{"paper_id":"23b59548-a40d-4c8d-b2f9-ef10351f8bb9","body_text":"Behavioral phenotyping identifies autism-like repetitive stereotypies in a Tsc2 haploinsufficient rat model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Behavioral phenotyping identifies autism-like repetitive stereotypies in a Tsc2 haploinsufficient rat model Antonia Ramme, Mirjam Zachow, Bettina Habelt, Iveta Vojtechova, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6006061/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jul, 2025 Read the published version in Behavioral and Brain Functions → Version 1 posted 12 You are reading this latest preprint version Abstract Besides deficits in social communication and interaction, repetitive behavior patterns are core manifestations of autism spectrum disorder (ASD). Phenotypes are heterogeneous and can range from simple lower-order motor stereotypies to more complex higher-order cognitive inflexibility and fixated interests. Due to ASD’s multifaceted etiology, animal models are often generated from monogenic diseases associated with ASD, such as Tuberous Sclerosis Complex (TSC), and are expected to copy behavioral core deficits to increase the model´s translational value for ASD disease research and novel treatment development. The global haploinsufficient Tsc2 +/− Eker rat model has been shown to display ASD core symptoms in the social domain. However, the presence and extent of aberrant repetitive behavior patterns in the Eker rat remain to be investigated. Thus, the present study applied a set of behavioral tests to determine the repetitive behavioral profile in Tsc2 +/− Eker rats and used brain-region-specific neurotransmitter analysis to support findings on a molecular level. Tsc2 +/− animals demonstrated lower-order repetitive behavior in the form of excessive self-grooming and nestlet shredding under non-stressful conditions that co-occurred alongside social interaction deficits. However, no higher-order repetitive behavior was detected in Tsc2 +/− rats. Interestingly, Tsc2 +/− rats exhibited increased levels of homeostatic dopamine in the prefrontal cortex, supporting the link between aberrant cortical dopaminergic transmission and the appearance of lower-order repetitive phenotypes. Together, our results support the Tsc2 +/− Eker rat as a model of ASD-like behavior for further investigation of ASD-related development and neurobiology. Autism spectrum disorder tuberous sclerosis complex repetitive behavior Eker rat Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder defined by deficits in two core behavioral domains: social interaction and repetitive behavior patterns ( 1 , 2 ). Despite a high global prevalence of approximately 1% ( 3 ), efficacious drugs are missing, and early behavioral interventions remain the most promising treatment option ( 4 ). The biggest impeding factor for drug development remains ASD´s complex and incompletely understood etiology. Disease pathogenesis arises from a multifaceted interaction between a range of genetic and environmental factors that together give rise to a wide symptom heterogeneity across ASD patients. Moreover, ASD is linked to over 100 candidate genes and a myriad of genetic risk factors like chromosomal rearrangements and copy number variations with mainly small to moderate effect sizes ( 5 – 9 ), thus posing additional challenge to generate disease models with high translational accuracy. Similarly, different transgenic ASD rodent models exhibit unique sets of behavioral alterations that fit the high aetiologic and symptomatic heterogeneity in humans ( 10 – 12 ). While social behavior is well-described in most ASD rodent models, characterizations of repetitive behavior profiles are less commonly reported. Repetitive behaviors in ASD patients include a range of motor patterns divided into a) “lower order” (stereotyped or repetitive motor movements), b) more complex “higher order” responses (compulsions, insistence on sameness, cognitive inflexibility and adherence to routine) and c) highly restricted interests such as unusual object fixation ( 1 , 13 ). In rodent ASD models, strain-specific repetitive patterns can be revealed by applying combinations of behavioral tests that cover multiple response categories. Commonly used models are often based on single-gene disorders associated with ASD such as Tuberous Sclerosis (TSC) and Fragile X syndrome ( 14 , 15 ), or high-effect risk genes like SHANK3 , NLGN3 , and NLGN4 , as they offer high construct validity and thereby increase relevance ( 12 , 16 – 18 ). Among genetic aberrations reported in ASD, the TSC2 gene remains one of the major contributors ( 19 ). TSC is an early-onset, monogenic disorder caused by loss-of-function mutations in the Tsc1 or Tsc2 gene. Patients present with autism-like neurobehavioral phenotypes including social deficits and epileptic seizures ( 20 – 22 ), and are co-diagnosed with ASD in up to 69% of cases, suggesting genetic linkage of ASD and TSC ( 19 , 23 ). While the connection between genetic and anatomical alterations is not yet fully understood, molecularly, Tsc1 and Tsc2 mutations are known to result in overactive mTORC1 (mammalian target of rapamycin complex 1), thereby interfering with metabolic processes including cell growth, proliferation, differentiation, and protein synthesis ( 24 ). Hyperactive mTOR signaling is associated with impaired neurodevelopment, synaptic plasticity, and signaling, and thus strongly correlates to neurodevelopmental diseases like epilepsy and ASD ( 25 , 26 ). The Tsc2 +/− Eker rat model carries a spontaneous germ line mutation of the Tsc2 gene ( 27 , 28 ), making it a valuable tool for studying TSC etiology and its implications in ASD ( 21 , 29 – 31 ). Reportedly, Eker rats recapitulate brain abnormalities and cellular pathology present in human TSC ( 21 , 32 – 34 ), show altered synaptic plasticity ( 33 ), and present with mild but consistent autism-like social impairments ( 35 – 37 ). The observed social deficits were found to improve upon selective inhibition of mTORC1 in Tsc2 +/− animals, supporting hyperactive mTORC1 signaling as a potential driver of behavioral manifestations in ASD ( 37 ). However, along with concerns about absent spontaneous seizure activity, the lack of repetitive behavior characterization sparks debate about the suitability of the Eker rat as an ASD model ( 21 ). Therefore, the present study aimed to provide an extended characterization of ASD-like repetitive behavior in Tsc2+/- Eker rats by exposing animals to a test battery addressing both lower and higher order repetitive phenotypes to strengthen the model’s translational value in ASD research. Methods Animals All experiments were performed in the heterozygous Eker rat strain with Tsc2 +/− mutation on a Long-Evans background, RRID:RGD_625624 and their wild-type (wt, Tsc2 +/+ ) littermates which were used as control. Animals were bred at and obtained from the National Institute of Mental Health in Klecany, Czechia at 8 weeks of age. Acclimatization and daily handling were performed for 4 weeks prior to behavioral experiments. The animals were housed in pairs of 2–3 in standard housing cages (Makrolon®, Type IV-S, Tecniplast Deutschland GmbH, Hohenpeißenberg, Germany) in a controlled environment of 21–24°C and an average humidity of 55% under an automated 12 h/12 h day-night cycle. Food and water were provided ad libitum . Experiments took place during the late-day phase and animals’ weight was controlled twice a week. Behavioral testing Behavioral experiments started when animals were 12 weeks of age, in accordance with relevant literature on social behavior of Tsc2 +/− Eker rats ( 35 ). A total of 11 Tsc2 +/− and 11 wt rats was used and subjected to the behavioral testing sequence and following post-mortem analysis. Sample-size was calculated a-priori using G*Power. Genotype groups included both male ( n = 6) and female ( n = 5) animals, respectively. Experiments were conducted in two separate birth cohorts, with sex and gender being counterbalanced across cohorts. Experimental testing and analysis were performed by trained and blinded experimenters. A broad behavioral test battery assessing both lower (marble burying test, nestlet shredding test, light-sound-confinement test, forced swim test) and higher order (water-T-maze test) repetitive behavior was used. Additional readouts for social behavior (social recognition test), reward function (sucrose consumption test) and cognition (water-T-maze test) were collected to report repetitive behavior alterations in the context of already known abnormalities. Behavioral tests were performed in a fixed sequence depicted in Fig. 1 , allowing for a minimum of 24 h resting period between tests to limit stress and potential carryover effects. Nestlet shredding test The nestlet shredding test was used to detect lower-order repetitive behavior in non-stressful conditions ( 38 ). Animals were placed individually in a standard cage containing bedding material, fresh nestlet material, and a square tissue paper (5 cm × 5 cm, weighing 2.5 g) in the opposite corner. The test was conducted for 30 minutes without food or water supply before the animals were returned to their home cage. The remaining unshredded nestlet material was then weighed ( 38 , 39 ). Marble burying test The marble burying test was used to detect lower-order motor stereotypy in form of repetitive digging behavior in non-stressful conditions ( 38 , 40 ). Animals were placed individually in a standard cage with bedding material filled up to 5 cm height, in which 9 clean, commercially available glass marbles (approx. 