Assessing behavioral phenotypes in mice with heterozygous deletion of

Autism spectrum disorder (ASD), despite occurring relatively frequently compared to most neurodevelopmental disorders, has remained challenging to study due to its multifaceted pathogenesis and resulting difficulties around modeling the disease. Despite this, a promising mouse model does exist that takes advantage of the TSC1 gene. Mutations in this gene follow an autosomal dominant pattern of inheritance in humans and lead to faulty mTORC1 signaling, leading to tuberous sclerosis, a debilitating condition that affects multiple systems of the body. Over 50% of humans with tuberous sclerosis also happen to meet diagnostic criteria for ASD, making mutations of this gene a critical tool for potentially modeling ASD behaviors. There exists a mouse model with targeted deletion via a Cre recombinase system of the TSC1 gene in cerebellar Purkinje neurons, and homozygous mutants have been shown to suffer motor coordination and sociability deficits when compared to wild type mice. However, there have been conflicting findings on the heterozygous mutant for this mouse model when compared to wild types, warranting further investigation into the autosomal dominant nature of this mutation in the mouse model. Thus, in this study, we utilized an open field and three-chamber social task hybrid assay and an elevated balance beam test to investigate deficits in the heterozygous mouse model. We found that the heterozygous mice exhibit virtually no differences in sociability when compared to wild type mice, and exhibit motor coordination that is equal to or even better than wild type mice. Our findings strongly indicate that the autosomal dominance in the TSC1 gene mutations that is observed in humans does not translate to the mouse model, and caution is advised when drawing conclusions about the condition in humans from the heterozygous mice.

Research Questions

Do heterozygous mutant (Tsc1mut/wt Cre+) mice exhibit behavioral phenotypes consistent with impairments caused by autosomal dominant mutations?

Methods

Test subjects:

This study utilized the L7-Pcp2 Cre/Tsc1 floxed animal model in Mus musculus with a C57BL/J6 genetic background for the mutant groups and a non-modified Mus musculus C57BL/J6 strain for the wild-type (WT) group (Jackson Laboratories). Mice were housed in Miami University’s Laboratory of Animal Resources facility on a 12:12 light cycle starting at 7:00 AM under controlled humidity and temperature. Mice had access to water and a standard rodent chow diet ad libitum (Lab Diet 5,001, Cincinnati Lab Supply Inc. Cincinnati, OH, USA). The student involved in testing underwent standard training and all experimentation was done under approval of the Institutional Animal Care and Use Committee of Miami University (protocol #1044). Cre- negative Tsc1flox/flox mice were crossed with Cre-positive Tsc1flox/wt to get Cre- positive Tsc1flox/wt, which will be referred to as “Tsc1mut/wt Cre+ mice”, and Cre- negative Tsc1flox/flox mice, which will be referred to as “Tsc1mut/mut Cre-” mice.

Balance beam apparatus:

The balance beam apparatus was adapted from that in Carter et al., (2001) and utilized three different balance beams: an 80 cm-long flat beam that was 10 mm in width, one that was 8 mm in width, and an 80 cm-long rounded beam with a 5 mm diameter. During testing, mice traversed the beam to reach an end platform that had an opaque plastic cover as well as some food. A cartoon graphic is given in Figure 1 as a visual.

Balance beam training protocol:

Mice were given two minutes to freely explore the beam without any cover or food, and another two minutes to explore the platform with the cover as well as some food and bedding. After acclimating, the beam was wiped with cleaning solution and mice were taken from the sheltered area and were placed at the other end of the beam to complete a practice crossing of the beam. Hesitant mice were gently tapped on the tail during training to complete the practice crossing. This process (not including the two minutes to acclimate to the sheltered area, as that remained unchanged between balance beam set ups) was repeated for each balance beam (the 10 mm and 8 mm flat beams, and the 5 mm rounded beam.) This training was performed for three consecutive days prior to testing. A 70% ethanol solution was used for cleaning the beams and platform in between mice.

