Behavioral Characterization of an Angelman Syndrome Mouse Model

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 individuals^3,4. 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'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^+/− heterozygous mice. For face validation of the model, we used animals from crosses of UBE3A^+/− females and wild-type males to gain UBE3A^+/- progeny (strain named C57BL/6NCrl-UBE3A<em1(IMPC)Ccpcz>/Ph and later assigned as UBE3A^mGenedel/+) 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' cognitive functions, as AS patients also suffer from intellectual disability⁶. However, we found no cognitive impairments in UBE3A^mGenedel/+ 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 validity^8,9,10. 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'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's step patterns is a sensitive evaluation of neuromuscular impairments connected to many neurogenerative movement disorders^13,14,15. 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's well-being^16,17.

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.

All animals and experiments used in this study underwent ethical review and were conducted in accordance with the European Directive 2010/63/EU. The study was approved by the Czech Central Commission for Animal Welfare. Mice were housed in individually ventilated cages and maintained at a constant temperature of 22 ± 2 °C with a 12 h light/dark cycle. The mice were provided with mouse chow and water ad libitum. The mice were housed in groups of three to six animals per cage. No handling other than weighing was performed prior to testing. See the Table of Materials for details regarding all materials and equipment used in this protocol.

1. General considerations preceding and during testing

NOTE: For the sake of clarity and comprehensibility, general comments are presented before the description of the individual tests. This applies to each test, with the obvious exception of the nestlet shredding test, which is carried out in a housing room and does not require the use of any experimental equipment.

1. Accommodate animals in the research facility for at least 14 days prior to testing to minimize any stress resulting from transport and changes in environment.
2. Record animal weights before testing, as weight is a common confounding factor in behavioral research.
3. Leave the animals to acclimate in the experimental room for at least 1 h after transport from their housing room to minimize transport stress, whenever such transport takes place (i.e., every test described below except nestlet shredding, which is performed in the housing room).
4. Label each animal on the tail with a nontoxic, water-based marker to allow rapid identification during the experiment.
5. Remove all urine and feces deposited by the animals in the experimental apparatus during testing after each trial.
6. Wipe all experimental apparatus with 75% alcohol before and after each tested animal. The cleaning removes olfactory traces deposited during testing and helps to retain stable experimental conditions.
7. Transport the animals from their home cage to the experimental apparatus with as much care as possible, preferably in a small opaque container, and then freely release them unless other manipulations are needed.
8. Place each animal into a temporary holding cage after testing to prevent them from influencing the untested animals in the home cage.
9. Test males and females on consecutive days. Alternate the order of different genotypes during testing to counterbalance unpredictable environmental factors between the experimental groups.
10. Place the animals back into their home cage after all the animals have been tested and return them back to the housing room.
11. In the case of repeated testing of animals, maintain at least a 1 day interval between each test.

