Name: Открытый полевой тест на мышиной модели с синдромом Ангельмана
Uploaded: 2023-10-20T12:10:00
Description: Watch this Scientific Journal Video about Open Field Test in an Angelman Syndrome Mouse Model at JoVE.com

open field test in an angelman syndrome mouse model

Валидация мышиной модели синдрома Ангельмана с помощью поведенческих тестов

Angelman syndrome (AS) is a rare neurodevelopmental disease. The most common genetic origin of AS is a large deletion of the 15q11-q13 region of the maternally-derived chromosome, which is found in nearly 74% of patients1. Deletion of this region causes the loss of UBE3A, the main causative gene of AS that encodes an E3 ubiquitin ligase. The paternal allele of the UBE3A gene in neurons is silenced in a process known as imprinting. As a consequence, paternal imprinting of the gene allows only maternal expression in the central nervous system (CNS)2. Therefore, UBE3A gene deletion from the maternally-derived chromosome leads to the development of AS symptoms. In humans, AS manifests at around 6 months of age, with developmental retardation that persists throughout all developmental stages and results in severe debilitating symptoms in affected individuals3,4. The core symptoms of the disorder include the deficit of fine and gross motor skills, including jerky ataxic gait, serious speech impairment, and intellectual disability. Approximately 80% of AS patients also suffer from sleep disturbances and epilepsy. To date, the only available treatment are symptomatic drugs, which reduce epileptic seizures and improve sleep quality1. Therefore, the development of robust animal models with reproducible behavioral phenotypes alongside refined phenotyping analysis will be essential to elucidate the pathophysiological mechanisms of the disorder and discover effective medications and treatments.
The complexity of the human disorder affecting the CNS demands model organisms to possess a comparable genome, physiology, and behavior. Mice are popular as a model organism due to their short reproductive cycle, small size, and relative ease of DNA modification. In 1984, Paul Willner proposed three basic disease model validation criteria: the construct, face, and predictive validity, which are used to determine the model&#39;s value5. Simply, construct validity reflects the biological mechanisms responsible for the disorder development, face validity recapitulates its symptoms, and predictive validity describes the model response to therapeutic drugs.
To adhere to the above principles, we have chosen the most common genetic etiology, a large deletion of the maternal 15q11.2-13q locus including the UBE3A gene, to create AS model mice. We used the CRISPR/Cas9 technique to delete a 76,225 bp long region spanning the entire UBE3A gene, encompassing both the coding and non-coding elements of the gene, in mice from a C57BL/6N background6. We then bred the animals to obtain UBE3A+/&#8722; heterozygous mice. For face validation of the model, we used animals from crosses of UBE3A+/&#8722; females and wild-type males to gain UBE3A+/- progeny (strain named C57BL/6NCrl-UBE3A&#60;em1(IMPC)Ccpcz&#62;/Ph and later assigned as UBE3AmGenedel/+) and control littermates. We tested their fine and gross motor skills, emotionality, and affect to recapitulate core AS symptoms. In a previous article, we also evaluated the animals&#39; cognitive functions, as AS patients also suffer from intellectual disability6. However, we found no cognitive impairments in UBE3AmGenedel/+ mice, perhaps due to the young age of the animals at the time of testing7. Later examination of the older animals, around 18 weeks old, revealed a deficit in behavioral flexibility during reversal learning in the place preference paradigm. However, the complexity of the employed equipment for this analysis requires a separate methodological module and it is not included here.
The behavioral tests presented here belong to the common phenotyping tools in genetic research, thanks to their high predictive value and sufficient construct validity8,9,10. We used these tests to validate a mouse model of AS by recapitulating core symptoms of the human disease in a reproducible, age-independent manner. The emotionality of the animal was evaluated in the elevated plus maze and open field tests. Both of these tests are based on the approach-avoidance conflict, where animals explore a new environment in search of food, shelter, or mating opportunities while simultaneously avoiding anxiogenic compartments11. Additionally, the open field test is used to test a mouse&#39;s locomotor activity8. The tail suspension test is widely used in depression research to screen for new antidepressant drugs or depressive-like phenotypes in mouse knockout models12. This test evaluates the despair that animals develop over time in an inescapable situation. Motor learning and detailed gait characteristics were determined on the rotarod and in DigiGait, respectively. Animal endurance on the accelerating rod characterizes its balance and movement coordination skills, while detailed analysis of a mouse&#39;s step patterns is a sensitive evaluation of neuromuscular impairments connected to many neurogenerative movement disorders13,14,15. The nestlet shredding test is part of the standard methodology for detecting impulsive behavior in rodents, and since it utilizes natural rodent building behavior, it indicates the animal&#39;s well-being16,17.
The size of the experimental groups was a result of a compromise to meet the 3R rule demands and efficient usage of colony breeding performance. However, to obtain statistical power, the groups had no fewer than 10 individuals, due to the establishment of a sufficient amount of breeding pairs. Unfortunately, breeding performance did not always result in a sufficient number of animals.