15 mm diameter) were evenly distributed on half of the cage´s surface. Animals were allowed to explore for 30 minutes without food and water supply, after which they returned to their home cage and the number of buried marbles that were at least 2/3 covered in bedding material was counted ( 38 , 39 ). Light-sound-confinement test A light-sound-confinement test (LSC) was performed to assess lower-order repetitive digging and grooming behavior in stressful conditions through the application of combined auditory and visual stress. The animals were placed in a plexiglass tube (30 cm height and 12 cm diameter) (Stoeltus Co., Ireland) fixed on a standard cage and the tube was fitted with a commercially available loudspeaker (SoundCore Select 2 Bluetooth Loudspeaker, Anker, Germany) and a light source (KL 1600 LED, Schott AG, Germany). Behavior of the rats was filmed from two sides using ManyCam (Visicom Media Inc.) for 30 min in total. For the first 10 min, activity of the animals was recorded without audiogenic or visual stressors (pre-test phase). During the following 10 min test phase, animals were exposed to both visual and auditory stressors. Light was applied at a maximum of 1400 lux combined with white noise bursts (100 dB SPL, 50 ms) (programmed with Audacity Version 2.3.0 and Matlab Version R2019a), played with random interstimulus intervals ranging between 10–20 s. After removal of both stressors, the activity of the animals was recorded for another 10 min (post-test phase). Total grooming time and digging time were analyzed for each phase ( 41 – 43 ). Forced swim test The forced swim test (FST) was conducted to measure active coping strategies in stress response ( 44 ). Further, response behavior was assessed for repetitive patterns. For habituation, animals were placed in a glass cylinder (60 cm height, 24 cm diameter) filled with 23 ± 1°C water to 30 cm height for 15 min individually. Afterward, animals were dried under a red-light source (SIL-6 red light lamp Beurer, Hans Dinslage GmbH, Germany) and placed back in their home cage. After 24 h, animals were tested in a water height of 40 cm and activity was recorded for 5 min. The total time of immobility per animal was determined using the software EthoVision® XT (Noldus Information Technology, Netherlands) ( 45 , 46 ). Additionally, potential repetitive climbing patterns were manually assessed by a trained experimenter and potential uninterrupted repeated circular swimming was evaluated in EthoVision´s heat map feature. Water-T-maze The water-T-maze (WTM) test was conducted to detect higher-order repetitive behavior as well as assess learning and memory ( 47 ). The experiment was conducted in a T-shaped pool (Maze Engineers, Conduct Science©, USA) filled with 23 ± 1°C opaque water (XSL titanium white, Kremer pigments, Germany), which consisted of a long stem (L 70 cm, W 15.5 cm, H 46 cm) that bifurcates into two arms (L 121 cm × W 15.5 cm × H 46 cm). A transparent platform (15.5 cm x 15.5 cm, 24 cm height) was randomly placed in one of the two arms of the T-maze and was submerged 1 cm below water level. On day one (acquisition phase), animals were trained to find the platform position in a fixed arm. Animals were placed in the starting arm and allowed to choose either direction after reaching the end of the stem. Once an animal reached either arm within a test period of 60 s, the arm was closed with a plexiglass door (47 cm × 20 cm), confining the animal for 5 s before removal to a holding cage for 10 s and subsequent test repetition. The training was repeated until the animal chose the correct arm, and escaped onto the platform, in five consecutive trials. On day two, 24 h later, animals were retrained by repeating the previous day´s test procedure (repetition phase). Once position learning was completed, the platform was switched to the opposite arm of the T-maze and the test procedure was repeated as described (reversal test). During each holding period outside of the maze, animals were dried under a red-light source (SIL-6 red light lamp Beurer, Hans Dinslage GmbH, Germany). The number of attempts required by each animal to make five consecutive correct decisions was recorded; the maximum number of trials was limited to 25 per day. Sucrose consumption test The sucrose consumption test was performed to assess reward function and depressive episodes as reported previously ( 48 ). Animals were habituated to a standard bottle of sweetened condensed milk (Nestlé, Milchmädchen, (1:3)) in their home cage for 30 min. After 24 h, animals were habituated to the single housing standard cage type III (Makrolon®, Tecniplast Deutschland GmbH, Germany) for 30 min and subsequently food restricted for 21 h (15 g per animal). On the day of testing, animals were exposed to the bottle containing sucrose solution for 10 min. Bottle weight was recorded before and after the test phase. Animals were weighed on each test day as well as the day after sucrose consumption ( 49 ). Social recognition test The social recognition test was used to evaluate sociability towards an unknown partner rat. Animals were kept in isolation for three consecutive days and were familiarized with the test box (45 cm × 45 cm, LE802S Panlab square arena, S.L.U. Panlab, Spain) for 1 h every day. On the test day, each experimental animal was presented with an unfamiliar sex- and age-matched conspecific of the same strain and placed in a locked startle box (Grid Rod Animal Holder, OCB Systems Ltd., United Kingdom) with openings for nose-to-nose contact. Experimental animals were allowed to explore or interact for a test period of 60 s in a total of four trials with the same social-partner animal in intervals of 10 min. In a fifth trial, experimental animals were presented with a novel social partner animal. Following each trial, the social-partner animal was returned to their home cage for the 10 min interval time, and the startle box was disinfected. All trials were videotaped and the examination time spent in close proximity was manually determined ( 50 ). Amphetamine-induced stereotypic behavior test An amphetamine-induction test was conducted to characterize the effect of amphetamine on repetitive behavior stereotypies. Animals were injected intraperitoneally with 2.0 mg amphetamine (Lipomed AG, Switzerland) per kg body weight, based on the emergence of amphetamine-induced stereotypy in wt animals above doses of 2 mg/kg ( 49 ). Rats were placed in a testing box (46.5 × 46.5 cm × 44 cm) and video-recorded for 2 h. Behavior following amphetamine application was analyzed in 5 min intervals, and the most prominent behavior was scored for each interval. Scoring was performed based on an adapted protocol described in Kelly et al. (1975) ( 51 ) by dividing behavior in (0) no locomotor activity (sleeping, residing without apparent sniffing) ( 1 ) limited exploratory activity (discontinuous sniffing, rearing or grooming) ( 2 ) locomotion (frequent rearing, sniffing) and ( 3 ) stereotypic behavior (repetitive grooming, rearing and licking). Scoring was performed manually by a single trained and blinded experimenter. Blinding was achieved by number-coding animals for video-analysis to eliminate genotype-bias. Post-mortem High-Performance Liquid Chromatography Animals were intraperitoneally anesthetized using pentobarbital (60 mg/kg) and decapitated. Whole brains were extracted and frozen in methylbutane for 2 min at -20 to -40°C and stored at -80°C. Bilateral micro-punches (Ø 1 mm) of prefrontal cortex (PFC) and striatum (caudate putamen, CPu) tissue were homogenized in 500 µl 0.1 M perchloric acid by ultrasonication that was applied for 3 × 10 s on ice. After protein quantification (PierceTM 660 nm Protein Assay; Thermo Fisher Scientific