Strategies for Assessing Autistic-Like Behaviors in Mice

Ziying Huang; Shane Wiebe; Laura Marsal-Garc&#237;a; Ilse Gantois; Nahum Sonenberg

doi:10.3791/66846

JoVE Journal > Behavior

Comportamento

Strategies for Assessing Autistic-Like Behaviors in Mice

Published: September 20, 2024

doi:

10.3791/66846

Ziying Huang¹, Shane Wiebe¹, Laura Marsal-García¹, Ilse Gantois¹, Nahum Sonenberg¹

¹Rosalind and Morris Goodman Cancer Institute, Department of Biochemistry,McGill University

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 interests¹^,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 2000⁴^,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 syndromes²^,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 enrichment⁷^,⁸^,⁹^,¹⁰, as well as genetic mutations such as Shank, Fmr1, Mecp2, Pten, and Tsc mutant¹¹^,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 study¹⁴^,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-walking¹⁸^,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 grooming²⁰^,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 behavior²⁴^,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 impairments¹⁸^,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 behaviors¹⁷^,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 strain³²^,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 therapeutics²⁹^,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

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 novelty³⁷^,³⁸.
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.
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.
Transfer mice to the animal behavioral testing facility for a minimum of 1 week prior to experiments³⁹^,⁴⁰.
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

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.
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).
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.
Set up overhead or front-facing cameras on tripods for video recordings. Make sure that the cameras are fully charged and have sufficient storage.
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.
Use privacy blinds, such as white plastic boards, which can be placed around the apparatus to eliminate external room cues.
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

Perform handling procedures for three consecutive days prior to the start of the behavioral experiments.
Conduct handling sessions in the same testing room at a consistent time, ideally in the morning when mice are more active.
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.
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.
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.
Continue handling until the mouse becomes comfortable staying on the hand. Always change the gloves before introducing hands to the next cage.
After handling each cage of mice, gently place them into their home cage, and then repeat the procedure.
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.
Handle the stranger mice in the same way.
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)

Perform all behavioral experiments between 7:00 am-3:00 pm.
Switch on dimmed lights, as described above. Clean the table and set up the room.
Prepare each test cage with a thin layer of fresh bedding (as described in step 2.2).
Put 1-2 cages on the table, separated from each other and the room environment by white plastic blinders.
Place a camera in front of each cage to capture the test mouse.
Transfer all mice into the room and cover the cages with a fabric sheet during the transfer process to avoid stress.
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.
At the start of the test, write down the mouse identification number and testing information on a whiteboard (as described in step 2.7).
Start recording and place the test mouse into the test cage.
Leave the room during the 20 min recording session.
For each test, consider the first 10 min as habituation. Use the following 10 min to observe and score grooming behavior (see section 6).
After 20 min, return to the room. Present the whiteboard to the camera and stop recording.
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.
After experiments are finished, return the mice to their housing room.

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

Perform all experiments between 7:00 am-3:00 pm.
Clean the table and turn on the lights (dimmed lights).
Place the three-chamber plexiglass instrument on the table surrounded by privacy blinds.
Place one camera overhead, ensuring that all three chambers are in the recording frame of the camera.
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.
Leave the mice in the room for at least 30 min with dimmed lighting before the start of the experiment.
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.
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.
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

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.
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.
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.
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.