2. Behavioral tests

1. Elevated plus maze (EPM)
   NOTE: Both sexes of C57BL/6NCrl and UBE3A^mGenedel/+ mice strains were tested for this study at 9-12 weeks of age. The weights of the animals ranged from 22 to 36 g for males and 18 to 28 g for females at the time of testing.
   1. Place the plus-shaped maze on the testing platform just below the camera. Using the potentiometer on the wall, set the light intensity to 70 lux at its center with the help of a luxometer, with its sensor placed at the center of the maze during adjustment.
   2. Open the software by double-clicking on the Viewer software icon and load the configuration for EPM testing by clicking on the icon on the upper left side of the Configuration tab. Load the EPM plugin from the File menu. Fill out the animal information using the computer keyboard-animal ID, genotype, sex, and the experiment information (date, light intensity)-in the corresponding fields of the Experiment tab. Check if the zone's position, open arms, and closed arms are properly configured. With the help of a visual control and computer mouse, ensure that the virtual outlined zones match the corresponding EPM zones on the video preview.
   3. The EPM is a test used to evaluate an animal's general anxiety, which is based on approach-avoid conflict. Rodents naturally tend to avoid well-lit unprotected areas (open arms), in a favor of safer ones (closed arms). As this fully automated test is based on a video tracking system, let the software automatically calculate the time spent in each zone as well as the number of entrances.
   4. During testing, record the animals on video via an industrial, infrared light-sensitive camera. Allow the software to detect the position of the animal in real time during recording. Following this, let the software automatically evaluate the animal's tracks to calculate all parameters describing the animal's behavior in the maze. Use the time spent in the anxiogenic open arms and the percentage of open arms visits to evaluate the level of anxiety-like behavior in animals.
      NOTE: The custom-made maze is made of infrared light-permeable material and is placed on a light emitting diode (LED) infrared light source platform.
   5. Place the mouse cursor on the arrow icon on the upper left side of the Acquisition tab. Remove an animal from the home cage by hand and place it gently in the center of the EPM. Start the protocol by left-clicking on the computer mouse and leave the experimental room immediately.
   6. Once the recording protocol finishes after 5 min of free maze exploration, save the recorded data by clicking OK on the window that appears after protocol termination, name the file appropriately, and click Save. Export the results to a .csv file for each tested animal for off-line analysis by clicking on the icon on the left vertical panel of the Data analysis tab.
   7. Remove the animal from the maze by hand and put it into the temporary holding cage. Proceed with testing of all animals in the same manner. Copy the results for all tested animals to a Notepad file for off-line analysis by clicking on the Copy Results icon in the Elevated Plus maze plugin’s results tab.
      NOTE: The software and hardware may differ, and the relevant manuals must be followed. Additionally, the experimental setup, such as lighting or computer placement, may vary depending on the construction of the animal facility.
2. Open field (OF) test
   NOTE: The open field test assesses an animal's overall movement, which is triggered by exploratory behavior in a novel environment. Additionally, it is commonly used as a screening tool to detect general anxiety in an unprotected, well-lit space. This is a fully automated test that utilizes a video tracking system, which was also used in the previous test.
   1. Place the four OF test boxes on the testing platform just below the camera. Using the potentiometer on the wall, set 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 adjustment.
   2. Open the software by double-clicking on the Viewer software icon and load the configuration for OF testing by clicking on the icon on the upper left side of the Configuration tab. Fill out the animal information using the computer keyboard-animal ID, genotype, sex, and experiment information (date, light intensity)-in the corresponding fields of the Experiment tab. Check if the zone's position (center and periphery) matches the OF test boxes and adjust them if needed. With the help of a visual control and computer mouse, ensure that the virtual outlined center and periphery zones match the corresponding OF test zones on the video preview.
   3. During the testing, record the animals on video via an industrial, infrared light-sensitive camera. Allow the software to detect the position of the animal in real time during recording and automatically evaluate the animal's tracks to calculate all parameters describing the animal's behavior in the OF test box. The distance walked, average speed, and resting time are parameters used to evaluate animal activity in a new environment, while the number of center entries and duration in the center describe anxiety-like behavior in animals.
      NOTE: The custom-made maze is made of infrared light-permeable material and is placed on an LED infrared light source platform.
   4. Place the mouse cursor on the arrow icon on the upper left side of the Acquisition tab. Remove four animals from the home cage by hand and place them gently in the corner of each OF test box. Start the protocol by left-clicking on a computer mouse and immediately leave the experimental room.
