Strategies for Assessing Autistic-Like Behaviors in Mice

Summary

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Abstract

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Introduction

Autism spectrum disorder (ASD) is a developmental brain disorder that manifests social communication or interaction impairments and restricted, repetitive patterns of behaviors or interests^1,2. In 2022, approximately 1 in 100 children were diagnosed with ASD globally³. According to the Centers for Disease Control and Prevention (CDC, USA), the prevalence of ASD has increased by 30% since 2008 and is up more than 2-fold since 2000^4,5. Individuals with ASD may also exhibit co-morbidities, such as intellectual disability (ID) (35.2%, IQ ≤ 70), attention-deficit/hyperactivity disorder (ADHD) (50%-70%), and other genetic syndromes^2,4,6.

The use of animal models in ASD research, especially rodents, has provided significant insights into the impact of various environmental factors, including diet, drugs, exercise, and enrichment^7,8,9,10, as well as genetic mutations such as Shank, Fmr1, Mecp2, Pten, and Tsc mutant^11,12,13, on ASD symptoms. Mouse models are commonly used to investigate ASD due to their social nature and shared genetic, biochemical, and electrophysiological features with humans. For instance, by deletion of a specific gene (such as Shank3, Fmr1, Cntnap2, and Pten), aberrant social and repetitive behaviors can be recapitulated, providing strong validity of the study^14,15,16. Here, we provide protocols for studying parallels between animal genetic models and human ASD symptoms¹⁷. We describe the self-grooming and three-chamber social interaction test, which reflect two core symptoms in ASD patients, namely restricted, repetitive patterns of behavior and social interaction (communication) impairments, respectively.

Based on the DSM-V (Diagnostic and Statistical Manual of Mental Disorders of the American Psychiatric Association 5^th Edition) and ICD-11 (International Classification of Diseases 11th Revision), ASD patients engage in restricted, repetitive, and stereotyped behavior patterns, in particular, non-functional body-focused repetitive behaviors (BFRBs), such as rocking, stimming, nail-biting, hair pulling, skin picking, or toe-walking^18,19. In animals, repetitive behavior is manifested by prolonged and repetitive self-grooming. Grooming is one of the most common innate activities among rodents, with approximately 40% of their wake time spent on grooming^20,21. It is instinctive for mice to lick their skin or fur to remove foreign dirt from the body surface, which serves to maintain body cleanliness, prevent injury, remove parasites, and regulate temperature. Grooming is categorized into two types: social grooming (allo-grooming), involving grooming by another mouse, and self-grooming. Self-grooming shows a stereotyped and conserved sequencing pattern consisting of four stages (mostly discrete and non-sequential)^22,23. In stage I (Elliptical stroke), mice initiate grooming by first licking both paws and then grooming around the nose with their paws. In stage II (Unilateral stroke), mice use their paws to wipe their face asymmetrically. In stage III (Bilateral stroke), mice symmetrically wipe their head and ears. In stage IV (Body licking), mice transition to body licking by moving their head backward and may extend grooming to the tail and genitals. When mice are individually placed in a clear cage, self-grooming behavior can be readily recognized and observed. Mice increase self-grooming behavior when faced with stress, pain, or social disruption, rendering the self-grooming test crucial when researching neurological disorders²². Different mouse models of ASD, including those with genetic mutations (such as Fmr1^−/y, Shank3B^−/-, NL1^−/−), pharmacological interventions (such as DO34, PolyI:C), and specific inbred strains (like BTBR and C58/J), have demonstrated excessive repetitive self-grooming behavior^24,25,26,27.