Representative 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 homolog on chromosome ten (PTEN), the tuberous sclerosis complex (TSC1/2), and the fragile X mental retardation protein (FMRP)⁴⁴^,⁴⁵^,⁴⁶^,⁴⁷^,⁴⁸^,⁴⁹. Deletion of these proteins in mouse models induces ASD-like behaviors, including social deficits and repetitive behaviors⁴⁵^,⁴⁶^,⁴⁷. Excessive synaptic protein synthesis, driven by increased eukaryotic initiation factor 4E (eIF4E)-dependent mRNA translation, particularly through mTORC1, has been linked to enhanced synaptic connectivity and ASD phenotypes⁴⁸^,⁴⁹^,⁵⁰. The eIF4E protein, in conjunction with the helicase eIF4A and the scaffolding protein eIF4G, assembles into the eIF4F cap-binding complex, which facilitates mRNA translation initiation⁵⁰. The eIF4E binding proteins (4E-BPs) inhibit translation by binding to eIF4E and not allowing eIF4F complex formation⁵¹. mTORC1 promotes translation by phosphorylating 4E-BPs, thereby freeing eIF4E to facilitate eIF4F formation⁵¹. Of the three 4E-BP paralogs in mammals, 4E-BP2 (encoded by Eif4ebp2) is most predominant in the brain⁵². Behavior tests using wildtype littermates and 4E-BP2 knockout mice have demonstrated the utility of this protocol: Previous research from our lab showed ASD-like repetitive behavior in homozygote Eif4ebp2 full-body knockout (Eif4ebp2 KO) mice and social deficits in Eif4ebp2 KO and inhibitory neuron conditional knockout models⁵³^,⁵⁴. To show natural animal behaviors, here, we present the results of self-grooming, and the three-chamber social interaction tests conducted on C57BL/6J mice. We conducted the self-grooming test on Eif4ebp2 KO and compared them to wild type littermate mice. For the three-chamber test, 4E-BP2 conditional KO (Eif4ebp cKO) mice were used, which were crossed with Camk2α-Cre mice and Gad2-Cre mice. As control mice (Eif4ebp2 CTL), we used the Camk2α-Cre and Gad2-Cre littermates of the same sex and age. The following findings serve as representative results to validate the protocols for assessing ASD-like behavior on genetic mice models.

Self-grooming test is presented in Figure 1. Schematic and actual setup shows the habituation and testing stages of the self-grooming test (Figure 1A). Mouse grooming was categorized into two main types: rostral grooming and caudal grooming (Figure 1B). Among the 10 videos recorded from C57BL/6J male mice during the test, we observed that mice typically initiated grooming with paws/nose grooming and then spent large amounts of the grooming time licking their bodies, especially focusing on washing the belly and hind limbs (Figure 1C). The means for different grooming areas are as follows: paws/nose 13.1 ± 4.7 s, face/head 5.2 ± 1.2 s, body/tail 39.3 ± 8.5 s. Grooming of ears and tails was rarely observed. Additionally, individual bouts (Figure 1D) represent each time when the test mouse switched areas for grooming (for example, from paws and nose to face/head or to body/tail), with body grooming being the most frequently observed self-grooming behavior. The means for different grooming areas are as follows: paws/nose 2.4 ± 0.4 bouts, face/head 2.5 ± 0.4 bouts, body and tail 5.4 ± 0.7 bouts. Subsequently, we analyzed videos of Eif4ebp2 KO mice and their wildtype control littermates (CTL) of both sexes. Based on previous research from our lab investigating male Eif4ebp2 KO mice, we anticipated an increase in grooming, indicative of abnormal repetitive behavior⁵³. As expected, we observed significant differences in self-grooming time and number of bouts. A significant difference was found in self-grooming time between male control mice (Figure 1E) (n = 7, mean = 25.61 ± 5.8 s) and Eif4ebp2 KO mice (n = 5, mean = 46.57 ± 7.3 s) (t(23)= 2.286, p = 0.0454; unpaired t-test), as well as between female control mice (n = 5, mean = 21.86 ± 7.8 s) and Eif4ebp2 KO mice (n = 6, mean = 81.70 ± 21.2 s) (t(23) = 2.440, p = 0.0373; unpaired t-test). Moreover, male Eif4ebp2 KO mice (n = 5, mean = 7.2 ± 1.4 bouts) initiated more grooming bouts than the control group (Figure 1F), (n = 7, mean = 3.4 ± 0.3 bouts) (t(23) = 2.683, p = 0.0476; Welch's t-test). A significant difference was also observed between female Eif4ebp2 KO mice (n = 6, mean = 7.5 ± 0.9 bouts) and the control group (n = 5, mean = 2.2 ± 0.5 bouts) (t(23) = 4.770, p = 0.0010; unpaired t-test). In conclusion, both female and male mice with 4E-BP2 KO exhibit an increased repetitive self-grooming phenotype.