   5. When the protocol finishes after 10 min of free maze exploration, save the data by clicking OK on the window that appears after protocol termination, name the file appropriately, and click Save. Export the results to a .csv file for each tested animal for off-line analysis by clicking on the icon on the left vertical panel of the Data analysis tab.
   6. Remove the animals from the maze by hand and put them in the temporary holding cage. Proceed with testing of all animals in the same manner. Analyze the exported data.
      NOTE: The software and hardware may differ, and the relevant manuals must be followed. Additionally, the experimental setup, such as lighting, number of mazes, or computer placement, may vary depending on the construction of the animal facility.
3. Tail suspension test (TST)
   NOTE: Three mice are tested simultaneously with the automated tail suspension apparatus.
   1. Maintain the room light intensity at 100-120 lux.
   2. Connect the TST system with the computer via a USB cable. Insert the USB dongle into the computer and start the software by double-clicking on the BIO-TST software icon. In the Settings tab under Global, adjust the acquisition duration to 360 s. In the Experiment tab, select 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.
   3. Start the run by clicking Start run | continue in the Acquisition tab. Prepare the animals for the test by wrapping single-sided adhesive tape, such as the transpore medical tape, around 3/4 of the animal's tail, starting from the base.
   4. Pass the suspension hook through the tape and suspend the animal on it. Start acquiring data for each animal individually immediately after hanging it on the hook by clicking the Start icon under the visualized position for each animal and observe animals continuously during the test.
   5. After completion of the acquisition for the first set of animals, click Initiate the next run, remove the animals from the hook, detach the adhesive tape from their tails, carefully cutting the tape with scissors along the tail, and put the animals in the temporary holding cage.
   6. Clean the apparatus with 75% alcohol and paper tissues and proceed with the rest of the animals as described above. In the Analysis tab, select the last 4 min of the acquisition for analysis, then select all valid runs in the Analysis period, click Analyze selected subjects, choose the desired data format, and click Export selected data to export the collected data for further analysis.
      NOTE: The test lasts 6 min. During the first 2 min, the animals will struggle vigorously, but as the despair reaction becomes prevalent during the remaining 4 min, the immobility time during this period is taken for the analysis. The software and hardware may differ, and the relevant manuals must be followed. Additionally, the equipment itself may vary (e.g., number of testing positions).
4. Gait analysis
   1. Turn on the treadmill and manually set the belt speed to 20 cm/s on the equipment panel by clicking the + or - symbol situated next to the speed indicator. Turn the apparatus light on by turning the knob clockwise. Launch the DigiGait Imager software by double-clicking the software icon and set the shutter speed to 100 for albino mice or 130 for black/dark mice in the field for shutter speed.
   2. Remove the first animal from the home cage by hand and gently place it onto the treadmill belt. Close the door to the animal compartment. Visually inspect to ensure that the animal's tail is not stuck between the door and the frame.
   3. Allow the mouse to explore the treadmill belt prior to recording. Ascertain that the animal is able to perform the test by setting the treadmill to a slow walking speed for ~3 s and then stopping it, observing the animal continuously.
   4. Start the belt by pressing the Start button on the equipment panel and record for approximately 10 s. Ensure that a clear and fluent locomotion of at least 10-15 steps is observable. Stop the belt by pressing the Stop button on the equipment panel and return the mouse to the temporary holding cage by hand.
   5. 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. Choose 10-15 fluent movements by manually writing their starting and ending frame numbers into the relevant fields (From frame# for the first frame and To for the last frame). Fill out the animal's information-animal ID, date of birth, sex, weight, belt speed, and belt angle-and comment when needed in the relevant fields. Save the file for further analysis by clicking Save.
   6. Clean the belt with water and proceed with the rest of the animals the same way. Choose CAMERA to proceed with recording the next animal walking. When recordings are acquired for all the animals, proceed to the analysis.
      NOTE: Animals that are not able to walk at a set speed of the belt are excluded from testing. Based on our experience, we observe that older animals (over 50 weeks) experience more difficulties with walking on the treadmill, with a variable frequency between 2% to 50% depending on the genotype. Animal waste is collected in trays on either the front or rear of the treadmill. The trays are emptied after each study and washed with warm soapy water. The belt is wiped with a damp cloth.
   7. Perform gait analysis based on a fully automated analysis of video recordings of animal footprints. Adjust the data in DigiGait Analysis software.