В центре внимания автора: батарея высоковоспроизводимых поведенческих тестов для валидации мышиной модели синдрома Ангельмана 

Поведенческая характеристика мышиной модели синдрома Ангельмана

Our goal is to elucidate the physiological roles of genes using mice as an experimental model. We also focus on developing mouse models of specific diseases that scientists can use to investigate disease causation and develop effective treatments. An example of such activity is the mouse model of Angelman syndrome.Arguably, the most promising new technology with potential is integrating artificial intelligence based on deep learning techniques in animal research. This approach can be used to monitor animal behavior in the home cage directly without need for the experimenter to disturb the animals. The biggest challenge in behavioral studies currently is reproducibility.Therefore, we introduce a battery of tests that validate the Angelman syndrome of model in a way that does not depend on the model's age or species. We use established behavioral tests to validate our AS model. However, with the novel approach for the model development closely resembling the human condition with a large deletion.Using CRISPR technology, we deleted the entire UBE3A gene from the mouse genome. In contrast to other models where the deletion is limited to 3KB. We're working on developing a genetic therapy that could restore a symptoms in a mouse model.

Watch this Scientific Journal Video about Open Field Test in an Angelman Syndrome Mouse Model at JoVE.com

Открытый полевой тест на мышиной модели с синдромом Ангельмана  

Поместите четыре тестовых коробки открытого поля (OF) на испытательную платформу под камерой. С помощью потенциометра, висящего на стене, отрегулируйте интенсивность света до 200 люкс в центре каждого теста OF с помощью люксометра с датчиком, расположенным в центре каждого бокса во время регулировки. Откройте программу, дважды щелкнув по значку программы просмотра, и загрузите конфигурацию для тестирования EF, нажав на иконку в верхней левой части вкладки конфигурации.Введите информацию о животном в соответствующие поля вкладки эксперимента. Убедитесь, что положение зоны совпадает с тестовыми коробками OF, и при необходимости отрегулируйте их. С помощью визуального управления и компьютерной мыши убедитесь, что виртуальный контурный центр и периферийные зоны совпадают с соответствующими тестовыми зонами OF.Наведите курсор мыши на значок стрелки в верхней левой части вкладки приобретения. Извлеките четырех животных из домашней клетки и поместите их в угол каждого тестового ящика. Остановите эксперимент, щелкнув левой кнопкой мыши по компьютерной мыши.В тесте OF материнские гетерозиготные животные с мутантным Ube3a были значительно гипоактивны, что отражалось в более коротком пройденном расстоянии, более низкой средней скорости и более длительном времени отдыха. Тем не менее, ни один из параметров, связанных с тревожным поведением, не был изменен у мутантов Ube3a.

open-field-test-in-an-angelman-syndrome-mouse-model

Research

JoVE Journal

Behavior

Open Field Test in an Angelman Syndrome Mouse Model

Elevated Plus Maze Test in an Angelman Syndrome Mouse Model

Тест в лабиринте с повышенным плюсом на мышиной модели с синдромом Ангельмана  

To begin, place the plus shaped maze on the testing platform just below the camera. Using the potentiometer on the wall, adjust the light intensity to 70 luxe at its center with the help of a luxometer with its sensor placed at the center of the maze during the adjustment. Open the software by double clicking on the viewer software icon and load the configuration for Elevated Plus Maze or EPM testing by clicking on the icon on the upper left side of the configuration tab.Enter the animal information into the corresponding fields of the experiment tab including animal id, genotype, gender, and experiment information. Verify that the zone's position, open arms, and closed arms are correctly configured. Using a visual control and computer mouse, ensure that the virtual outline zones match the corresponding EPM zones.Place the mouse cursor on the arrow icon on the upper left side of the acquisition tab. Remove the animal from the home cage and place it gently in the center of the EPM. Start the experiment by left clicking on the computer mouse.After five minutes, save the recorded data by clicking okay. Name the file and click save. Export the results to a csv file for offline analysis by clicking on the icon on the left vertical panel of the data analysis tab.In the EPM test, none of the parameters related to anxiety-like behavior were altered in maternal heterozygous Ube3A mutants.