The three-chamber social interaction test is depicted in Figure 2. Schematic and actual setup shows the overall picture of the three-chamber social test, which includes habituation, social approach, and preference for social novelty (Figure 2A). We conducted the three-chamber test initially on C57BL/6J mice (Figure 2B; n = 12, male). Mice typically preferred to interact with S1 over a novel inanimate object, with a mean interaction time of 124.2 ± 10.6 s for S1 and 60.01 ± 3.9 s for E (t(23) = 5.665, P < 0.0001; Welch's test). Similarly, C57BL/6J mice displayed more interest in a newly introduced S2 compared to a familiar S1 (Figure 2C), with a mean interaction time of 66.07 ± 7.3 s for S2 and 36.21 ± 4.6 s for S1 (t(23) = 3.468, P = 0.0027; unpaired t-test). Figures 2D–G present data from a previous publication by Dr. Wiebe in PNAS, three-chamber test of Eif4ebp2 cKO mice⁵⁴. These mice had 4E-BP2 conditionally deleted in CamkIIα⁺ expressing excitatory neurons (Eif4ebp2^Ex cKO) and Gad2⁺ expressing inhibitory neurons (Eif4ebp2^IncKO). Both Eif4ebp2^Ex cKO (n = 14) and control littermates Eif4ebp2^Ex CTL (n = 11) spent a longer time interacting with S1 over E (Figure 2D), (main effects of chamber, F(1,23) = 28.90, p < 0.0001, Bonferroni post hoc mean: Eif4ebp2^Ex cKO, S1 84.2 ± 7.4 s, E 49.7 ± 4.4 s, t(23) = 3.544, p = 0.0035; Eif4ebp2^Ex CTL S1 94.1 ± 5.7 s, E 49.7s ± 5.6, t(23) = 4.042, p = 0.0010; no main effect of genotype, F(1,23) = 1.328, p = 0.2610; no chamber x genotype interaction effect, F(1,23) = 0.4548, p = 0.5068; 2-way ANOVA). A similar result was observed in the preference for the social novelty stage, where both groups showed more interaction with S2 over S1 (Figure 2E), (main effect of chamber, F(1,23) = 35.28, p < 0.0001, Bonferroni post hoc: Eif4ebp2^Ex cKO, S1 42.8 s ± 5.1, S2 75.7 s ± 5.5 t(23) = 4.076, p = 0.0009 and Eif4ebp2^Ex CTL , S1 38.3 ± 4.3 s, S2 77.6 ± 6.8 s, t(23) = 4.325, p = 0.0005; no main effect of genotype, F(1,23) = 0.07213, p = 0.7907; no chamber by genotype interaction effect, F(1,23) = 0.2839, p = 0.5993; 2-way ANOVA). Interestingly, Eif4ebp2^IncKO mice did not display social approach for S1 over E compared to the wild type littermates Eif4ebp2^Ex CTL (Figure 2F), (chamber by genotype interaction effect, F(1,30) = 8.624, p = 0.0063, Bonferroni post hoc: Eif4ebp2^IncKO, S1 60.1 ± 5.8 s, E 59.6 ± 6.8 s, t(30) = 0.04905, p > 0.9999; Eif4ebp2^Ex CTL, S1 63.4 ± 6.7 s, E 26.4 ± 3.0 s, t(30) = 4.667, p = 0.0001; main effect of chamber, F(1,30) = 9.074, p = 0.0052; main effect of genotype, F(1,30) = 7.994, p = 0.0083; 2-way ANOVA). Both Eif4ebp2^IncKO and Eif4ebp2^Ex CTL mice demonstrated similar preference for social novelty (Figure 2G), (main effect of chamber, F(1,30) = 19.56, p = 0.0001, Bonferroni post hoc: Eif4ebp2^IncKO, S1 29.5 ± 4.3 s, S2 42.9 ± 5.6 s, t(30) = 2.441, p = 0.0415 and Eif4ebp2^InCTL, S1 25.5 ± 5.4 s, S2 43.7 ± 5.0 s, t(30) = 3.986, p = 0.0008; no main effect of genotype, F(1,30) = 0.05985, p = 0.8084; no chamber by genotype interaction effect, F(1,30) = 0.4351, p = 0.5145; 2-way ANOVA). Taken together, these findings suggest that Eif4ebp2 cKO in Gad2⁺ interneurons, but not Camk2IIα⁺ excitatory neurons, results in a decreased social approach. The representative data further demonstrate the effectiveness of our protocols in evaluating the social behaviors of transgenic mice.