Alterations in social behavior serve as one of the criteria for assessing ASD. According to the DSM-V and the ICD-11, ASD patients display persistent social communication and social interaction impairments^18,19. These may manifest in verbal and nonverbal communication deficits (i.e., abnormal eye contact, gestures, and facial expression), lack of sharing interest and emotions with others, unawareness of social contextual cues, or difficulties developing relationships. In line with the social impairment symptoms, various behavioral tasks have been designed and optimized to assess social interactions in mice, such as the direct social interaction test, the three-chamber social approach and preference for social novelty test, and analysis of ultrasonic vocalizations (USVs)^16,28. The three-chamber social interaction test is an extensively used experiment for evaluating ASD-related behaviors^17,29,30,31. The apparatus comprises three connected chambers; the left and right chambers contain a wire cage that may be either empty or occupied by a mouse, enabling the test mouse to interact freely with both cages. Two measurements help assess different aspects of social behavior in the test mouse during the three-chamber experiment. First, the test mouse is scored for the time spent interacting with the empty cage (novel object) versus a cage that contains a novel mouse. This part of the task provides insight into the mouse's sociability. Next, an unfamiliar mouse is placed into the previously empty wire cage. The time difference in interaction of the test mouse between the unfamiliar and familiar mouse measures the preference for social novelty. In this part of the task, a control mouse prefers to interact with an unfamiliar rather than the previously encountered mouse, which was already present in the sociability part of the test. Deficits in social interaction and decreased motivation of interacting with novel mice are generally found in mouse model of ASD. The three chamber test has proven robust since its invention. It has been used to study social phenotypes in various mouse models of ASD, including Fmr1^−/−, Shank3B^−/-, Cntnap2^−/−, and the BTBR inbred strain^{32,33,34,35,36}.

The two tests utilize naturally occurring, spontaneous behavior of mice as meritorious tools for studying ASD-like behavior. Since they are considered low-stress tests, it is feasible to conduct both tests within the same group of mice to measure ASD-like behavior, with the self-grooming test being performed first and the three-chamber social interaction test on subsequent days. The protocols we provide present an essential tool for the assessment of ASD-like behavior and the development of new therapeutics^29,30,31. Ultimately, they would contribute to improving outcomes for individuals affected by ASD.

Protocol

All procedures and experiments involving animal subjects were approved by the Facility Animal Care Committee (FACC) regulations, which follow the guidelines established by the Canadian Council on Animal Care, the McGill University Animal Care Committee, and the NIH Office of Laboratory Animal Welfare (OLAW). The Public Health Service (PHS) Assurance number for McGill University is F-16-00005(A5006-01).

1. Animal preparation

1. Test mice: Select 2-3 month (8 to 13 week) old male and/or female mice. All mice used in the experiments here are from C57BL/6J background. For self-grooming behavior and the three-chamber social interaction assays, the generally acceptable range of mice per group suggested is n = 10-15, with a minimum of n = 8.
   1. For genetically modified mice, always add wildtype littermates as control. Match the control group with the experimental group in terms of genetic background, age, sex, and housing conditions. The homozygous transgenic mice used in this study are normal in size and do not display any gross physical abnormalities.
      NOTE: In some more complex cases, researchers may be interested in evaluating more than two genotypes (i.e., wild type, heterozygous, homozygous), investigating the effects of drugs (i.e., treatment, vehicle treatment, non-treatment), or comparing males and females. When working with female mice, it is crucial to take into account their estrous cycles. While female estrous cycles do not significantly influence self-grooming behaviors, studies have indicated that these cycles can impact social approach and preference for social novelty^37,38.
2. Stranger mice: Use at least 8 wild type mice for the three-chamber test. Use 2 cages, 4 mice per cage, matching age, sex, and the same genetic background (C57BL/6J) of the test mice. Ensure that the stranger mice did not have prior interaction with the test mice.
3. Housing: Group-house 3-5 mice per home cage (7½" W x 11½" L x 5" H mouse plastic cages) and maintain on a 12 h light/dark cycle. Provide mice with access to food and water in the cage ad libitum.
   NOTE: In the housing rooms in this study, lights switch on at 7:00 am.
4. Transfer mice to the animal behavioral testing facility for a minimum of 1 week prior to experiments^39,40.
5. Handling and behavioral testing: Conduct the test at a consistent time of day. Adjust the start time depending on the light cycle in the mouse housing rooms.First, transfer the mice into the testing room and allow them to habituate to the testing room for a minimum of 30 min under dimmed lighting before starting the behavioral tests.
   NOTE: In this study, mouse experiments were routinely performed between 7:00 am-3:00 pm (relative to 7:00 am light on and 7:00 pm light off). Hence, the actual initiation of the behavior test occurs around 8 am.

