Studying the function of acetylcholine receptors in zebrafish
The purpose of this application is to study the function of one gene: the acetylcholine receptor 4 (chrna4) in epilepsy. Mutations in this gene were identified in patients suffering from a genetic form of epilepsy known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Here we will generate zebrafish disease models of ADNFLE and study the mechanism leading to epilepsy in human patients. In this project we want to get familiar with this disease model in order to pursue future studies with clinical applications.
First, we will generate zebrafish models of ADNFLE by mutating the 2 orthologues of chrna4 (chrna4a/b) and chrna2 (chrna2a/b). We will inactivate the gene (knock-out) or introduce the human mutation (S280F, knock-in) in the zebrafish genome. In total 7 different lines will be generated: chrna4a knock-out, chrna4b knock-out, chrna4a-S280F, chrna4b-S280F, chrna2a knock-out, chrna2b knock-out and UAS:humanChrna4-S280F transgenic. To ensure the specificity of the genetic manipulation, 2 mutations per gene (mut#1 and mut#2) will be selected. Thus, we will raise and monitor 12 genotypes (with 60 fishes/line). The 71 genotypes are: chrna4a+/mut#1, chrna4amut#1/mut#1, chrna4a+/mut#2, chrna4amut#2/mut#2, chrna4b+/mut#1, chrna4bmut#1/mut#1, chrna4b+/mut#2, chrna4bmut#2/mut#2, chrna4a+/S280F, chrna4aS280F/S280F, chrna4b+/S280F, chrna4bS280F/S280F, chrna2a+/-, chrna2a-/-, chrna2b+/-, chrna2b-/- and UAS:hchrna4S280F. In total, we will monitor 720 animals +400 animals.
Mutations in chrna4 gene may affect brain activity, behavior and may lead to spontaneous seizures. Previous work in rodent indicates that Chrna4 knock-out mice are viable, fertile and show slightly elevated anxiety. Thus we predict zebrafish chrna4a/chrna4b knockout to be viable and have mild behavioral phenotypes. ADFLNE mutations resulted in epilepsy in some of the existing rodent models. Thus we expect that knock-in zebrafish may develop spontaneous seizures.
All our experiments are non-invasive. Our animals' health is important. To ensure the wellbeing of our animals, larvae, juvenile and adult zebrafish will be monitored daily by experienced staff. In addition we will perform video recording at 2 stages during development. This allow us to identify abnormal behaviors, stress levels and occurrences of epileptic seizures. Using zebrafish, we replace mammalian animal models such as the mouse or rats. All our experiments consider studying the function of the vertebrate brain in health and disease; hence these experiments require working with living animals.
In this pilot study, we will generate zebrafish ADNFLE models by introducing genetic mutations in the chrna4a/4b and chrna2a/b genes and monitor their behavior and occurrence of epileptic seizures. The aims are:
1) to get familiar with ADFNLE disease model, identify if and when mutant animals develop seizure and/or other severe neurological disorders
2) to optimize our breeding strategy in order to reduce the impact on the wellbeing of our animals (e.g. if homozygous mutant develop seizure but not heterozygous, we will maintain a heterozygous colony)
We will put a strong emphasis on studying the function of chrna4a/b and chrna2a/b genes in epilepsy in larval zebrafish (less than 5days old), which are considered as a replacement to animal experimentation. This implies that we will maintain an actively breeding colony with heterozygous and homozygous mutants (if animals are healthy and fertile).
We anticipate that our results will go beyond zebrafish brain and will inspire novel potential treatment to prevent epileptic seizures in humans.
First, we will generate zebrafish models of ADNFLE by mutating the 2 orthologues of chrna4 (chrna4a/b) and chrna2 (chrna2a/b). We will inactivate the gene (knock-out) or introduce the human mutation (S280F, knock-in) in the zebrafish genome. In total 7 different lines will be generated: chrna4a knock-out, chrna4b knock-out, chrna4a-S280F, chrna4b-S280F, chrna2a knock-out, chrna2b knock-out and UAS:humanChrna4-S280F transgenic. To ensure the specificity of the genetic manipulation, 2 mutations per gene (mut#1 and mut#2) will be selected. Thus, we will raise and monitor 12 genotypes (with 60 fishes/line). The 71 genotypes are: chrna4a+/mut#1, chrna4amut#1/mut#1, chrna4a+/mut#2, chrna4amut#2/mut#2, chrna4b+/mut#1, chrna4bmut#1/mut#1, chrna4b+/mut#2, chrna4bmut#2/mut#2, chrna4a+/S280F, chrna4aS280F/S280F, chrna4b+/S280F, chrna4bS280F/S280F, chrna2a+/-, chrna2a-/-, chrna2b+/-, chrna2b-/- and UAS:hchrna4S280F. In total, we will monitor 720 animals +400 animals.
Mutations in chrna4 gene may affect brain activity, behavior and may lead to spontaneous seizures. Previous work in rodent indicates that Chrna4 knock-out mice are viable, fertile and show slightly elevated anxiety. Thus we predict zebrafish chrna4a/chrna4b knockout to be viable and have mild behavioral phenotypes. ADFLNE mutations resulted in epilepsy in some of the existing rodent models. Thus we expect that knock-in zebrafish may develop spontaneous seizures.
All our experiments are non-invasive. Our animals' health is important. To ensure the wellbeing of our animals, larvae, juvenile and adult zebrafish will be monitored daily by experienced staff. In addition we will perform video recording at 2 stages during development. This allow us to identify abnormal behaviors, stress levels and occurrences of epileptic seizures. Using zebrafish, we replace mammalian animal models such as the mouse or rats. All our experiments consider studying the function of the vertebrate brain in health and disease; hence these experiments require working with living animals.
In this pilot study, we will generate zebrafish ADNFLE models by introducing genetic mutations in the chrna4a/4b and chrna2a/b genes and monitor their behavior and occurrence of epileptic seizures. The aims are:
1) to get familiar with ADFNLE disease model, identify if and when mutant animals develop seizure and/or other severe neurological disorders
2) to optimize our breeding strategy in order to reduce the impact on the wellbeing of our animals (e.g. if homozygous mutant develop seizure but not heterozygous, we will maintain a heterozygous colony)
We will put a strong emphasis on studying the function of chrna4a/b and chrna2a/b genes in epilepsy in larval zebrafish (less than 5days old), which are considered as a replacement to animal experimentation. This implies that we will maintain an actively breeding colony with heterozygous and homozygous mutants (if animals are healthy and fertile).
We anticipate that our results will go beyond zebrafish brain and will inspire novel potential treatment to prevent epileptic seizures in humans.