Inc., USA), homogenates were centrifuged for 15 min at 13,000 g at 4°C. The supernatant was separated via HPLC (1260 Infinity II LC System, OpenLab LC ChemStation Software, Agilent Technologies, USA) on a PRONTOSIL 120-5-C18SH (VDS Optilab, Germany) analytical column followed by electrochemical detection (Coulochem III, Thermo Fisher Scientific Inc., USA) of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels as µg/g protein. Dopamine turnover was calculated from concentration levels, adjusted to the protein content of the sample, as neurotransmitter to metabolite ratio (DA/(DOPAC + HVA)). Statistical Analysis Statistical analysis was performed using GraphPad Prism 9.5.1. All behavioral tests as well as HPLC measures were analyzed using a two-way analysis of variance (ANOVA) for the main effect of genotype (wt, Tsc2 +/− Eker) and sex (male, female). In consideration of our small dataset we did not test for normality but relied on the visual inspection of qq-plot for residuals and the robustness of ANOVA, to violations of normality, especially when the sample sizes are small and equal across groups ( 52 ). Tukey’s HSD was used to adjust for multiple post hoc comparisons. No animals were excluded from analysis. Statistical significance was set at p < 0.05. Results Tsc2 haploinsufficiency increases lower-order repetitive behavior patterns To determine if the Tsc2 +/− genotype induces ASD-associated behavior in rats, a set of behavioral tests was performed on the Eker model to reveal repetitive behavior patterns. First, lower-order restricted behavior was observed under non-stressful conditions using the nestlet shredding and marble burying test. Both behavioral tests are commonly used to reveal stereotypic repetitive behavior ( 38 , 40 ). While no differences in marble-burying behavior were observed between wild-type (wt) and Tsc2 +/− animals (Fig. 2 A), Tsc2 +/− rats displayed significantly increased nestlet shredding behavior ( F ( 1 , 18 ) = 16.39, p = 0.001) (Fig. 2 B), indicating lower-order repetitive behavior in Eker rats. Since repetitive behavior represents a strategy to deal with stress and anxiety, next, stereotyped movements were assessed under stressful conditions. The Light-Sound-Confinement test was utilized to investigate repetitive digging and grooming behavior under the application of multiple stressors. First, animals were subjected to the pre-test phase, in which they adjusted to the novel test environment in the absence of additional stressors. Tsc2 +/− rats showed significantly increased grooming behavior compared to wt (Fig. 2 C, C´; F ( 1 , 18 ) = 23.47, p = 0.001), further suggesting repetitive behavioral abnormalities in the Eker rat model. Conversely, digging time was significantly reduced in Tsc2 +/− rats compared to wt in the pre-test phase (Fig. 2 D, D´; F ( 1 , 18 ) = 13.43, p = 0.002). Notably, a significant sex effect was observed in the case of digging in the pre-test phase (Fig. 2 D, D´; F ( 1 , 18 ) = 47.92, p = 0.001), as female rats displayed higher levels of digging behavior than males. Next, animals were subjected to the test phase, where unpredictable visual and acoustic stressors were introduced to elevate stress levels. Neither genotype demonstrated grooming behavior nor digging behavior during stressor application (Fig. 2 C, D). Interestingly, a sex and genotype interaction for grooming was observed during recovery in the post-test phase as female Tsc2 +/− rats displayed significantly increased grooming behavior compared to male Tsc2 +/− and wt animals (Fig. 2 C, C´; F ( 1 , 18 ) = 11.22, p = 0.004). In contrast, digging behavior did not significantly differ between genotypes or sex during recovery in the post-test phase (Fig. 2 D, D´). However, animals might need more time to recover from the stress phase, thus the occurrence of a genotype effect at later timepoints cannot be excluded. In conclusion, while Tsc2 +/− animals were found to display lower-order repetitive behavior in the form of increased grooming in non-stressful conditions, high-stress application in the Light-Sound-Confinement (LSC) test did not exacerbate repetitive behavior patterns in the Eker rat. However, female Tsc2 +/− rats might recover faster following high-stress levels and showed higher compulsive grooming behavior compared to wt. No repetitive digging behavior was found in the Eker rat model. To test whether Tsc2 +/− rats show altered mobility as a stress response, animals were subjected to the forced swim test (FST). The total time of immobility did not differ significantly between Tsc2 +/− and wt animals (Fig. 2 E), suggesting that stress-coping responses involving mobility are not altered in the Eker rat model. Additionally, no repetitive behavior that could present as repetitive circling or climbing was detected in Tsc2 +/− rats. The presence of higher-order repetitive behavior was investigated using the water T-maze test (WTM), as its reversal learning paradigm reveals an animal´s ability to switch behavioral strategies. Higher order repetitive behavior would have presented as perseverative behavior by not successfully completing the task criterion, a consistent choice of novel platform location, thus, animals would reach the maximum number of trials on day 2 of the WTM. Instead, both wt and Tsc2 +/− rats completed the task successfully with no significant difference (Fig. 2 F). Both recalling of position habit learning (repetition phase) and finding an unknown platform position (reversal phase) showed no significant difference in attempts needed to fulfill criterion (Fig. 3 D, E). Thus, unaltered performance in the WTM suggests that the Eker rat does not demonstrate a significant lack of behavioral response variability and therefore does not display higher-order repetitive behavior. Stress-induced repetitive behavior is thought to act as anxiety relief by activating the dopaminergic reward system, thereby representing an important strategy for emotional regulation in ASD and obsessive-compulsive disorder models ( 53 ). In fact, abnormal lower-order repetitive stereotypies are associated with overactivation of the dopaminergic cortical basal ganglia circuitry ( 54 ). Accordingly, treatment with the indirect dopamine agonist amphetamine exacerbates repetitive motor stereotypies and can reveal underlying changes in the dopamine (DA) system in rodent models ( 55 ). Therefore, we opted to investigate whether administering amphetamine at a dosage of 2 mg/kg would reveal signs of repetitive behavior in Eker rats. To determine if the Eker rat model presents with altered DA function and connected occurrence of repetitive behavior, an amphetamine challenge protocol was applied. Behavioral categorization using an adapted scoring protocol from Kelly et al. (1975) ( 51 ) revealed that both wt and Tsc2 +/− animals mainly presented with limited ( 1 ) and frequent exploratory behavior ( 2 ) (rearing, sniffing, grooming) but did not exhibit repetitive stereotypic behavior ( 3 ) (repetitive rearing and grooming) (Fig. 2 G). A mean behavioral categorization score was calculated to show the overall locomotive activity of sex and genotype. Repeated ANOVA measures did not reveal significant effects of sex on behavior categories, so data from males and females were combined. No significant effect on the extent and type of locomotion was found between wt and Tsc2 +/− animals after amphetamine injection (Fig. 2 G, G´). Thus, the application of amphetamine provoked unaltered responses in Tsc2 +/− animals and did not induce repetitive behavior. In conclusion, the Eker rat model was found to display lower-order repetitive behavior alterations under low-stress conditions in the form of excessive nestlet shredding and grooming. Surprisingly, the application of stressors did not exacerbate repetitive behavior patterns. Furthermore, no higher-order repetitive behavior was detected, indicating sufficient behavioral flexibility in Tsc2 +/− rats. Lastly, the Eker model seems to display wt-like behavioral responses following manipulation of the dopamine system through amphetamine administration, suggesting no major alterations of the DA system to be present. Repetitive behavior alterations in the Eker rat are observed in a context of impaired social behavior Apart from repetitive stereotypies, ASD presents with deficits in social behavior and communication, as well as cognitive impairment and behavioral inflexibility ( 1 ). While autistic-like social behavior has been reported in Eker rats, learning and memory performance was found to be unaltered, suggesting normal cognitive function ( 35 ). To validate that Tsc2 +/− rats show previously reported deficits in social interaction, first, animals