2. Room and equipment preparation

1. Prepare a small room with overall floor areas between 16-36 ft (5-12 m²; 6 m² room in this study). Maintain consistent temperature, lighting (adjustable dimmed lighting), and noise levels (ideally soundproof) for all the experiments.
   1. If using standing lamps (bulbs 23 W, 120 V), place them in symmetrical locations and on each side, at least 1 m away from the three-chamber apparatus, to illuminate the two side chambers equally.
2. Place the apparatus on a portable white table. For the self-grooming experiment, use clean, transparent cages (7½" W x 11½" L x 5" H mouse plastic cages); use one cage per test mouse. Fill cages with approximately 1 cm of fresh bedding without nesting material. Use the same type of bedding as in the home cage (here, fresh corncob bedding ([1/4" Corncob bedding]).
   NOTE: The cages should be the same size as the home cages, with a filter lid placed on top, but without a metal grid (which is normally used for holding bottles and food delivery).
3. For the social experiment, use a three-chamber apparatus set⁴¹. Each chamber is 20 cm x 40 cm x 22 (h) cm and surrounded by Plexiglas walls. Each side chamber is accessible from the center by transparent sliding doors that can be removed (5 x 8 cm). Use two wire cages (diameter: 7 cm, height: 15 cm; with cover lids (empty or holding stranger mice). The cage is elevated and enclosed by smooth stainless steel wire bars (bar diameter: 3 mm, bar spacing: 7 mm). Do not include sitting on top of the cages as interaction time.
   1. If male and female mice are both included in the experiment, label each wire cage for its dedicated function to minimize cross-contamination.
4. Set up overhead or front-facing cameras on tripods for video recordings. Make sure that the cameras are fully charged and have sufficient storage.
5. In between each test mouse experiment, clean the three-chamber setup and wire cages thoroughly with an odorless cleaning agent (here, Versa-Clean). Dilute the cleaning agent in tap water at a ratio of 1:40, apply it to the soiled surface, and dry the surfaces with a paper towel before the next test to minimize residual odorant from the previous mouse.
   NOTE: Ethyl alcohol (70%) or other disinfectants with odors should not be used to clean the apparatus during social interaction tests, as they may impact rodent social behavior.
6. Use privacy blinds, such as white plastic boards, which can be placed around the apparatus to eliminate external room cues.
7. Prepare a small whiteboard and a marker. Record essential information on the whiteboard, including the mouse number, the experiment title, the date, and the time of the experiment.
   NOTE: The whiteboard is presented to the camera at the end of the recording to prevent the scoring person from knowing the mouse's number before assessing the mouse's behavior. The study ensured that the individual scoring behavior remained blind to the experimental groups to minimize potential bias.

3. Handling

1. Perform handling procedures for three consecutive days prior to the start of the behavioral experiments.
2. Conduct handling sessions in the same testing room at a consistent time, ideally in the morning when mice are more active.
3. On each of the 3 days, bring the mice to the testing room and leave them undisturbed for 30 min in dim light to acclimatize.
4. After a 30 min acclimatization period, open the cage and introduce a gloved hand into the cage. Allow mice to explore the hand for 1 min.
5. Gently scoop the mice onto the hand (or use a small cardboard tube to pick them up), holding them loosely. Allow mice to explore the hand and wrist for 1-2 min. In instances where a mouse is jumpy or aggressive, place it on the grid and allow it to compose itself.
6. Continue handling until the mouse becomes comfortable staying on the hand. Always change the gloves before introducing hands to the next cage.
7. After handling each cage of mice, gently place them into their home cage, and then repeat the procedure.
8. Put on new gloves and handle stranger mice when all the experimental and control mice have been handled to avoid affecting testing mice with scented messages.
9. Handle the stranger mice in the same way.
10. Optional: Habituate the stranger mice to the wire cage and the three-chamber apparatus for 10-15 min.
    NOTE: This habituation session helps reduce agitation and aberrant behaviors in the stranger mice during the three-chamber test. However, for the three-chamber test, this study only considers social approach behavior (sniffing) of the cage initiated by the test mouse, whether a stranger mouse is present or not. Reciprocate sniffing by the stranger mouse is not reliably observable in this assay and, therefore, is not counted. Due to this limitation of the three-chamber test and to minimize cross-contamination of chemical signals (such as smells), in this case, the stranger mice did not have prior experience with the wire cage and the apparatus before the start of the experiment.