Figure 1: Self-grooming behavior and effects of 4E-BP2 deletion on self-grooming behavior. (A) Schematic illustrating the timeframe and setup utilized to evaluate self-grooming behavior. Figures show the test mouse in an empty cage with fresh bedding for a total of 20 min, with 10 min allocated for habituation and 10 min for scoring self-grooming behaviors. The actual setup of the self-grooming test: a camera is placed in front of a clean cage (with fresh bedding). (B) Examples of different stages of mouse self-grooming to assess ASD-like repetitive behavior. The image on the left depicts the rostral grooming of paws, nose, and face; the photo on the right illustrates caudal grooming of the belly and hind limb. (C) Manual analysis of video recordings of self-grooming time of C57/BL6J male mice (2-3 months old, n = 10), and (D) number of individual bouts of self-grooming. (E) Grooming time of Eif4ebp2 knockout mice (2-3 months old), and (F) grooming bouts in both Eif4ebp2 knockout male (blue) and female (pink) mice compared to their respective Eif4ebp2 wildtype littermates (light blue and light pink). For statistical analysis, unpaired Student's t-test was performed in (E,F). Data are displayed as mean ± sem. **p < 0.01; *p < 0.05; ns, p > 0.05, not significant. Please click here to view a larger version of this figure.

Figure 2: Three-chamber social interaction behavior and effects of 4E-BP2 conditional knockout mice on social interaction. (A) The schematic illustrates the three stages of the three-chamber social interaction test. In the habituation stage, the test mouse is placed in the center of an empty three-chamber apparatus for 10 min. The actual setup of the three-chamber apparatus and wire enclosures for stimuli. A camera is fixed above the apparatus. Manual analysis of overhead video recordings of C57BL/6J male mice (2-3 months old, n = 12) during the (B) social approach and (C) the preference for social novelty phases. C57BL/6J mice spent a longer time interacting with S1 compared to E, as well as with S2 compared to S1. (D) The social approach of 2-3 months old male Eif4ebp2^Exconditional knockout (Eif4ebp2 ^flx/flx:Camk2a-Cre, purple, n = 14) and Eif4ebp2^Exwildtype (Eif4ebp2 ^+/+:Camk2a-Cre, white, n = 11) mice. (E) Preference for the social novelty of 2-3 months old male Eif4ebp2^Exconditional knockout (Eif4ebp2 ^flx/flx:Camk2a-Cre, purple, n = 14) and Eif4ebp2^Exwildtype (Eif4ebp2 ^+/+:Camk2a-Cre, white, n = 11) mice. Bar graphs indicate similar interaction time differences for social approach and preference for social novelty in Eif4ebp2^Exconditional knockout compared to its control littermates. (F) Social approach of 2-3 months old male Eif4ebp2^Inconditional knockout (Eif4ebp2 ^flx/flx:Gad2-Cre, green, n = 13) and Eif4ebp2^Inwildtype (Eif4ebp2^+/+:Gad2-Cre, white, n = 19) mice. (G) Social approach of 2-3 months old male Eif4ebp2^Inconditional knockout (Eif4ebp2 ^flx/flx:Gad2-Cre, green, n = 13) and Eif4ebp2^Inwildtype (Eif4ebp2^+/+:Gad2-Cre, white, n = 19) mice. Bar graphs indicate impaired social approach but no difference in the preference for social novelty in Eif4ebp2^Inconditional knockout compared to its wildtype littermates. For statistical analysis, Welch's t-test and unpaired Student's t-test were performed in (B) and (C), respectively; two-way ANOVA with Bonferroni post hoc test was performed in (D–G). Data are displayed as mean ± sem. ****p < 0.0001; ***p < 0.001; **p < 0.01; ns (p > 0.05): not significant. The data for panels D-G has been obtained with permission from Wiebe et al.⁵⁴. Please click here to view a larger version of this figure.