were challenged with a social recognition test in which exploration time with an unfamiliar rat was assessed. Eker rats showed overall less interaction duration compared to wt during three out of five trials (Fig. 3 A), with a significantly decreased interaction time during the second trial (Fig. 3 A´, F ( 1 , 18 ) = 12.47, p = 0.003), confirming the previously reported general decrease in social interest. Wt animals expectedly displayed variety in social exploratory behavior, with a gradual decrease in interaction during the first four trials and an increase in interaction upon the introduction of a novel actor rat in trial 5. In contrast, Tsc2 +/− animals were found to maintain similar levels of interaction time across all trials, independent of actor rat familiarity or novelty (Fig. 3 A), suggesting restricted social interest in Eker rats. Tsc2 haploinsufficiency was found to cause changes in activity-dependent hippocampal synaptic plasticity ( 33 ). To assess whether Eker rats show signs of impaired spatial memory, which is dependent on hippocampal learning, animals were challenged with the water T maze test (Fig. 3 B-E). During the acquisition phase, a significant interaction of sex and genotype was observed ( F ( 1 , 18 ) = 4.353, p = 0.012) as male Tsc2 +/− rats needed significantly fewer trials than wt males to learn the correct arm position (Fig. 3 C; p = 0.001). However, no negative effect of the Tsc2 +/ genotype on learning speed was observed, suggesting that learning is not impaired in Eker rats. In the repetition test, 24 hours later, wt and Tsc2 +/− animals found the correct arm position equally well (Fig. 3 D). Similarly, no significant differences in number of trials needed to criterion were detected between genotypes in the reversal test (Fig. 3 E). These results indicate that spatial memory formation is not affected in Eker rats and are in line with previous reports ( 35 ). To test if alterations in reward function might influence behavioral test results, a sucrose preference test was performed. No difference between genotypes nor interaction effects have been observed (Fig. 3 F), indicating that Tsc2 +/− animals show intact reward function and do not develop anhedonic tendencies. In summary, Eker rats show signs of social behavior impairment in the form of abnormal social interest. On the contrary, spatial learning and memory as well as reward function remained unaffected by Tsc2 haploinsufficiency. Herein, we provide evidence that the Tsc2 +/− animal model of ASD presents with an overt repetitive behavior pattern that occurs alongside social behavior impairments (Fig. 3 G, G´). Eker rats show evidence for impaired PFC-dependent dopaminergic signaling Next, we opted to explore neurochemical alterations in the brain of Tsc2 +/− rats by determining DA levels in post-mortem brain tissue. Despite absent changes in amphetamine response after systemic administration, the dopaminergic pathway remains a promising target due to its strong connection to repetitive behavior formation and general movement modulation ( 53 , 56 , 57 ). An important DA target is the cortico-striatal-thalamo-cortical (CSTC) pathway, a neuronal circuit that is crucial for movement selection and initiation ( 57 – 59 ) and is implicated in influencing stereotyped behavior such as repetitive self-grooming and digging in rodents ( 60 , 61 ). Thus, we analyzed neurotransmitter levels in the striatum (CPu) and prefrontal cortex (PFC), as both are key areas regulating and modulating CSTC activity and function and are linked to stereotyped behavior initiation ( 62 – 64 ). Therefore, region-specific HPLC for dopamine (DA), and downstream DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was performed. While no significant alteration of DA level was found in Tsc2 +/− rat CPu tissue (Fig. 4 A), significantly increased levels of DA were present within the PFC of Tsc2 +/− animals compared to wt (Fig. 4 B; F ( 1 , 18 ) = 4.478, p = 0.0485). Similarly, no changes in DA turnover were observed in CPu tissue (Fig. 4 C). In the PFC, a significant sex x genotype interaction revealed a significantly decreased DA/(DOPAC + HVA) ratio in male Tsc2 +/− rats compared to female Tsc2 +/− and wt rats (Fig. 4 D; F ( 1 , 18 ) = 8.5, p = 0.01), suggesting significantly increased DA turnover in male Tsc2 +/− animals. Overall, a significant effect of genotype was found, revealing an increased DA turnover rate in Tsc2 +/− Eker rats compared to wt ( F ( 1 , 18 ) = 15, p = 0.001). Thus, alterations in PFC-specific dopamine metabolism might contribute to social and repetitive behavioral alterations in the Tsc2 haploinsufficient Eker rat. Discussion Restricted repetitive behavior is one of the core pathological features of ASD and presents with high phenotypic symptom heterogeneity. Animal models of ASD are expected to offer construct validity, e.g., in the form of associated genetic mutations, and recapitulate characteristic key manifestations. Here, using a series of behavioral tests accounting for a spectrum of stereotyped behavior, we sought to characterize lower-order and higher-order repetitive behavior of the Tsc2 +/− haploinsufficient Eker rat. Importantly, we show that Tsc2 +/− Eker rats demonstrate a moderate phenotype of ASD-like behavior in the repetitive behavior domain involving increased lower-order motor stereotypies but no higher-order repetitive behavior, which was accompanied by a PFC-specific increase in DA levels. While the Eker rat strain has been postulated as a model for ASD and ASD-like behavior ( 29 , 35 , 36 , 65 ), to our knowledge, this is the first study to evaluate the repetitive behavior pattern in Tsc2 +/− Eker rats. Since the Tsc2 +/− Eker strain offers high construct validity and was indicated to present with social deficits, we hypothesized that Eker rats additionally develop repetitive stereotypies that are typically exacerbated under stress. Reported deficits include reduced novel object recognition and environmental exploration in the open field paradigm, as well as decreased social exploration in the form of non-anogenital ( 35 ) and anogenital exploration ( 37 ). Interestingly, Petrasek et al. (2021) ( 37 ) further reported alterations in ultrasonic vocalization in Tsc2 +/− pups, suggesting phases of impaired social communication which is consistent with ASD characteristics in humans. A study on hippocampal plasticity reported a significant decrease in synaptic modification capacity in the form of reduced LTP and LTD amounts ( 33 ). While this finding implies an impact on cognitive function, Tsc2 +/− animals were found to display unimpaired learning abilities in a Morris water maze paradigm ( 35 ), suggesting that molecular and functional changes are subtle and do not elicit a major behavioral impact ( 32 ). Other TSC rodent models have been more thoroughly assessed regarding ASD-like behavior. Tsc1 +/− mice showed deficits in social interaction and impaired hippocampal learning revealed through the Morris water maze task and a fear conditioning test ( 66 ). In a mouse model with Purkinje cell-specific Tsc2 +/− heterozygous genotype, repetitive digging behavior (marble burying test) and decreased social exploration, but no alterations in learning and memory were detected ( 67 ). Interestingly, ASD symptom manifestations overall seem to be highly strain-dependent, as several heterozygous TSC rodent models present with specific combinations of social deficits, cognitive impairment and repetitive behavior. However, while social interaction and learning are commonly assessed domains in TSC rodent models, repetitive behavior analyses remain less frequently reported. ASD-related rodent studies have focused on fixated action patterns such as obsessive self-grooming, digging, and nestlet shredding to quantify lower-order repetitive behavior ( 38 , 40 , 59 , 68 ). Here, Tsc2 +/− Eker rats were found to present with lower-order stereotypies in the form of increased nestlet shredding and self-grooming behavior. Conversely, no excessive digging was revealed in the marble burying test or occurred spontaneously. Instead, Eker rats spent significantly less time digging than wt rats. This contradictory finding fits the highly heterogeneous behavioral profiles of both human patients and rodent ASD models ( 57 ). Some do not manifest repetitive behavior at all ( 69 ), others show anomalies only in specific behavioral tests. Shank3 knockout mice, as an established ASD model, manifest repetitive behavior in the form of increased grooming and nestlet shredding but show decreased digging and marble-burying behavior ( 70 – 72 ). Similar patterns can be found in Shank2 mutant lines ( 57 , 73 ). Genetic factors exert great influence on innate digging behavior, thus, strain-variations in digging responses are common ( 60 , 74 ). Although excessive digging is commonly used as a measure for repetitive stereotypies, it can also be interpreted as an indicator of exploratory drive, depending on the motivational background ( 60 ). Thus, reduced digging, especially in the habituation setting of this study, may correspond to a decrease in explorative behavior, which has been previously reported in Tsc2 +/− Eker rats ( 35 ). Acute stress is known to play a role in altering repetitive behavior patterns, such as increasing grooming behavior ( 53 , 75 – 77 ). Thus, repetitive behavior was evaluated under stressful conditions in a here-described LSC test and the FST. Interestingly, the application of stress did not exacerbate repetitive behavior patterns in Tsc2 +/− Eker rats. Further, we report the absence of higher-order repetitive abnormalities in Tsc2 +/− Eker rats. Higher-order repetitive behaviors are often modeled using reversal learning paradigms such as the water T-maze or Morris water maze test ( 35 , 47 ). Animals are trained to prefer correct-rewarded over incorrect-unrewarded choices, after which the choice and reward relation is reversed. The number of trials needed to switch to the new correct-reward association is meant to reflect cognitive flexibility and insistence on sameness ( 11 ). Together, Eker rats were found to present with mild changes in stereotypic behavior but seem to lack a higher-order repetitive behavior domain. Several ASD rodent models (C58 and BTBR T + tf/J mice) do not manifest with apparent cognitive inflexibility as they show unaltered responses in classical reversal learning paradigms. However, only when using a probabilistic reversal learning test approach with a reward rate of 80% instead of 100%, which is commonly used to model cognitive inflexibility in ASD patients, significant differences became apparent ( 78 – 80 ). Importantly, we found that alterations in lower-order repetitive behavior in the Eker rat occur alongside previously reported social and cognitive behavioral patterns. Here, Tsc2 +/− animals showed mild impairments in the social recognition test, confirming deficits in the social domain of ASD manifestations ( 35 , 37 ). Interestingly, Tsc2 +/− rats maintained similar interaction time throughout all trials, while wt rats expectedly decreased the interaction time during the first four trials with the same partner and increased interaction time upon the introduction of a novel partner. Changes in the social recognition test can arise for several reasons, such as olfactory system impairments or locomotion deficits ( 81 – 83 ). Additionally, decreased social interaction could be attributed to generalized alterations in exploratory drive that need to be specifically addressed. Potential cross-target effects should be excluded to increase the robustness of social deficit findings in Tsc2 +/− Eker rats. The social recognition test is thought to reflect both social interest and short-term recognition memory in rodent studies ( 81 ). Thus, our results can be interpreted as both impaired social interest and target discrimination in Eker rats, as well as altered short-term recognition memory suggesting an impact on cognitive function. Here, we show that the Eker model does not present significant changes in spatial learning and memory, as assessed in the WTM test. Our results fit previous reports showing typical learning and spatial memory in Tsc2 +/− animals in the Morris water maze, radial maze, and conditioned taste aversion test ( 35 , 84 ). Contrary, Tsc2 +/− animals surprisingly show increased performance compared to wt upon modification to a delayed matching-to-place task meant to model episodic-like memory by adding a 2 h interval between trials ( 84 ). However, improved performance in an episodic memory task can also be interpreted as a cognitive disadvantage; that is, Eker rats might perform better in finding novel platform locations due to a lack of memory formation of previous platform locations ( 84 ). The entirety of observed brain malformations, cellular aberrations, and impairments of neuroplasticity suggest cognitive deficits to be present in the Tsc2 +/− Eker rat ( 21 ). However, behavioral assessments so far have failed to show robust impairments in cognition. As discussed previously, applying higher task difficulty in the form of a probabilistic reversal learning test might be more sufficient to reveal higher-order repetitive behavior and cognitive inflexibility. Given the growing understanding of sex discrepancies in ASD symptom manifestation ( 85 , 86 ), sex effects are important to consider in ASD models. Thus, repetitive behavior alterations were assessed in both male and female Tsc2 +/− Eker rats. While little is known about sex-specific differences in ASD models, available data remains inconsistent with variance between animal strains and specific tasks ( 87 , 88 ). For example, the BTBR ASD model was found to present with repetitive grooming and marble burying in male but not female mice ( 89 ), while repetitive grooming in Nlgn4 ASD mice appears in female animals exclusively ( 90 ). Here, we report a significant effect of sex before stress application in the LSC test, with increased digging behavior in female compared to male animals. Additionally, Tsc2 +/− females were found to show increased grooming behavior compared to both wt and males. In a Tsc2 +/− mouse model, female animals exhibited deficits in habituation response and following decreases in anxiety ( 91 ), suggesting that observed differences in repetitive behavior during the LSC test may arise from an impaired habituation response to the novel environment in female Tsc2 +/− rats. Further, Tsc2 +/− males presented with improved performance in the acquisition phase of the WTM. In line with our finding, Tsc2 +/− males are reported to show increased performance in an episodic memory task ( 84 ), suggesting altered memory formation in the Eker rat. Interestingly, this phenotype does not seem to occur in female Eker rats, suggesting sex differences in cognition; however, a confirmation and extension to other cognitive domains needs to be made before drawing meaningful conclusions. The here reported sex effects should be interpreted carefully, as male and female animal numbers are too small to draw meaningful conclusions. Overall, the discussed sex effects are considered to be of small relevance regarding the main concern of genotype effects in the Eker ASD model. While the Tsc2 +/− Eker model presents with both social impairment and repetitive behavior, symptom expression remains mild. Deviations from human pathology in the Tsc2 +/− rat model have been proposed to originate from its genetic basis. Homozygous deletion is embryonically lethal in both mice and rats, thus heterozygous TSC models are used ( 92 , 93 ). However, heterozygous animal models do not always accurately mirror typical neuropathy. In case of TSC, spontaneous seizure development as a hallmark manifestation cannot be recapitulated in animal models so far ( 21 ). Often, “second hit paradigms” are therefore applied to model neurodevelopmental diseases with complex gene-environmental etiology, as they are thought to recapitulate human disease development more closely ( 21 ) and have been reported to induce ASD-like phenotypes ( 94 ). Chemical “second hit” induction has been used to generate epileptic seizures in the Tsc2 +/− rat model and was found to increase ASD-associated behavior in mutant animals ( 35 , 37 ). Thus, introducing a “second hit” paradigm may exacerbate behavioral alteration in the Eker rat model also in respect to its repetitive behavior profile. Finally, pharmacological testing of mTOR inhibitors or dopamine receptor antagonists and their effect on repetitive phenotypes as well as lifespan analysis of stereotypies, especially considering early symptom onset in ASD, may be applied in the Eker model to challenge its translational accuracy and support predictive validity. Various brain networks and dopamine-dependent signaling pathways have been linked to repetitive behavior formation ( 56 , 57 ). Here, we report alterations in PFC but not striatal DA levels of Tsc2 +/− animals, both of which are components of the CSTC pathway that regulates motor activity ( 57 – 59 , 95 ). The PFC has been shown to mediate repetitive behavior patterns ( 96 , 97 ), is thought to be involved in goal-directed reinforcement learning ( 80 , 98 ) and shows disrupted function in various animal models of ASD-linked risk genes and ASD patients ( 57 ). Projections from the PFC into the