4. Method 1: Self-grooming for repetitive behavior (Figure 1A)

1. Perform all behavioral experiments between 7:00 am-3:00 pm.
2. Switch on dimmed lights, as described above. Clean the table and set up the room.
3. Prepare each test cage with a thin layer of fresh bedding (as described in step 2.2).
4. Put 1-2 cages on the table, separated from each other and the room environment by white plastic blinders.
5. Place a camera in front of each cage to capture the test mouse.
6. Transfer all mice into the room and cover the cages with a fabric sheet during the transfer process to avoid stress.
7. Remove the sheet once the mice are placed in the testing room, leaving them in the room for at least 30 min with dimmed lights before the start of the experiment.
8. At the start of the test, write down the mouse identification number and testing information on a whiteboard (as described in step 2.7).
9. Start recording and place the test mouse into the test cage.
10. Leave the room during the 20 min recording session.
11. For each test, consider the first 10 min as habituation. Use the following 10 min to observe and score grooming behavior (see section 6).
12. After 20 min, return to the room. Present the whiteboard to the camera and stop recording.
13. Gently place the test mouse back into the home cage and repeat the experiment.
    NOTE: Counterbalance the order of tests between experimental and control groups to reduce various outside influences.
14. After experiments are finished, return the mice to their housing room.

5. Method 2: Three-chamber social interaction test (Figure 2A)

1. Perform all experiments between 7:00 am-3:00 pm.
2. Clean the table and turn on the lights (dimmed lights).
3. Place the three-chamber plexiglass instrument on the table surrounded by privacy blinds.
4. Place one camera overhead, ensuring that all three chambers are in the recording frame of the camera.
5. Transfer the test and stranger mice into the room. Cover the cages with a fabric sheet during the transfer process and remove the sheet once in the room.
6. Leave the mice in the room for at least 30 min with dimmed lighting before the start of the experiment.
7. Habituation
   1. Keep the three chambers empty during the habituation phase.
   2. To start, select a test mouse and write the identification number on the whiteboard.
   3. Then, gently place the test mouse in the center compartment while the sliding doors are still closed.
   4. Start recording and open the doors to allow the test mouse to explore the three empty chambers.
   5. Exit the room and let the test mouse habituate to the apparatus for 10 min.
   6. After 10 min habituation is complete, return to the room. Guide the test mouse gently to the center compartment and close the sliding doors.
   7. Display the whiteboard and stop the camera.
8. Social approach (novel wire cage object versus novel stranger mouse 1)
   1. Position two wire cages in the left and right chambers. Place these cages diagonally opposite each other in the corners of each chamber, ensuring a 5 cm clearance from the wall, allowing the test mouse to run around the cages. Additionally, ensure that the cages do not directly face the chamber doors.
   2. Choose one stranger mouse and introduce it into one of the wire cages, while no mouse is present in the other wire cage.
   3. To start the test, press record and remove both sliding doors. Leave the room.
   4. Allow the test mouse to explore an empty wire cage in one chamber and a cage containing a stranger mouse (novel stranger mouse 1 or S1) in the other chamber for 10 min.
   5. After 10 min, return to the room. Display the whiteboard to the camera and end the recording.
   6. Re-introduce the mouse to the center compartment and close the interconnecting doors.
      NOTE: Do not remove the wire cages from the setup after finishing the social approach test so they stay in the same position during the whole task.
9. Preference for social novelty (novel stranger mouse 2 versus novel mouse 1)
   1. Place a new, never-encountered mouse to the previously empty wire cage (novel stranger mouse 2 or S2).
      NOTE: S2 must be from a different home cage than S1.
   2. Start recording and open the interconnected doors to allow the test mouse to explore the apparatus for 10 min.
   3. Leave the room. The test mouse will explore two wire cages: one with the previously encountered S1 mouse from the social approach stage and the other with the newly introduced S2 mouse.
   4. After 10 min, return to the room. Display the whiteboard to the camera and conclude the recording.
   5. Return all the mice (test and novel stranger mice) to their home cages.
   6. Thoroughly clean the chambers and wire cages with odor-free disinfectant. Make sure the apparatus is dried before using it for the next experiment.
   7. Repeat the experiment for all remaining test mice.
      NOTE: Alternate the placement of the cages with S1 and S2 mice between the left and right chambers for the different test mice. The counterbalance prevents a biased preference towards a particular chamber of the apparatus.
   8. After testing, return the mice to the housing facility.
   9. Between each part of the test, stop and restart the camera, resulting in 3 times 10 min videos per test mouse.