      NOTE: Gait analysis provides not just a measure of motor coordination, but also a detailed kinematic description based on the analysis of dynamic gait signal, representing the temporal history of paw placement through sequential strides. The following parameters are automatically measured by the software: swing duration, percentage of stride duration in swinging, braking duration, percentage of stride duration in braking, propulsion duration, percentage of stride in propulsion, stance duration, percentage of stride in stance, stride duration, braking percentage of the stance, propulsion percentage of the stance phase, swing to stance ratio, stride length, stride frequency, paw angle, paw angle variability, stance width, step angle, stride length variability, stride width variability, step angle variability, coefficient of variation of stride length, coefficient of variation of stance width, coefficient of variation of step angle, coefficient of variation of swing duration, paw area at peak stance, paw area variability at peak stance, hind limb shared stance duration, percentage of shared stance, ratio of left and right rear stance durations, gait symmetry, maximal rate of change of paw area in contact with the treadmill belt during the braking phase, maximal rate of change of paw area in contact with the treadmill belt during the propulsion phase, tau-propulsion, paw overlap distance, paw placement positioning, ataxia coefficient, midline distance, axis distance, and paw drag. The software allows for a small correction of step trace noise, which should be completed prior to statistical analysis. The software and hardware may differ, and the relevant manuals must be followed.
5. Rotarod
   NOTE: The rotarod test is used to assess rodent motor functions-balance and motor coordination. The test requires a mouse to walk on a rotating rod of a fixed diameter (5 cm), with the rotation accelerating over a given period of time (5 min) until the animal can no longer stay on.
   1. Switch on the rotarod equipment by pressing the on/off switch on the equipment and launch the software by double-clicking the Rod software icon. Initialize a new file in the File tab and save it an under 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 s, initial speed to 4 rpm, and terminal speed to 40 rpm.
   2. Prepare a schedule for the tested animals in the Animal field, and assign each animal to its position on the rod. The positions are not indicated in the software explicitly, but they correspond to the list line; for example, the first line would indicate the first position of the rod, the fifth line would indicate the fifth position of the rod, and so on. Remember to counterbalance each rod position between the experimental groups.
      NOTE: Five animals can be tested simultaneously.
   3. Close the Setup panel by clicking Close and open the measuring panel by clicking Measure. Start the initial rotation of the rod at 4 rpm by clicking Start/Stop and place the first five animals onto their assigned positions.When all the animals are on the rod, start the testing protocol by clicking Start Profile, and the rod will gradually accelerate to 40 rpm over 5 min. If an animal falls off the rod, return it to the rod before the protocol begins.
      NOTE: Animals usually do not stay on the rod long enough to place all the mice on it at once during the first attempt. It is important to be patient when placing animals on the rod with the constant speed of rotation at the start. The purpose of the test is not to determine the animal's endurance on the rod at a fixed rotation speed, but to find the speed at which the animal is unable to stay on the rod. The speed of the rod is proportional to the latency of staying on it; thus, it is used to express the animal's balance.
   4. Move the animals to the temporary holding cage after all of them have fallen from the rod or after 5 min have passed. Remove any animal waste and clean the rod and tray with alcohol.
   5. Click Animals -> to proceed with the next group of animals in the same manner. After testing all the animals, close the Measure window by clicking Close and click Show to display the collected data. Export the acquired data in .csv file format for further analysis by clicking Export CSV.
   6. Test each animal on the rod three times with 15 min intertrial intervals. Use the averaged value of the latency to fall over the three trials for further statistical analysis. Evaluate the animal's motor learning by repeating the test for 5 consecutive days.
      NOTE: The software and hardware may differ, and the relevant manuals must be followed. Additionally, the equipment itself may vary, for example, in number of testing positions, overall construction, and rod dimension.
6. Nestlet shredding-nest building
   1. Separate the animals into single polycarbonate mouse cages with standard equipment (bedding, food mesh, and water supply) for 1 week. Take approximately 12 g of cotton nestlet using forceps, record its weight manually using scales, and place it randomly in a cage, but at the opposite side to the water supply. Return the cages with the animals to the housing room.
   2. Weigh each nestlet at the same time every day for the next 4 days manually using scales. Record the weights on paper or in a premade spreadsheet. Make sure each nestlet is dry when weighed; if not, dry on a heating pad and return all the nestlets to their assigned cages at the same time in the place where the mouse made its nest. If the nestlet is torn into several parts, weigh the largest one.
   3. For data analysis, express the decrement in nestlet weight on each day relative to the initial weight and present it as a percentage of the used material.
      NOTE: Bringing males back to a common cage may lead to increased aggression and unwanted injuries among the animals. Therefore, the nestlet shredding test should be scheduled toward the end of the testing regimen to avoid compromising animal welfare.

