This research was supported by the Czech Academy of Sciences RVO 68378050, LM2018126 Czech Centre for Phenogenomics provided by MEYS CR, OP RDE CZ.02.1.01/0.0/0.0/16_013/0001789 (Upgrade of the Czech Centre for Phenogenomics: developing toward translation research by MEYS and ESIF), OP RDE CZ.02.1.01/0.0/0.0/18_046/0015861 (CCP Infrastructure Upgrade II by MEYS and ESIF), and OP RDI CZ.1.05/2.1.00/19.0395 (higher quality and capacity for transgenic models by MEYS and ERDF). In addition, this study received funding from the NGO &#34;Association of Gene Therapy (ASGENT)&#34;, Czechia (https://asgent.org/) and LM2023036 Czech Centre for Phenogenomics provided by Ministry of Education, Youth and Sports of the Czech Republic.

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

The authors have no conflicts of interest to disclose.

AS models created in different murine strains are commonly validated with tests of animal emotional state, motor functions, and cognitive abilities to facilitate comparison to human symptoms31,32. A motor deficit in AS models is the most consistent finding across laboratories, followed by an unchanged emotionality state of mutants and difficulties building nests31,32,33....

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cages, individually ventilated</td>
			<td>Techniplast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DigiGait</td>
			<td>Mouse Specifics, Inc., 2 Central Street Level 
			Unit 110 
			Framingham, MA 01701, USA</td>
			<td>Equipment was tendered, no catalogue&#160; number was provided, nor could be find on company&#39;s web site</td>
			<td>Detailed analysis of mouse gait, hardware and software provided.&#160;</td>
		</tr>
		<tr>
			<td>FDA Nestlet squares</td>
			<td>Datesand Ltd., 7 Horsfield Way, Bredbury, Stockport SK6, UK</td>
			<td>Material was bought from Velaz vendor via direct email request. Velaz do not provide any catalogue no.</td>
			<td>Cotton nestlets for nest building test. Nestlet discription: 2-3 g each, with diameter around 5 x 5 x 0.5cm.</td>
		</tr>
		<tr>
			<td>Mouse chow</td>
			<td>Altramion</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotarod</td>
			<td>TSE Systems GmbH, Barbara-McClintock-Str.4 
			12489 Berlin, Germany</td>
			<td>Equipment was tendered, no catalogue&#160; number was provided, nor could be find on company&#39;s web site</td>
			<td>Rotarod for 5 mice, hardware and software provided. Drum dimensions: Diameter: 30 mm, width per lane: 50 mm, falling distance 147 mm.</td>
		</tr>
		<tr>
			<td>Tail Suspension Test</td>
			<td>Bioseb, In Vivo Research Instruments, 13845 Vitrolles 
			FRANCE</td>
			<td>Reference: BIO-TST5</td>
			<td>Fully automated equipment for immobility time evaluation of 3 mice hanged by tail, hardware and software provided</td>
		</tr>
		<tr>
			<td>Transpore medical tape</td>
			<td>Medical M, Ltd.</td>
			<td>P-AIRO1291</td>
			<td>The tape used to attach an animal to the hook by its tail.</td>
		</tr>
		<tr>
			<td>Viewer - Video Tracking System</td>
			<td>Biobserve GmbH, Wilhelmstr. 23 A 
			53111 Bonn, Germany</td>
			<td>Equipment with software were tendered, no catalogue&#160; number was provided, nor could be find on company&#39;s web site</td>
			<td>Software with custom made hardware: maze, IR base, IR sensitive cameras. Custom-made OF dimensions: 42 x 42 cm area, 49 cm high wall, central zone area: 39 cm2. A custom-made EPM was elevated 50 cm above the floor, with an open arm 79 cm long,&#160; 9 cm wide, and closed arm 77 cm long, 7.6 cm wide.&#160;</td>
		</tr>
	</tbody>
</table>

behavioral characterization of an angelman syndrome mouse model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

Behavioral Characterization of an Angelman Syndrome Mouse Model

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.

Elevated plus maze and open field tests 
The EPM and OF tests use the natural tendency of rodents to explore new environments18,19. The exploration is governed by an approach-avoidance conflict, where rodents choose between the exploration of a new environment and avoidance of possible danger. Animals explore unknown places in search for shelter, social contact, or foraging. However, new places may involve risk factors such as predators or c...

Watch this Scientific Journal Video about A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model at JoVE.com

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

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Elevated Plus Maze Test in an Angelman Syndrome Mouse Model

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.

Open Field Test in an Angelman Syndrome Mouse Model

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

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.

Gait Analysis in an Angelman Syndrome Mouse Model

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.

Rotarod Test in an Angelman Syndrome Mouse Model

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.

Nestlet Shredding: Nest Building Test in an Angelman Syndrome Mouse Model

Take a cotton nestlet with forceps and record its weight manually using scales. Place the nest lit randomly in a cage but on the opposite side of the water supply. Weigh each nestlet for the next four days manually using scales.Ensure that the nestlet is dry when weighed. If not dry on a heating pad. If the nestlet is torn into several parts, weigh the largest part.Record the weights on paper or in a pre-made spreadsheet. The UBE3A mutant animals used significantly less material to build their nests and this difference was particularly prominent between transgenic females and their control counterparts.

1.6K Views.  Institute of Molecular Genetics of the Czech Academy of Sciences.  This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Summary