substantia nigra pars compacta regulate the majority of DA release in the striatum. Striatal overactivation is further implicated in repetitive behavior development ( 64 ). The Scn1a +/− ASD mouse model, for example, exhibits hyperactivity and excessive self-grooming upon increased excitation in the PFC ( 99 ). Observed increases in PFC DA levels occurring alongside increases in DA turnover, thus, fit with our observation of repetitive behavior formation in Tsc2 +/− Eker rats. Interestingly, DA/(DOPAC/HVA) rates showed differences between sexes in Tsc2 +/− animals, suggesting that PFC-dependent DA increase causes compensatory changes through elevation of DA turnover in male Tsc2 +/− rats. While observed alterations in PFC-DA turnover may partially explain sex differences in repetitive behavior, extended mechanistic analysis of DA receptor and enzyme function are necessary to build this hypothesis and connect repetitive phenotypes to dysfunction of the dopaminergic PFC system. Considering that we did not find changes in homeostatic CPu DA levels, both regions possibly exert different effects on specific types of repetitive behavior, as the striatum for example is involved in maintaining and flexibly changing choice patterns, thereby influencing reversal learning and behavioral flexibility ( 100 ). Further, we recognize that other brain regions have been implicated in repetitive behavior formation, including the hippocampus, amygdala, and ventral tegmental area ( 57 ), which may be addressed to characterize region-specific alterations of DA in the Eker rat. Additionally, the usage of animals for HPLC analysis that have undergone behavioral assessment to reduce animal numbers and comply with ethical standards raises the possibility of interference. To minimize this, a single low dose of amphetamine, established to not generate behavioral stereotypies in wt animals, was used for behavioral testin,g and tissue was sampled after a one-day rest, circumventing acute DA alterations as well as long-term effects which manifest after 1–3 weeks ( 101 ) or after repeated treatment ( 102 ). Lastly, alterations in dopaminergic signaling in the PFC are further known to result in changed social behavior and social recognition in ASD-associated animal models ( 103 , 104 ). This study lacks experimental evidence to specifically connect alterations in DA level to behavioral alterations; however, we demonstrate that changes in PFC-dependent DA levels occur simultaneously with ASD-like alterations in the social and repetitive behavior domain in Tsc2 +/− Eker rats. Conclusion For the first time, we characterized the repetitive behavior profile of the Tsc2 +/− Eker rat model. In summary, we report the presence of lower-order repetitive behaviors that occurred alongside decreased social interaction in Tsc2 +/− Eker rats. Importantly, we showed that DA homeostasis is altered in the PFC of Tsc2 +/− rats, suggesting a contribution to ASD-like repetitive and social behavior manifestation. We propose that our findings add translational value to the usage of the Tsc2 +/− Eker rat model in preclinical ASD research. Abbreviations ASD autism spectrum disorder CPu striatum (caudate putamen) CSTC cortico-striatal-thalamo-cortical DA dopamine DOPAC 3,4-dihydroxyphenylacetic acid FST forced swim test HVA homovanillic acid LSC light-sound-confinement test LTD long-term depression LTP long-term potentiation mTORC1 mammalian target of rapamycin complex 1 PFC prefrontal cortex TSC tuberous sclerosis complex wt wild-type WTM water-T-maze Declarations Ethics approval All experiments were conducted in accordance with the European Union guidelines on care and use of laboratory animals (EU directive 2010/63/EU) and were approved by the local ethics committees (Landesdirektion Sachsen, No. 10/2018, Institutional Animal Care and Use Committee No. 66/2016). Funding The work reported in this study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project-ID 518530049, Project-ID 521379614 – SFB/TRR 393 and Project-ID 454245598 – GRK2773/1. Author Contribution M.Z. behavioral experiments. B.H. Audacity/Matlab programming, HPLC analysis. T.P., I.V., breeding, genotyping, and provided materials. N.B., T.P., R.W. study conceptualization and design. N.B. data and statistical analysis. A.R., N.B manuscript draft. N.B., A.R., figure design. N.B. funding acquisition. All authors contributed to revisions of the manuscript draft. Acknowledgement We acknowledge the valuable technical assistance provided by Kristin Wogan. Data Availability All data is provided within the manuscript. References American Psychiatric Association. Diagnostic and statistical manual of mental disorders: DSM-5. 5th edition. American Psychiatric Association. 2013. Available from: https://doi.org/10.1176/appi.books.9780890425596 Christensen DL, Bilder DA, Zahorodny W, Pettygrove S, Durkin MS, Fitzgerald RT, et al. Prevalence and Characteristics of Autism Spectrum Disorder Among 4-Year-Old Children in the Autism and Developmental Disabilities Monitoring Network. J Dev Behav Pediatr. 2016;37(1):1–8. Zeidan J, Fombonne E, Scorah J, Ibrahim A, Durkin MS, Saxena S, et al. Global prevalence of autism: A systematic review update. Autism Res. 2022;15(5):778–90. McCracken JT, Anagnostou E, Arango C, Dawson G, Farchione T, Mantua V, et al. Drug development for Autism Spectrum Disorder (ASD): Progress, challenges, and future directions. Eur Neuropsychopharmacol. 2021;48:3–31. Gogate A, Kaur K, Khalil R, Bashtawi M, Morris MA, Goodspeed K, et al. The genetic landscape of autism spectrum disorder in an ancestrally diverse cohort. npj Genom Med. 2024;9(1):1–22. Willsey HR, Willsey AJ, Wang B, State MW. Genomics, convergent neuroscience and progress in understanding autism spectrum disorder. Nat Rev Neurosci. 2022;23(6):323–41. Satterstrom FK, Kosmicki JA, Wang J, Breen MS, De Rubeis S, An JY, et al. Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism. Cell. 2020;180(3):568–e58423. Gaugler T, Klei L, Sanders SJ, Bodea CA, Goldberg AP, Lee AB, et al. Most genetic risk for autism resides with common variation. Nat Genet. 2014;46(8):881–5. Geschwind DH. Genetics of Autism Spectrum Disorders. Trends Cogn Sci. 2011;15(9):409–16. Kazdoba TM, Leach PT, Crawley JN. Behavioral phenotypes of genetic mouse models of autism. Genes Brain Behav. 2016;15(1):7–26. Möhrle D, Fernández M, Peñagarikano O, Frick A, Allman B, Schmid S. What we can learn from a genetic rodent model about autism. Neurosci Biobehavioral Reviews. 2020;109:29–53. Kas MJ, Glennon JC, Buitelaar J, Ey E, Biemans B, Crawley J, et al. Assessing behavioural and cognitive domains of autism spectrum disorders in rodents: current status and future perspectives. Psychopharmacology. 2014;231(6):1125–46. Whitehouse CM, Lewis MH. Repetitive Behavior in Neurodevelopmental Disorders: Clinical and Translational Findings. Behav Anal. 2015;38(2):163–78. Sundberg M, Sahin M. Cerebellar Development and Autism Spectrum Disorder in Tuberous Sclerosis Complex. J Child Neurol. 2015;30(14):1954–62. Niu M, Han Y, Dy ABC, Du J, Jin H, Qin J, et al. Autism Symptoms in Fragile X Syndrome. J Child Neurol. 2017;32(10):903–9. Jamain S, Radyushkin K, Hammerschmidt K, Granon S, Boretius S, Varoqueaux F, et al. Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc Natl Acad Sci U S A. 2008;105(5):1710–5. Betancur C, Buxbaum JD. SHANK3 haploinsufficiency: a common but underdiagnosed highly penetrant monogenic cause of autism spectrum disorders. Mol Autism. 2013;4:17. Radyushkin K, Hammerschmidt K, Boretius S, Varoqueaux F, El-Kordi A, Ronnenberg A, et al. Neuroligin-3-deficient mice: model of a monogenic heritable form of autism with an olfactory deficit. Genes Brain Behav. 2009;8(4):416–25. Specchio N, Pietrafusa N, Trivisano M, Moavero R, De Palma L, Ferretti A, et al. Autism and Epilepsy in Patients With Tuberous Sclerosis Complex. Front Neurol. 2020;11:639. Crino PB, Nathanson KL, Henske EP. The New England Journal of Medicine. Massachusetts Medical Society; 2006 [cited 2025 Jan 21]. The Tuberous Sclerosis Complex. Available from: https://www.nejm.org/doi/full/ 10.1056/NEJMra055323 Kútna V, O’Leary VB, Newman E, Hoschl C, Ovsepian SV. Revisiting Brain Tuberous Sclerosis Complex in Rat and Human: Shared Molecular and Cellular Pathology Leads to Distinct Neurophysiological and Behavioral Phenotypes. Neurotherapeutics. 2021;18(2):845–58. Jeste SS, Varcin KJ, Hellemann GS, Gulsrud AC, Bhatt R, Kasari C, et al. Symptom profiles of autism spectrum disorder in tuberous sclerosis complex. Neurology. 2016;87(8):766–72. Gadad BS, Hewitson L, Young KA, German DC. Neuropathology and Animal Models of Autism: Genetic and Environmental Factors. Autism Res Treat. 