Elevated plus maze and open field tests
The EPM and OF tests use the natural tendency of rodents to explore new environments^18,19. 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 competitors. Both the OF test and the EPM consist of safe and risky compartments-the periphery and center in the OF test and closed and open arms in the EPM, respectively. Rodents naturally prefer dark, enclosed compartments compared to open, elevated, and brightly lit areas. Thus, reduced exploration of the risky/anxiogenic parts, expressed as a decrement in the number of visits and visit duration, or as increased latency to the first visit, characterize animal anxiety-like behavior^8,11. Resting time, average speed, and total traversed distance deliver additional information about the spontaneous activity of the animals. None of the parameters related to anxiety-like behavior were altered in UBE3A^mGenedel/+ mutants in either the OF test or the EPM (Figure 1D-G). However, UBE3A^mGenedel/+ animals were significantly hypoactive, as reflected by a shorter traversed distance, lower average speed, and longer resting time in the OF test (Figure 1A-C).

Figure 1: Spontaneous activity and anxiety response to a new environment in the EPM and OF test. (A-E) Exploration of the open field. The UBE3A^mGenedel/+ animals walked a shorter distance (A) with a lower average speed (B) and prolonged resting time (C). The number of visits and duration in the center did not differ between animals (D,E). A two-way ANOVA revealed a significant main genotype effect with no significant interaction between genotype and sex (genotype effect: p < 0.01; genotype/sex interaction: p > 0.7). The percentage of visits to open and closed arms did not depend on genotype (F), nor did the time spent in the anxiogenic open arms differ between experimental groups (G). A two-way ANOVA did not reveal significant main effects or genotype/sex interaction (genotype effect: p > 0.9; genotype/sex interaction: p > 0.9). Data depicted in the boxplot show the median value, inter quartile range, and range of values. Significant post-hoc test results are indicated as *. Data for control animals (female n = 10, male n = 11) are presented in red, and mutants (female n = 9, male n = 10) in blue. This figure was adapted from Syding et al.⁶. Abbreviations: EPM = elevated plus maze; OF = open field. Please click here to view a larger version of this figure.

Tail suspension test
The TST measures animal despair developed in an inescapable situation. When suspended by the tail, rodents become rapidly immobile after an initial period of vigorous activity. The duration of the immobility indicates the magnitude of the "despair". Numerous laboratories have shown that a wide range of clinically active antidepressant drugs reduce the immobility duration^9,20,21. This uncomplicated test has become commonly used for screening for potential antidepressant substances, and it may also be utilized to characterize the phenotype of various animal strains, as well as transgenic murines, in studies exploring the neurobiological basis of depressive states^9,21. UBE3A^mGenedel/+ animals were immobile significantly longer than their control littermates, indicating their depression-like behavior (Figure 2).