Balance beam testing protocol:

Before the mice were put on the beam for testing, the opaque plastic cover along with some food were placed on the platform, which the mice were accustomed to running toward by the end of training. The stopwatch timer was started for the mice when their hind feet crossed the green starting line on the beam, and the timer was stopped when the mice reached and touched the green finish line. Mice stayed in the sheltered area while beams were switched out and cleaned, and mice were tested on each of the three beams consecutively (10 mm, then 8 mm, then the 5 mm). Mice were normally tested for five consecutive days, though some exceptions did occur, which are detailed in figure legends.

Three chamber assay set up:

The Open field and social tasks were assessed using the three-chamber social interaction task apparatus. The apparatus is composed of three connected rectangular (20 cm x 40 cm and 22 cm high) plexiglass chambers with removable plexiglass walls that can separate the three chambers. A cartoon depiction of the three-chamber assay is provided in Figure 3.

Three chamber assay conduct:

The three-chamber social task was split into four phases during this experiment, which is modified from the original set up in Yang et al. (2011). The first phase, or adaptation phase, was used as an open field assay (see “Open field assay” below). After those 10 minutes, the second phase began, and walls separating the other two chambers were lifted and laid along the top of the apparatus (preventing mice that attempted to escape by jumping), allowing the mice to explore all three chambers freely for 10 minutes. Once complete, the social approach phase (SA) began. The mouse was coaxed back into the middle chamber while stranger mouse #1 was placed in one of the chambers and an empty, upside-down wire cup was placed in the other chamber with another cup on top to prevent climbing by the test mouse. The walls were lifted again, and the test mouse was allowed to freely explore all three chambers for 10 minutes. The fourth and final phase, social novelty (SN) then began, and the test mouse was coaxed again into the middle chamber while stranger mouse #2 was added to the previously empty cup (stranger mouse #1 is now considered “familiar.”) Once stranger mouse #2 was placed, the walls were lifted and the test mouse could freely explore for 10 minutes, which concluded the assay. “Direct contacts” were defined as any instance of interaction with either of the cups that persisted for more than half of a second. All “interactions” were defined as any instance involving sniffing or paw contact with a given cup.

Open field assay

The open field assay was combined with the first phase of the three-chamber social task. During this phase, mice were placed in the middle chamber with walls preventing access to the nearby chambers and were allowed to freely explore for 10 minutes. Repetitive grooming and time spent in the thigmotaxic (perimeter) zone was observed and measured via a stopwatch during this time. A red rectangle was drawn using a marker on the outside of the bottom of the chamber by drawing parallel lines to the four walls 4 cm into the middle chamber. This rectangle defined the “center” versus the “perimeter” of the chamber. A mouse was considered in the perimeter of the chamber when either 3 or more feet had crossed the line towards the walls or 2 feet had crossed the line and the mouse was directly perpendicular to the wall (this was included because some of the bigger mice’s hind feet would not cross the line when directly pointing and touching the wall, whereas smaller mice could have the exact same orientation but would have had all four feet across the line.)

Stranger mice training protocol:

Stranger mice were the same sex as the tested mice, were no more than two months (sixty days) older or younger than the tested mice, were no less than six weeks old, and were Cre-. Before being used as stranger mice, they would be placed in one of the sides of the three-chamber assay in their wire cup, just like testing, for fifteen minutes and were observed for disruptive behavior. If disruptive behavior (e.g. excessive self-grooming or bar-biting) remained frequent by the end of the fifteen-minute period, the mouse would be disqualified for the next week. Mice underwent this training a week before being used in three chamber testing and would undergo this training again if reused. Stranger mice were used a maximum of two times per day of testing to prevent excessive tiring and separation from their cage and food source.

Testing conditions:

All tests were conducted between the hours of 8 AM to 5 PM and were all performed and scored by the same undergraduate researcher to reduce personal variation.

Statistical Analyses:

All Statistical analyses were performed using GraphPad Prism 10.6.0 Software (GraphPad Software Inc., La Jolla, CA, USA). Data from elevated balance beam across genotypes and days were assessed using multiple comparisons one-way ANOVAs. Time spent in each chamber during the three-chamber task used two-way ANOVAs to compare between genotype and the content within each chamber and their respective impacts on time spent in each chamber. Other three-chamber comparisons (number of direct contacts and percent investigation times) and open field findings (percent of time spent in perimeter, total/average time spent self-grooming) utilized student’s t-tests to compare between genotypes. p < 0.05 constituted statistical significance for all tests.