2013;2013:731935. Palavra F, Robalo C, Reis F. Recent Advances and Challenges of mTOR Inhibitors Use in the Treatment of Patients with Tuberous Sclerosis Complex. Oxidative Med Cell Longev. 2017;2017(1):9820181. Switon K, Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J. Molecular neurobiology of mTOR. Neuroscience. 2017;341:112–53. Lipton JO, Sahin M. The Neurology of mTOR. Neuron. 2014;84(2):275–91. Eker R, Mossige J. A Dominant Gene for Renal Adenomas in the Rat. Nature. 1961;189(4767):858–9. Yeung RS, Xiao GH, Jin F, Lee WC, Testa JR, Knudson AG. Predisposition to Renal Carcinoma in the Eker Rat is Determined by Germ-Line Mutation of the Tuberous Sclerosis 2 (TSC2) Gene. Proc Natl Acad Sci USA. 1994;91(24):11413–6. Granak S, Tuckova K, Kutna V, Vojtechova I, Bajkova L, Petrasek T, et al. Developmental effects of constitutive mTORC1 hyperactivity and environmental enrichment on structural synaptic plasticity and behaviour in a rat model of autism spectrum disorder. Eur J Neurosci. 2023;57(1):17–31. Chi OZ, Wu CC, Liu X, Rah KH, Jacinto E, Weiss HR. Restoration of Normal Cerebral Oxygen Consumption with Rapamycin Treatment in a Rat Model of Autism–Tuberous Sclerosis. Neuromolecular Med. 2015;17(3):305–13. Chi OZ, Liu X, Fortus H, Werlen G, Jacinto E, Weiss HR. Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis. Neuromolecular Med. 2024;26(1):10. Kútna V, Uttl L, Waltereit R, Krištofiková Z, Kaping D, Petrásek T, et al. Tuberous Sclerosis (tsc2+/-) Model Eker Rats Reveals Extensive Neuronal Loss with Microglial Invasion and Vascular Remodeling Related to Brain Neoplasia. Neurotherapeutics. 2020;17(1):329–39. Von Der Brelie C, Waltereit R, Zhang L, Beck H, Kirschstein T. Impaired synaptic plasticity in a rat model of tuberous sclerosis. Eur J Neurosci. 2006;23(3):686–92. Takahashi DK, Dinday MT, Barbaro NM, Baraban SC. Abnormal Cortical Cells and Astrocytomas in the Eker Rat Model of Tuberous Sclerosis Complex. Epilepsia. 2004;45(12):1525–30. Waltereit R, Japs B, Schneider M, de Vries PJ, Bartsch D. Epilepsy and Tsc2 Haploinsufficiency Lead to Autistic-Like Social Deficit Behaviors in Rats. Behav Genet. 2011;41(3):364–72. Schneider M, de Vries PJ, Schönig K, Rößner V, Waltereit R. mTOR inhibitor reverses autistic-like social deficit behaviours in adult rats with both Tsc2 haploinsufficiency and developmental status epilepticus. Eur Arch Psychiatry Clin Neurosci. 2017;267(5):455–63. Petrasek T, Vojtechova I, Klovrza O, Tuckova K, Vejmola C, Rak J, et al. mTOR inhibitor improves autistic-like behaviors related to Tsc2 haploinsufficiency but not following developmental status epilepticus. J Neurodev Disord. 2021;13:14. Angoa-Pérez M, Kane MJ, Briggs DI, Francescutti DM, Kuhn DM. Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice. J Vis Exp. 2013;(82):50978. Kozlova AA, Rubets E, Vareltzoglou MR, Jarzebska N, Ragavan VN, Chen Y, et al. Knock-out of the critical nitric oxide synthase regulator DDAH1 in mice impacts amphetamine sensitivity and dopamine metabolism. J Neural Transm (Vienna). 2023;130(9):1097–112. Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology. 2009;204(2):361–73. Campeau S. Apparatus and General Methods for Exposing Rats to Audiogenic Stress. Bio Protoc. 2016;6(21):e1994. Valsamis B, Schmid S. Habituation and Prepulse Inhibition of Acoustic Startle in Rodents. J Vis Exp. 2011;(55):3446. Bouwknecht JA, Spiga F, Staub DR, Hale MW, Shekhar A, Lowry CA. Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors; relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus. Brain Res Bull. 2007;72(1):32–43. Commons KG, Cholanians AB, Babb JA, Ehlinger DG. The Rodent Forced Swim Test Measures Stress-Coping Strategy, Not Depression-like Behavior. ACS Chem Neurosci. 2017;8(5):955–60. Porsolt RD, Anton G, Blavet N, Jalfre M. Behavioural despair in rats: A new model sensitive to antidepressant treatments. Eur J Pharmacol. 1978;47(4):379–91. Slattery DA, Cryan JF. Using the rat forced swim test to assess antidepressant-like activity in rodents. Nat Protoc. 2012;7(6):1009–14. Guariglia SR, Chadman KK. Water T-maze: A useful assay for determination of repetitive behaviors in mice. J Neurosci Methods. 2013;220(1):24–9. Meyerolbersleben L, Winter C, Bernhardt N. Dissociation of wanting and liking in the sucrose preference test in dopamine transporter overexpressing rats. Behav Brain Res. 2020;378:112244. Hadar R, Edemann-Callesen H, Reinel C, Wieske F, Voget M, Popova E, et al. Rats overexpressing the dopamine transporter display behavioral and neurobiological abnormalities with relevance to repetitive disorders. Sci Rep. 2016;6(1):39145. Winslow JT, Camacho F. Cholinergic modulation of a decrement in social investigation following repeated contacts between mice. Psychopharmacology. 1995;121(2):164–72. Kelly PH, Seviour PW, Iversen SD. Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Res. 1975;94(3):507–22. Blanca MJ, Alarcón R, Arnau J, Bono R, Bendayan R. Non-normal data: Is ANOVA still a valid option? Psicothema. 2017;29(4):552–7. Sun J, Yuan Y, Wu X, Liu A, Wang J, Yang S, et al. Excitatory SST neurons in the medial paralemniscal nucleus control repetitive self-grooming and encode reward. Neuron. 2022;110(20):3356–e33738. Muehlmann AM, Lewis MH. Abnormal repetitive behaviours: shared phenomenology and pathophysiology. J Intellect Disabil Res. 2012;56(5):427–40. Moy SS, Riddick NV, Nikolova VD, Teng BL, Agster KL, Nonneman RJ, et al. Repetitive behavior profile and supersensitivity to amphetamine in the C58/J mouse model of autism. Behav Brain Res. 2014;259. 10.1016/j.bbr.2013.10.052 . Kim H, Lim CS, Kaang BK. Neuronal mechanisms and circuits underlying repetitive behaviors in mouse models of autism spectrum disorder. Behav Brain Funct. 2016;12(1):3. Gandhi T, Lee CC. Neural Mechanisms Underlying Repetitive Behaviors in Rodent Models of Autism Spectrum Disorders. Front Cell Neurosci. 2021;14:592710. Parr-Brownlie LC, Hyland BI. Bradykinesia Induced by Dopamine D2 Receptor Blockade Is Associated with Reduced Motor Cortex Activity in the Rat. J Neurosci. 2005;25(24):5700–9. Kalueff AV, Stewart AM, Song C, Berridge KC, Graybiel AM, Fentress JC. Neurobiology of rodent self-grooming and its value for translational neuroscience. Nat Rev Neurosci. 2016;17(1):45–59. de Brouwer G, Fick A, Harvey BH, Wolmarans DW. A critical inquiry into marble-burying as a preclinical screening paradigm of relevance for anxiety and obsessive-compulsive disorder: Mapping the way forward. Cogn Affect Behav Neurosci. 2019;19(1):1–39. Taylor GT, Lerch S, Chourbaji S. Marble burying as compulsive behaviors in male and female mice. Acta Neurobiol Exp (Wars). 2017;77(3):254–60. Vaccarino F, Franklin KBJ. Self-stimulation and circling reveal functional differences between medial and lateral substantia nigra. Behav Brain Res. 1982;5(3):281–95. Abbott AE, Linke AC, Nair A, Jahedi A, Alba LA, Keown CL, et al. Repetitive behaviors in autism are linked to imbalance of corticostriatal connectivity: a functional connectivity MRI study. Soc Cogn Affect Neurosci. 2018;13(1):32–42. Kim IH, Rossi MA, Aryal DK, Racz B, Kim N, Uezu A, et al. Spine Pruning Drives Antipsychotic-sensitive Locomotion via Circuit Control of Striatal Dopamine. Nat Neurosci. 2015;18(6):883–91. Chi OZ, Liu X, Fortus H, Werlen G, Jacinto E, Weiss HR. Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis. Neuromolecular Med. 2024;26(1):10. Goorden SMI, van Woerden GM, van der Weerd L, Cheadle JP, Elgersma Y. Cognitive deficits in Tsc1+/–mice in the absence of cerebral lesions and seizures. Ann Neurol. 2007;62(6):648–55. Reith RM, McKenna J, Wu H, Hashmi SS, Cho SH, Dash PK, et al. Loss of Tsc2 in Purkinje cells is associated with autistic-like behavior in a mouse model of tuberous sclerosis complex. Neurobiol Dis. 2013;51:93–103. Pearson B, Pobbe R, Defensor E, Oasay L, Bolivar V, Blanchard D, et al. Motor and cognitive stereotypies in the BTBR T + tf/J mouse model of autism. Genes Brain Behav. 2011;10(2):228–35. Shin W, Kweon H, Kang R, Kim D, Kim K, Kang M, et al. Scn2a Haploinsufficiency in Mice Suppresses Hippocampal Neuronal Excitability, Excitatory Synaptic Drive, and Long-Term Potentiation, and Spatial Learning and Memory. Front Mol Neurosci. 2019;12:145. Bauer HF, Delling JP, Bockmann J, Boeckers TM, Schön M. Development of sex- and genotype-specific behavioral phenotypes in a Shank3 mouse model for neurodevelopmental disorders. Front Behav Neurosci. 2023;16:1051175. Yoo T, Cho H, Lee J, Park H, Yoo YE, Yang E et al. GABA Neuronal Deletion of Shank3 Exons 14–16 in Mice Suppresses Striatal Excitatory Synaptic Input and Induces Social and Locomotor Abnormalities. Front Cell Neurosci [Internet]. 