Figure 2: Immobility time in the tail suspension test. UBE3A^mGenedel/+ animals showed a longer immobility during the tail suspension. A two-way ANOVA showed significant main effects but no significance in genotype/sex interaction (genotype effect: p < 0.001; sex effect: p < 0.001; genotype/sex interaction: p > 0.5). Data depicted in the boxplot show the median value, interquartile range, and range of values. Significant post-hoc test results are indicated as *. Data for control animals (female n = 10, male n = 14) are presented in red, and mutants (female n = 10, male n = 11) in blue. This figure was adapted from Syding et al.⁶. Please click here to view a larger version of this figure.

Rotarod and gait analysis
The history of rotarod testing in models of neuromotor deficits dates back to the mid-20^th century²². The rotarod is used to assess animal balance and movement coordination, since their impairments manifest in a significantly shorter latency to fall from the rotating rod¹⁴. Repeated testing on the rotarod is used to study animal motor learning capabilities. The rapid development of modern equipment and digital technologies have enabled automated, precise, and unbiased evaluation of rodent locomotor phenotypes based on the detailed descriptions of their gait²³. Automated gait analysis replaced footprint analysis, and is also more sensitive to neuromuscular deficits^14,24,25. Alterations of spatio-temporal characteristics of the animal gait are specific to the modeled nosological unit^26,27,28. UBE3A^mGenedel/+ mutants had a robust alternation of gait indices (Figure 3A-G), further confirmed by a reduced latency to fall from the rotarod (Figure 3H).

Figure 3: Detailed gait analysis and motor learning on the rotarod. (A-G) The gait indices of UBE3A^mGenedel/+ animals were altered. UBE3A^mGenedel/+ animals had a longer swing (A) and stance (B) that resulted in prolonged stride duration and length (C,D). Their hind limbs propulsion duration (E) and deceleration (F) were also increased. The analysis also revealed a larger paw area at the peak stance (G). Neither the animals' metric parameters nor weight differed (data not shown), indicating that observed differences were not due to differences in animal size. A two-way ANOVA with repeated measurements showed a significant main effect of genotype with no significant genotype/sex interaction (genotype effect: p < 0.001; genotype/sex interaction: p > 0.2). (H) Results of the rotarod performance show a shorter latency to fall in UBE3A^mGenedel/+ animals. A two-way ANOVA with repeated measurements revealed significant main effects without a significant interaction (genotype effect: p < 0.001; sex effect: p < 0.01; genotype/sex interaction: p > 0.1). Gait parameters depicted in the boxplot show the median value, interquartile range, and range of values. Significant post-hoc test results are indicated as *. Data of the latency to fall are presented in a line plot as mean ± SEM. Data for control animals (female n = 10, male n = 14) are presented in red, and mutants (female n = 10, male n = 11) in blue. This figure was adapted from Syding et al.⁶. Please click here to view a larger version of this figure.

Nestlet-shredding - nest-building
The nestlet shredding test is primarily used to detect stereotypical compulsive behavior in mice^29,30. However, mice show a natural tendency to tear provided material to build their nest. The inability to shred a cotton nestlet is thus used as an indicator of their wellbeing affected by neurodevelopmental impairment^16,31. The UBE3A^mGenedel/+ animals used significantly less material to build their nests, and this difference was particularly prominent between transgenic females and their control counterparts (Figure 4A).

Figure 4: Use of nestlet material for nest building. UBE3A^mGenedel/+ animals shredded less cotton material than their control littermates. The data were transformed to aligned ranks to satisfy the normality prerequisite. An analysis of variance with repeated measures revealed a significant genotype effect without a significance of genotype/sex interaction (genotype effect: p < 0.05; genotype/sex interaction: p > 0.4). Data depicted in the line plot show mean ± SEM. Significant post-hoc test results are indicated as *. Data for control animals (female n = 10, male n = 14) are presented in red, and mutants (female n = 10, male n = 11) in blue. This figure was adapted from Syding et al.⁶. Please click here to view a larger version of this figure.

Testing timescale
Each group (control and experimental) is subjected to the same tests on the same days. A break of 1 day between tests is employed to minimize potential carryover effects. If possible, females and males are tested on consecutive days; otherwise, females are tested after males have been tested (Figure 5)⁶.