Results/Findings and Discussion

Figure 1

7–10-week-old Tsc1mut/wt Cre+, Tsc1mut/mut Cre-, and WT mice had their crossing times (y-axis) evaluated on the balance beam apparatus across five days (x-axis) with three different difficulties, with points representing the average for the group and bars representing ±SEM. All p-values are adjusted p-values from one-way ANOVAs with multiple comparisons. For Tsc1mut/mut Cre-, N = 5 females and 3 males (on day five, N = 4 females). For Tsc1mut/wt Cre+ mice, N = 6 females and 5 males for days 1-2, and 5 females and 3 males for days 3-5. For WT mice, N = 4 females and 5 males for days 1-2, 2 females and 5 males for days 3-4, and 2 females and 4 males for day 5. Sample sizes were consistent across the three difficulties. (A) Cartoon depiction of the balance beam apparatus, demonstrating the basic set up and orientation of the mouse during testing. (B) The 10 mm balance beam revealed no significant differences between any of the genotypes in terms of their time to cross. (C) Tsc1mut/wt Cre+ crossed the 8 mm balance beam significantly quicker than the WT and Tsc1mut/mut Cre- mice (p = 0.0233 and 0.0010, respectively.) (D) Tsc1mut/mut Cre- mice crossed the 5 mm rounded beam significantly slower than the WT and Tsc1mut/wt Cre+ mice (p = 0.0016 and 0.0001, respectively.) (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Figure 2

7–10-week-old Tsc1mut/wt Cre+, Tsc1mut/mut Cre-, and WT mice had their number of hindlimb foot slips (y-axis) recorded on the balance beam apparatus across five days (x-axis) with three different difficulties, with individual points representing the group’s average and bars representing ±SEM. All p-values are adjusted p-values from one-way ANOVAs with multiple comparisons. For Tsc1mut/mut Cre-, N = 5 females and 3 males (on day five, N = 4 females). For Tsc1mut/wt Cre+ mice, N = 6 females and 5 males for days 1-2, and 5 females and 3 males for days 3-5. For WT mice, N = 4 females and 5 males for days 1-2, 2 females and 5 males for days 3-4, and 2 females and 4 males for day 5. Sample sizes were consistent across the three difficulties. (A) The 10 mm balance beam revealed no significant differences in the mean number of foot slips between the genotypes. (B) Similarly, there were no significant differences in the mean number of foot slips between any of the genotypes on the 8 mm balance beam. (C) Tsc1mut/mut Cre- were significantly more likely to experience hindlimb foot slips than the other two genotypes, with p < 0.0001 when compared to the other two genotypes. (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Figure 3

The adaptation phase of the three-chamber task also served as an open field assay. 7–10-week-old WT (N = 5 males and 2 females), Tsc1mut/wt Cre+ (N = 4 males and 6 females), and Tsc1mut/mut Cre- (N = 3 males and 4 females) mice had perimeter percent time (open field perimeter % time = (time spent in thigmotaxic zone / total time in assay (10 mins)), total time self-grooming, and average time spent self-grooming compared. Bar graphs display the mean value of all the mice within that genotype ±SEM. WT and Tsc1mut/mut Cre- were combined into a “control” group as they were not significantly different. Student’s t-tests were used to obtain p-values for open field measurements. (A) An illustration depicting the three-chamber apparatus is shown. The open field assay occurred for 10 minutes in the middle chamber (20 cm x 40 cm) while the other two chambers were walled off and empty. A red rectangle was used to define the “center” (12 cm x 32 cm) and was drawn in on the opposite side of the bottom panel, so the mouse could not smell or detect a marking. (B) Control and Tsc1mut/wt Cre+ mice displayed no significant difference in the amount of time that they spent in the perimeter of the chamber, with p = 0.2390. Control and Tsc1mut/wt Cre+ also did not significantly differ in the total (C) or average (D) amount of time spent self-grooming during the assay (p = 0.5196 and 0.7299, respectively.) ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Figure 4