6. Scoring and statistical analysis

1. Necessary equipment
   1. Use two stopwatches and a computer or laptop with the following software: Microsoft Excel, GraphPad Prism, and other statistical programs such as SPSS for scoring, plotting the graphs, and statistical analysis.
2. Self-grooming scoring
   NOTE: When a mouse is placed in a comfortable environment, low-stress spontaneous self-grooming behavior can be observed. Typically, mice initiate grooming by licking their paws around the nose and whiskers (stage I). This might be followed by stages II and III, where the mouse proceeds to wipe their whole face and head with its paws. In stage IV the mouse continues grooming its body and licking its tail¹⁴. Given the infrequency of grooming their ears (stage III) and tails (stage IV) within a 10 min test period, and aiming to enhance the clarity of manual analysis, self-grooming behavior was categorized into two primary types: rostal grooming and caudal grooming. Rostral grooming involves activities such as paw licking, nose grooming, and thorough washing of the face, ears, and head. Caudal grooming includes licking the body part covering areas such as the belly, back, hind limbs, and genital area, as well as the tail.
   1. Observe the grooming behavior carefully and record the bouts. An individual grooming session happens when the mouse engages in a single instance of self-grooming. Consider a grooming session from the moment it starts until it stops, regardless of the specific grooming behaviors involved.
   2. If the test mouse pauses its grooming for a few seconds without changing position, count it as part of the same bout. However, if the mouse stops grooming and begins to explore, consider that the grooming bout has concluded.
   3. Additionally, only record a full grooming session when the mouse grooms continuously from the front to the back (from stage I to stage IV).
   4. Document the total grooming time, the number of grooming bouts, and the number of full grooming bouts observed during the 10 min testing period, marking observations at 2 min, 5 min, and 10 min intervals.
3. Three-chamber social interaction test
   ​The analysis determines the time and frequency of sniffing interaction with each mouse (novel S1 and empty cage for social approach, and S1 and novel S2 for the preference for social novelty).
   1. Use two timers to record the interaction time with the mouse in each cage and the total time spent in each chamber. For each part of the test (social approach and preference for social novelty), record the number of times the test mouse enters each chamber (total entries).
   2. Determine the time in compartments (s) by the presence of the test mouse's body within one of the chambers (four paws must enter).
   3. Record interaction time (s) when the test mouse sniffs the novel stranger mouse (direct face contact or sniffing the tail of the stranger mouse) or sniffs the empty cage. Do not consider sitting on the top of the cage as interaction.
   4. Record at 2 min, 5 min, and 10 min. Do a time-dependent analysis.
   5. Compare the sociability discrimination index (DI) within the groups.
      NOTE: DI is calculated as: DI = (time interacting with S1 mouse - time interacting with the empty cage)/total interacting time x 100. During the social novelty part, control mice will interact more with S2 (unfamiliar mouse) than with S1 (familiar mouse). The social novelty (social memory) DI is calculated: DI = (time interacting with S2 mouse - time interacting with the familiar mouse (S1))/ total interacting time x 100.
4. Statistical analysis
   1. Use a two-tailed unpaired student's T-test to compute the p-values with a significant level set at p < 0.05 for the self-grooming time, bouts, and full bouts, where there are only two groups.
   2. For the three-chamber test, use a student's T-test to calculate the statistical significance of the total entries between two groups. When the assumption of the equal variance of data between groups is not met (Levene's test p < 0.05), perform a Welch's T-test to adjust the deviation and compare the means of independent groups.
      NOTE: Measuring total entries assesses the overall activity of mice. Potential concerns may arise (related to motor impairment and high anxiety levels of mice) if the experimental group explores the three-chamber apparatus significantly less than the control group. To address this, consider evaluating motor function and anxiety-like behavior with depressive-like behavioral tests, as diminished exploration could be attributed to these conditions⁴².
   3. Use a mixed-way ANOVA to compare the time in each chamber and the interaction time for each test. Depending on the number of groups and factors, use a mixed two-way ANOVA (2 factors: genotype and side of the chamber, or interacting with empty cage and S1, or S1 and S2), or a mixed three-way ANOVA (3 factors: genotype, treatment, and side of the chamber, or interacting with empty and S1, or S1 and S2). This analysis is particularly useful for revealing the statistically significant social interaction of mice between various factors.
      NOTE: These factors include the main effect of genotype (impact of different genetic backgrounds on social behaviors), the main effect of interaction with the cage (sniffing and preference for novel stranger mouse versus an empty cage or previously encountered mouse), the main effect of treatment (impact of various drug treatments on mouse social behaviors), and the genotype x treatment interaction (reveal how the applied treatment affects the genotype).
   4. Subsequently, use Bonferroni's post hoc test (α-level set at 0.05) to further assess the comparison between different groups.