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 behaviors¹⁸^,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. Nevertheless, three sets of criteria are essential for assessing behaviors in ASD animal models: face validity (the test recapitulates ASD endophenotypes in mouse models), construct validity (the utility of the test in measuring the real phenomenon), and predictive validity (where the performance of a model in the test predicts treatment effects in people with ASD)⁵⁶. Based on these criteria, we focus on two specific behavioral tests in mice that measure core behaviors observed in human ASD patients: repetitive behavior, measured by the self-grooming test, and social behavior, measured with the social approach and preference for social novelty test.

During the self-grooming test, a mouse is placed in its familiar and comfortable environment, specifically a cage with the same type of bedding as the home cage. Under this condition, natural self-grooming behavior can be observed. By comparing the experimental mice and their controls, repetitive behavior can be assessed, and it can be determined whether pharmacological treatment corrects this phenotype. The three-chamber test assesses sociability and preference for social novelty by enabling testing mice to freely explore and interact with stranger mice and a novel object (empty wire cage). Since this test limits direct social approach and direct physical contact between the test mouse and the novel-introduced mouse, this task minimizes stress levels caused by aggressive behavior between the mice and allows for a better assessment of the test mouse's social interaction preference⁵⁷. By comparing the interaction times between an empty cage and a stranger or between a familiar mouse and a novel stranger mouse, their sociability and social novelty are evaluated. It is important to emphasize that age, sex, and genetic backgrounds are critical factors in these social behavioral tests⁵⁸^,⁵⁹^,⁶⁰.

According to the Simons Foundation Autism Research Initiative (SFARI) gene database, 1,231 genes and 1,353 mouse lines (genetic, inbred, or agent-induced models) are associated with ASD. Firstly, mice with mutations in known ASD-linked genes are associated with aberrant self-grooming and social interaction phenotypes¹⁵^,²⁴. For example, deletion of any one of the three Shank genes causes increased self-grooming and social deficits³³^,⁶¹; specific deletion of isoforms in these genes might influence this specific ASD-like behavioral phenotype⁶². Aside from gene mutations, dysregulation of mRNA translation is associated with ASD-like behaviors. Deletion of the 4E-BP2 causes repetitive behavior and social interaction deficits⁵³^,⁵⁴. In addition to genetic factors, environmental factors like drugs can be used with this protocol to evaluate their impact on ASD-like behavior. For example, studies examined the effects of specific drugs such as Risperidone (an antagonist of the serotonin 5-HT2A receptor) and Fluoxetine (a selective serotonin reuptake inhibitor-SSRI) on ASD mouse models⁶³^,⁶⁴^,⁶⁵. Risperidone demonstrated efficacy in alleviating self-grooming behavior in BTBR mice (BTBR T⁺Itpr3^tf/J, a mouse strain used for studying ASD) but failed to correct social deficits in the three-chamber test³²^,⁶⁵. Unfortunately, the dose that was used for reducing the self-grooming behavior also induced sedation in mice. On the other hand, specific doses of Fluoxetine administered were able to increase sociability in the three-chamber test and reduce total grooming time and bouts in mice⁶³^,⁶⁴^,⁶⁵.

It is noteworthy that animal behavioral tests, such as self-grooming and three-chamber tests, only reflect some conserved underlying circuits and do not exactly mimic the complexity of behaviors in ASD patients. Self-grooming behaviors, for instance, are typical features of complex, repetitive, and self-directed behaviors. Prolonged self-grooming is associated not only with ASD-like behaviors but also with anxiety, ADHD, and obsessive-compulsive disorder (OCD)²². Therefore, including measures of anxiety and activity are necessary controls, such as the open field test, elevated plus maze, and the light/dark box. Specifically, self-grooming may be evoked by stress, so it is recommended to observe the grooming patterns to distinguish between low-stress self-grooming in our protocol and stress-induced self-grooming, which is related to anxiety and characterized by frequent bursts of short grooming activity⁶⁶. Assessing normal anxiety-related behavioral parameters in the elevated plus maze and light/dark box will help to explain the results. In addition, it is also important to consider the interest gene function and the possibility of motor impairments. The progressive loss of self-grooming is observed in various neural disorders, including Huntington's, Alzheimer's, and Parkinson's disease²²^,⁶⁷.