Тест в лабиринте с повышенным плюсом на мышиной модели с синдромом Ангельмана

Place the four open field, or OF, test boxes on the testing platform below the camera. Using the potentiometer on the wall, adjust the light intensity to 200 lux at the center of each OF test with the help of a luxometer with its sensor placed at the center of each box during the adjustment. Open the software by double clicking on the viewer software icon and load the configuration for EF testing by clicking on the icon on the upper left side of the configuration tab.Enter the animal information into the corresponding fields of the experiment tab. Verify that the zone's position matches the OF test boxes and adjust them if needed. Using a visual control and computer mouse, ensure that the virtual outline center and periphery zones match the corresponding OF test zones.Place the mouse cursor on the arrow icon on the upper left side of the acquisition tab. Remove four animals from the home cage and place them in the corner of each test box. Stop the experiment by left clicking on the computer mouse.In the OF test, the maternal heterozygous Ube3a mutant animals were significantly hypoactive as reflected by a shorter traversed distance, lower average speed, and longer resting time. However, none of the parameters related to anxiety-like behavior were altered in Ube3a mutants.

Tail Suspension Test in an Angelman Syndrome Mouse Model

Тест на подвешивание хвоста на мышиной модели с синдромом Ангельмана  

Connect the tail suspension cage to the computer using a USB cable. Insert the USB dongle into the computer and start the software by double clicking on the BioTSD software icon. In the settings tab on the global, ensure the acquisition duration is set to 360 seconds.In the experiment tab, select the new list of subjects and create a new experiment file and a new list of tested subjects by following the instructions in the opened tab. Prepare the animals for the test by wrapping single-sided adhesive tape around three-fourths of the animal's tail starting from the base. Start the run by clicking start run then continue in the acquisition tab.Immediately after hanging the animal on the hook, click the start icon under the visualized position to acquire data. After testing all the animals, in the analysis tab, select the last four minutes of the acquisition for analysis. Select all valid runs in the analysis period and click analyze selected subjects.Choose the desired data format and click export selected data to export the collected data. In the tail suspension test, Ube3a mutant animals were immobile for significantly longer than their control littermates, indicating their depression-like behavior.

Тест на подвешивание хвоста на мышиной модели с синдромом Ангельмана

Gait Analysis in an Angelman Syndrome Mouse Model

Анализ походки на мышиной модели с синдромом Ангельмана  

Turn on the treadmill and manually set the belt speed to 20 centimeters per second on the equipment panel before switching on the apparatus light. Launch the DigiGait Imager software and set the shutter speed to 100 for albino mice or 130 for black or dark mice in the field for shutter speed. Place the mouse onto the treadmill belt and confirm that the mouse can perform the test by setting it to a slow walking speed for around three seconds and then stopping it while observing the mouse continuously.Start the belt by pressing the start button and record for 10 seconds. Ensure that a clear and fluent locomotion of at least 10 to 15 steps is observable. Stop the belt by pressing the stop button and return the mouse to the temporary holding cage.Screen the recording for a sequence of images with fluent steps by clicking play and reviewing the recording with the visual control in edit mode. Select 10 to 15 fluent movements by manually writing their starting and ending frame numbers into the relevant fields. Enter the animal information, including animal ID, date of birth, sex, weight, belt speed, and belt angle.Save the file by clicking save. Next, open the DigiGait Analysis software and perform gait analysis based on a fully automated analysis of video recordings of animal footprints. The gait indices of UBE3A mutant animals were altered.UBE3A mutant animals had a longer swing and stance that resulted in prolonged stride duration and length. Mice's hind limbs, propulsion, duration, and deceleration were also increased. These findings also revealed a larger pore area at the peak stance.

Анализ походки на мышиной модели с синдромом Ангельмана

Rotarod Test in an Angelman Syndrome Mouse Model

Тест Ротарод на мышиной модели с синдромом Ангельмана  

Turn on the rotarod equipment and launch the software by double-clicking the rod software icon. Create a new file in the file tab and save it with an appropriate name. In the setup window, fill out the experiment details, such as the date, user's name, and any eventual comments.Set the speed profile to 300 seconds, initial speed to 4 RPM, and terminal speed to 40 RPM. Open the measuring panel by clicking Measure. Start the initial rotation of the rod at 4 RPM by clicking Start and place the first five animals in their assigned positions.Start the testing protocol by clicking start profile, and the rod will gradually accelerate to 40 RPM over five minutes. After all the animals have fallen from the rod, close the Measure window by clicking Close and click Show to display the collected data. Export the acquired data in csv format by clicking Export CSV.Rotarod performance showed a reduced latency to fall in UBE3A mutant animals.