Results

The mammalian target of rapamycin (mTOR) serves crucial roles in the central nervous system (CNS) by regulating de novo protein synthesis and repressing autophagy⁴³. Dysregulation of the mTOR pathway and synaptic protein synthesis has been implicated in ASD²⁸. Genome-wide studies on ASD patients have identified various ASD-associated gene mutations, including those affecting proteins involved in mTOR complex 1 (mTORC1) signaling, such as phosphatase and tensin homo...

Discussion

Most etiological causes, pathological changes, and biological markers of ASD are not known or available. ASD diagnosis is primarily based on two established sets of clinical symptoms: persistent deficits in social communication and excessive repetitive behaviors^18,19,55. Given that ASD is a spectrum disorder encompassing a wide range of symptoms, it is challenging to fully reproduce ASD symptoms in experimental animals. Neverthe...

Materials

Name	Company	Catalog Number	Comments
1/4'' Teklad Corncob Bedding	Harlan, TEKLAD	7092-7097	The raw stock for corncob bedding products  is 100% corncob. No other components or additives are used.
HD Video Recording Cameratraditional Video Camera	Sony	HDRCX405	50 Mbps XAVC S1 1920 x 1080 at 60P, AVCHD and MP4 codecs. 30x Optical / 60x Clear Image Zoom to get closer to the action. 26.8 mm wide angle ZEISS Lens.
Nitrile Powder Free Examination Gloves	Aurelia, Transform	ASTM D6319-00	Tested for use with Chemotherapy drugs per ASTM D6978
Rodent Plastic Cage Bottoms	Ancare	AN75PLF	AN75 Mouse 7½” W x 11½” L x 5” H
TÅGARP floor lamp and bulbs	IEKA	604.640.49	Bulbs are 23 W 120 V.
Ugo Basile Sociability Apparatus	Stoelting	60450	The Sociability Apparatus (3-chambered social test) is a valuable tool to study social behaviour in mice.
Versa-Clean	Fisherbrand	PVCLN04	Cleaning agent
Whiteboard and  Low Odor Dry Erase Marker	EXPO	NA	Dry erase markers in bold black