It is also important to acknowledge that distinct ASD mouse models may exhibit variable social impairments under different three-chamber protocols. The results could be influenced by several factors, including variations in trial duration (5 min-20 min), the presence or absence of a non-social object in the wire cage, and the estrous cycle in female mice. In the case of Shank3^+/ΔC, 16p11.2^dp/+, and Cul3^f/- mice, employing a three-chamber protocol with a non-social object (such as a paper ball) within the wire cage demonstrated enhanced sensitivity in detecting social deficits⁶⁰. However, this protocol requires an extended habituation period compared to the protocol used in this study. Specifically, the test mouse needs to undergo two habituation stages: first to the apparatus with two empty cages, and then to the apparatus with two identical objects in each cage. The following testing is similar, which contains a phase I involving an object and stranger 1 in each cage, followed by a phase II with stranger 1 and stranger 2 in each cage. The latter protocol can be applied as needed to ensure precise evaluation of social behavior in specific ASD mouse models. Furthermore, deficits in social interaction observed from the three-chamber test can provide insights into complex brain disorders besides ASD, such as depression and schizophrenia⁶⁸^,⁶⁹. Most animal models for these disorders exhibit a decrease in social interaction⁷⁰^,⁷¹. To further corroborate the social behavioral results, additional tests can be employed. For example, forced swimming test (FST), tail suspension test (TST), pre-pulse inhibition(PPI), and various memory tests can be applied. FST and TST are used to monitor depressive-like behaviors, which might potentially affect social behaviors. PPI is used for evaluating mouse schizophrenia-relevant behavior. Among the memory tests, Morris water maze (MWM) and novel object recognition and location (NOR and NOL) are commonly used in animal models of depression and schizophrenia⁷⁰^,⁷¹.

The protocol here focuses on assessing social interaction and repetitive behavior. Depending on the specific focus of the research, it is advisable to incorporate additional tests. First, we did not include assessments for social communication, which is another core symptom of ASD. Given that mice communicate through ultrasonic sounds, incorporating the ultrasonic vocalization (USV) test could provide valuable insights into their communication abilities. Second, the protocol presented here does not elaborate evaluations of sensory and motor behaviors. Individuals with ASD may exhibit signs of motor deficits due to alterations in motor circuits, particularly those within the cerebellum. Including motor tests, such as the rotarod test and gait analysis, will advance the interpretation of the results. Third, learning and memory tests, such as the T-maze, contextual fear conditioning, etc., could be added to study cognition.

The self-grooming test and three-chamber test in this protocol can also be monitored by tracking software for automated scoring⁵⁷^,⁷²^,⁷³. However, manual scoring can offer greater accuracy compared to automated methods, in which some limitations persist. For instance, in self-grooming assessments, most tracking programs fail to differentiate between grooming and other behaviors, such as chewing. On the other hand, in tasks such as the three-chamber test, tracking software adeptly measures total entries and time spent in each chamber. However, accurately considering total interaction time often necessitates manual scoring due to potential inconsistencies in software tracking, particularly in detecting subtle mouse movements and direct interactions with unfamiliar mice. Manual scoring requires special execution to maintain integrity. It is essential that video scoring be conducted by researchers unaware of the genotype or treatment to mitigate bias. Additionally, different observers may interpret behaviors differently, underscoring the importance of employing multiple observers for behavioral tests. Regular evaluations of inter-observer reliability are crucial for ensuring experiment accuracy and objectivity.

In conclusion, the protocols described here are useful for advancing the understanding of ASD-relevant behavioral outcomes of genetic mutations in mice.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

We thank Dr. Karim Nader (Department of Psychology, McGill University) for providing access to the animal behavior facility.

Materials

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

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