The social approach phase of the three-chamber social task revealed very few differences between the genotypes in terms of sociability. 7-10 week old WT (N = 5 males and 2 females), Tsc1mut/wt Cre+ (N = 5 males and 6 females), and Tsc1mut/mut Cre- (N = 3 males and 5 females) mice had their mean percent investigation time (time spent interacting with the novel mouse / total time spent interacting with either the novel mouse or novel object), time spent in each chamber, and number of direct contacts compared. Student’s t-tests were used to compare percent investigation time and number of direct contacts and two-way ANOVAs were used to compare chamber times. Individual values with the means ±SEM bars are demonstrated in the graphs. Due to lack of statistical difference, WT and Tsc1mut/mut Cre- were combined into one group (A) Mice had their time spent (y-axis) in each chamber (x-axis) compared during the 10-minute SA phase. Mice of all genotypes show a clear preference for spending time in the stranger mouse’s chamber, as the content in each chamber (e.g. stranger mouse vs. novel object) was a highly significant factor in predicting where mice spent time (p < 0.0001). The factor of genotype, on the other hand, was not a significant factor in predicting where mice would spend time (p = 0.6619). The genotypes (x-axis) had their percent investigation time (y-axis) measured and compared. and had their mean percent investigation time, revealing no significant difference with p = 0.6268. (C) Mice of different genotypes (x-axis) had their number of direct contacts (y-axis) compared, revealing no significant difference in number of direct contacts with the stranger mouse (p = 0.9932.) ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Figure 5

The social novelty phase of the three-chamber social task revealed no significant differences between the genotypes in terms of sociability. 7-10 week old WT (N = 5 males and 2 females), Tsc1mut/wt Cre+ (N = 5 males and 6 females), and Tsc1mut/mut Cre- (N = 3 males and 5 females) mice had their mean percent investigation time (time spent interacting with the unfamiliar mouse / total time spent interacting with either mouse), their time spent in each chamber, and their direct contacts evaluated and compared. WT and Tsc1mut/mut Cre- mice were combined to create a “control” group, as they were indistinguishable from each other. Means ±SEM are shown, and p-values were obtained via student’s t-tests for percent investigation time and direct contact comparisons and via two-way ANOVAs for chamber time. (A) The genotypes (x-axis) had their percent investigation time (y-axis) measured and compared. Like the social approach phase, genotype was not a significant predictor for where a mouse would spend time during the social novelty phase, with p = 0.6995. The content of the chamber was a significant factor though, with all genotypes displaying a preference for the unfamiliar mouse’s chamber compared to the familiar mouse’s (p = 0.0213). However, there were no statistically significant differences between genotypes (x-axis) in either their (B) percent investigation times (y-axis) or their (C) number of direct contacts with the unfamiliar mouse (y-axis), with p = 0.5327 and 0.3859, respectively. (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Conclusions and Future Study

-Tsc1mut/wt Cre+ mice do not exhibit motor coordination deficits, with performance being indistinct from that of the WT mice. Tsc1mut/mut Cre- mice may experience motor deficits in highly challenging motor tasks.

-Tsc1mut/wt Cre+ mice do not display differences in movement patterns or repetitive behaviors in the open field assay.

-Tsc1mut/wt Cre+ mice do not display different social interactions in the three chamber social tasks

-Taken together, the autosomal dominant inheritance pattern of tuberous sclerosis in humans does not appear to translate to the mouse model.

- Future studies will need to further investigate the motor deficits observed in the Cre- mice.

This poster was presented at the 2026 URF.

Acknowledgements

We would like to recognize Miami University's Undergraduate Summer Scholars program for their generous aid in funding this project during the summer of 2025. Without their aid this project would not have been possible.

References

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NACE Career Readiness Competencies

-Leadership: I gained tons of experience as a leader in terms of organizing and conducting a project of my own. I was given the opportunity to make major decisions for this project and offer my insights when organizing the project.

-Communication & Teamwork: Consistent meetings and discussions with Dr. Ransdell (PI) and Sam Brown (Grad student) were integral to the completion of this project. Without their help, this project would not be where it is today.

-Career Development: This project was the result of an Undergraduate Summer Scholars award and served as the foundation for what would become my Honors Thesis. These findings are likely to be published in the future!

Research Compliance Protocols

All tests were conducted under IACUC compliance protocol #1044

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