Figure 5: Testing timescale. UBE3A^mGenedel/+ animals and their controls were tested in two cohorts. The testing timescale for the first cohort is presented in the upper panel, and for the second cohort in the lower panel. The days on which males were tested are indicated in blue, while days on which females were tested are indicated in green. Days on which both sexes were tested are indicated in yellow. No testing was performed on weekends. Please click here to view a larger version of this figure.

The figures were adapted from Syding et al.⁶ in accordance with the MDPI license policy.

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 symptoms^31,32. A motor deficit in AS models is the most consistent finding across laboratories, followed by an unchanged emotionality state of mutants and difficulties building nests^31,32,33. In contrast, cognitive impairment is either mild or absent^7,31,33. Discrepancy in the cognitive phenotype seems to depend on the tested animals age, as shown by Huang et al.⁷. Therefore, for this paper, a battery of tests was chosen on the basis of their reproducibility, as well as age- and species-independence, as comparable results are observed in both mouse and rat AS models^6,31,32.

Critically, one should keep in mind that testing animals repeatedly in different experimental setups demands for their careful ordering, starting with the tests most sensitive to prior manipulation, and at the same time with minimal effect on the following tests, such as the EPM and OF tests³⁴. Additional concerns pertain to the nestlet shredding test, where animals are single-housed, which is known to be a stressful condition³⁵. Subsequently pooling males in a common cage often leads to increased aggression due to hierarchy establishment. Thus, the nestlet shredding test should conclude the testing schedule. It is also good practice to test males before females to avoid male behavior becoming influenced by trailing female olfactory traces. Alternating animals belonging to different experimental groups during testing is crucial in behavioral research to balance the effects of unpredictable factors on animal behavior. It is well known that handling animals before testing in the EPM influences their observed stress response. Therefore, the amount of handling must be consistent for all animals³⁶. It is also very important to maintain the housing conditions (single vs. group), lighting during testing, time of testing, and prior to testing experience for each animal, as all these factors influence a mouse's response in the EPM and OF test and may bias the results³⁷.

Despite the presented tests belonging to well-established screening tools in drug development and genetically modified mice phenotyping that yield reproducible results across laboratories, some tests can still be subject to minor modifications. As motor impairment is the main feature of an AS animal model's phenotype, the rotarod test could be limited to 1 day testing instead of 5 consecutive days. Additionally, parameters that describe the quality of a built nest could be incorporated into the nestlet shredding test³⁸.

One clear limitation of the presented results is the ambiguity of their interpretation. In particular, AS animals' motoric deficit can explain changes in locomotion-based tasks, such as the OF test and EPM. Analogically, a prolonged immobility time in the TST can be a result of the greater physical fatigue that AS animals develop during this demanding test, as opposed to depressive-like behavior. Also, in the nestlet shredding test, reduced cotton usage may be due to the neuromuscular phenotype rather than the loss of the nest building instinct. The interpretation of stride length changes is ambiguous, as shortening is observed in some mouse models of Parkinson's disease, while prolongation is observed in aging mice^39,40. However, we believe that an increase in total stride length is a consequence of a longer swing duration. Swing duration increases with pain and is prolonged in arthritis models, which implies that a longer swing duration in mice could potentially allow for proper positioning of the limbs before bearing weight^41,42. Propulsion duration refers to the duration of time required for an animal to initiate and maintain forward motion. Thus, a short duration in healthy animals may indicate greater strength and better control. These findings not only characterize this AS mice model but also indicate gait impairment. However, closer investigation is needed to elucidate the physiological basis of such impairment, such as determining muscle strength and examining neuromuscular connections/transmission.

Despite the interpretational dilemma, the presented battery of behavioral tests provide reproducible results consistent across laboratories and can serve as an elegant validation tool for new murine models of Angelman syndrome and new therapeutic approaches^{6,31,32,43,44,45}.