverecently emerged as an attractive in vivo model for epilepsy.Seven-day-old zebrafish larvae exposed to the GABA< /span style="font-size:8.0pt;font-family:"Calibri","sans-serif";color:windowtext;mso-ansi-language:EN-US" lang="EN-US">A < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";color:windowtext;mso-ansi-language:EN-US" lang="EN-US">antagonistpentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-likebehavior, and epileptiform electrographic activity. A previous study showedthat 12 out of 13 AEDs suppressed PTZ-mediated increases in larval movement,indicating the potential utility of zebrafish as a high-throughput in vivo modelfor AED discovery. However, a question remained as to whether an AED-induceddecrease in locomotion is truly indicative of anticonvulsant activity, as somedrugs may impair larval movement through other mechanisms such as generaltoxicity or sedation. We therefore carried out a study in PTZ-treated zebrafishlarvae, to directly compare the ability of AEDs to inhibit seizure-likebehavioral manifestations with their capacity to suppress epileptiformelectrographic activity. We re-tested the 13 AEDs of which 12 were previouslyreported to inhibit convulsions in the larval movement tracking assay, administeringconcentrations that did not, on their own, impair locomotion. In parallel, wequantified the parameters of open-field recordings on larval brains aftertreatment with each AED. For 10 of AEDs tested we obtained the same response inboth the behavioral and electrographic assays. Overall our data correlate wellwith those reported in the literature for acute rodent PTZ tests, indicatingthat the larval zebrafish brain is more discriminatory than previously thoughtin its response to AEDs with different modes of action. Our results underscorethe validity of using the zebrafish larval locomotor assay as a rapidfirst-pass screening tool in assessing the anticonvulsant and/or proconvulsantactivity of compounds, but also highlight the importance of performing adequatevalidation when using in vivo models. < /span style="font-size:11.5pt;font-family:"Calibri","sans-serif";color:windowtext;mso-ansi-language:EN-US" lang="EN-US">The other question explored was ifzebrafish could be used as a model organism for treatment-resistant seizures.Refractory seizures affect about a third of patients suffering from epilepsy.To fulfill the need for new medications targeting these difficult-to-treatcases, chronic rodent models offer the opportunity to study mechanisms of drugresistance. The use of such models, however, has been rather limited, as allhave proven to be time-consuming and labor-intensive. In this study, wecharacterize the allylglycine (AG) seizure model, a simple in vivo modelshowing a high level of treatment resistance. (D,L)-Allylglycine inhibitsglutamic acid decarboxylase – the key enzyme in GABA biosynthesis – leading toGABA depletion, seizures and neuronal damage. We performed a side-by-sidecomparison of mouse and zebrafish acute AG treatment including biochemical,electrographic and behavioral assessment. Interestingly, seizure progressionrate and GABA depletion kinetics were comparable in both species. Fivemechanistically diverse AEDs (valproate, diazepam, levetiracetam, phenytoin andtopiramate) were used. All tested AEDs showed a limited protective effect(mainly mortality delay) at doses close to TD< /span style="font-size:8.0pt;font-family:"Calibri","sans-serif";color:windowtext;mso-ansi-language:EN-US" lang="EN-US">50 < /span style="mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;color:windowtext;mso-ansi-language:EN-US" lang="EN-US">(doseinducing motor impairment in 50% of animals) in mice; experiments in fishdemonstrated a complete< /span style="font-size:11.5pt;line-height:115%;font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA" lang="EN-US"> protective effect ofVPA, while other AEDs were only partially or not active in the assays run.Having demonstrated cross-species similarities, we conclude that the use of AGin zebrafish could allow for high-throughput drug screening and thereforepropose it as a convenient model in the context of treatment-resistantseizures. 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