2018 Oct 9 [cited 2025 Jan 29];12. Available from: https://www.frontiersin.org/journals/cellular-neuroscience/articles/ 10.3389/fncel.2018.00341/full Peça J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature. 2011;472(7344):437–42. Jung S, Park M. Shank postsynaptic scaffolding proteins in autism spectrum disorder: Mouse models and their dysfunctions in behaviors, synapses, and molecules. Pharmacol Res. 2022;182:106340. Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology. 2009;204(2):361–73. García-Villamisar D, Rojahn J. Comorbid psychopathology and stress mediate the relationship between autistic traits and repetitive behaviours in adults with autism. J Intellect Disabil Res. 2015;59(2):116–24. Katz RJ, Roth KA. Stress induced grooming in the rat–an endorphin mediated syndrome. Neurosci Lett. 1979;13(2):209–12. Homberg JR, Van Den Akker M, Raasø HS, Wardeh G, Binnekade R, Schoffelmeer ANM, et al. Enhanced motivation to self-administer cocaine is predicted by self-grooming behaviour and relates to dopamine release in the rat medial prefrontal cortex and amygdala. Eur J Neurosci. 2002;15(9):1542–50. Amodeo DA, Jones JH, Sweeney JA, Ragozzino ME. Differences in BTBR T + tf/J and C57BL/6J mice on probabilistic reversal learning and stereotyped behaviors. Behav Brain Res. 2012;227(1):64–72. Whitehouse CM, Curry-Pochy LS, Shafer R, Rudy J, Lewis MH. Reversal learning in C58 mice: Modeling higher order repetitive behavior. Behav Brain Res. 2017;332:372–8. Solomon M, Smith AC, Frank MJ, Ly S, Carter CS. Probabilistic Reinforcement Learning in Adults with Autism Spectrum Disorders. Autism Res. 2011;4(2):109–20. Min JY, Park S, Cho J, Huh Y. The anterior insular cortex processes social recognition memory. Sci Rep. 2023;13(1):10853. Choleris E, Clipperton-Allen AE, Phan A, Valsecchi P, Kavaliers M. Estrogenic involvement in social learning, social recognition and pathogen avoidance. Front Neuroendocr. 2012;33(2):140–59. Kim SH, An K, Namkung H, Saito A, Rannals MD, Moore JR, et al. Anterior Insula–Associated Social Novelty Recognition: Pivotal Roles of a Local Retinoic Acid Cascade and Oxytocin Signaling. AJP. 2023;180(4):305–17. Waltereit R, Welzl H, Dichgans J, Lipp HP, Schmidt WJ, Weller M. Enhanced episodic-like memory and kindling epilepsy in a rat model of tuberous sclerosis. J Neurochem. 2006;96(2):407–13. Werling DM, Geschwind DH. Sex differences in autism spectrum disorders. Curr Opin Neurol. 2013;26(2):146–53. Leow KQ, Tonta MA, Lu J, Coleman HA, Parkington HC. Towards understanding sex differences in autism spectrum disorders. Brain Res. 2024;1833:148877. Jeon SJ, Gonzales EL, Mabunga DFN, Valencia ST, Kim DG, Kim Y, et al. Sex-specific Behavioral Features of Rodent Models of Autism Spectrum Disorder. Exp Neurobiol. 2018;27(5):321–43. Murta V, Seiffe A, Depino AM. Sex Differences in Mouse Models of Autism Spectrum Disorders: Their Potential to Uncover the Impact of Brain Sexual Differentiation on Gender Bias. Sexes. 2023;4(3):358–91. Bove M, Sikora V, Santoro M, Agosti LP, Palmieri MA, Dimonte S, et al. Sex differences in the BTBR idiopathic mouse model of autism spectrum disorders: Behavioural and redox-related hippocampal alterations. Neuropharmacology. 2024;260:110134. El-Kordi A, Winkler D, Hammerschmidt K, Kästner A, Krueger D, Ronnenberg A, et al. Development of an autism severity score for mice using Nlgn4 null mutants as a construct-valid model of heritable monogenic autism. Behav Brain Res. 2013;251:41–9. Saré RM, Lemons A, Figueroa C, Song A, Levine M, Beebe Smith C. Sex-Selective Effects on Behavior in a Mouse Model of Tuberous Sclerosis Complex. eNeuro. 2020;7(2):ENEURO.0379-19.2020. Onda H, Lueck A, Marks PW, Warren HB, Kwiatkowski DJ. Tsc2+/– mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J Clin Invest. 1999;104(6):687–95. Rennebeck G, Kleymenova EV, Anderson R, Yeung RS, Artzt K, Walker CL. Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development. Proc Natl Acad Sci U S A. 1998;95(26):15629–34. Zheng W, Wang M, Cui Y, Xu Q, Chen Y, Xian P, et al. Establishment of a two-hit mouse model of environmental factor induced autism spectrum disorder. Heliyon. 2024;10(9):e30617. Li W, Pozzo-Miller L. Dysfunction of the corticostriatal pathway in autism spectrum disorders. J Neurosci Res. 2020;98(11):2130–47. Morency MA, Stewart RJ, Beninger RJ. Effects of unilateral microinjections of sulpiride into the medial prefrontal cortex on circling behavior of rats. Prog Neuropsychopharmacol Biol Psychiatry. 1985;9(5):735–8. Kelly E, Meng F, Fujita H, Morgado F, Kazemi Y, Rice LC, et al. Regulation of autism-relevant behaviors by cerebellar–prefrontal cortical circuits. Nat Neurosci. 2020;23(9):1102–10. Doll BB, Jacobs WJ, Sanfey AG, Frank MJ. Instructional control of reinforcement learning: A behavioral and neurocomputational investigation. Brain Res. 2009;1299:74–94. Han S, Tai C, Jones CJ, Scheuer T, Catterall WA. Enhancement of Inhibitory Neurotransmission by GABAA Receptors Having α2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Model of Autism. Neuron. 2014;81(6):1282–9. O’Neill M, Brown VJ. The effect of striatal dopamine depletion and the adenosine A2A antagonist KW-6002 on reversal learning in rats. Neurobiol Learn Mem. 2007;88(1):75–81. Vanderschuren LJ, Schmidt ED, De Vries TJ, Van Moorsel CA, Tilders FJ, Schoffelmeer AN. A single exposure to amphetamine is sufficient to induce long-term behavioral, neuroendocrine, and neurochemical sensitization in rats. J Neurosci. 1999;19(21):9579–86. Taracha E, Czarna M, Turzyńska D, Maciejak P. Amphetamine-induced prolonged disturbances in tissue levels of dopamine and serotonin in the rat brain. Pharmacol Rep. 2023;75(3):596–608. Blum K, Bowirrat A, Sunder K, Thanos PK, Hanna C, Gold MS, et al. Dopamine Dysregulation in Reward and Autism Spectrum Disorder. Brain Sci. 2024;14(7):733. Sato M, Nakai N, Fujima S, Choe KY, Takumi T. Social circuits and their dysfunction in autism spectrum disorder. Mol Psychiatry. 2023;28(8):3194–206. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 03 Jul, 2025 Read the published version in Behavioral and Brain Functions → Version 1 posted Editorial decision: Revision requested 09 May, 2025 Reviews received at journal 08 May, 2025 Reviews received at journal 07 May, 2025 Reviews received at journal 06 May, 2025 Reviewers agreed at journal 01 May, 2025 Reviewers agreed at journal 01 May, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviewers agreed at journal 26 Apr, 2025 Reviewers invited by journal 26 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 23 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6006061\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":450775343,\"identity\":\"57321771-039c-4497-9a5c-923fdf1cee40\",\"order_by\":0,\"name\":\"Antonia Ramme\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital Carl Gustav Carus, Technische Universität Dresden\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Antonia\",\"middleName\":\"\",\"lastName\":\"Ramme\",\"suffix\":\"\"},{\"id\":450775346,\"identity\":\"9c026a4e-31ae-4b8f-a4c3-e6afab80524c\",\"order_by\":1,\"name\":\"Mirjam Zachow\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital Carl Gustav Carus, Technische Universität Dresden\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mirjam\",\"middleName\":\"\",\"lastName\":\"Zachow\",\"suffix\":\"\"},{\"id\":450775349,\"identity\":\"66f959c6-4857-46e2-b763-5a6e5617c22f\",\"order_by\":2,\"name\":\"Bettina Habelt\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital Carl Gustav Carus, Technische Universität Dresden\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Bettina\",\"middleName\":\"\",\"lastName\":\"Habelt\",\"suffix\":\"\"},{\"id\":450775353,\"identity\":\"551f20b4-0d19-456a-9400-a2c229d87374\",\"order_by\":3,\"name\":\"Iveta Vojtechova\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Institute of Mental Health\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Iveta\",\"middleName\":\"\",\"lastName\":\"Vojtechova\",\"suffix\":\"\"},{\"id\":450775354,\"identity\":\"f232f43c-53df-46f3-a2ea-e1d9a9c2eae3\",\"order_by\":4,\"name\":\"Tomas Petrasek\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Institute of Mental Health\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tomas\",\"middleName\":\"\",\"lastName\":\"Petrasek\",\"suffix\":\"\"},{\"id\":450775358,\"identity\":\"90c1704a-6377-4f38-acbb-2b7e7aa3c2a6\",\"order_by\":5,\"name\":\"Robert Waltereit\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universitätsmedizin Göttingen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Robert\",\"middleName\":\"\",\"lastName\":\"Waltereit\",\"suffix\":\"\"},{\"id\":450775360,\"identity\":\"49d251d8-b56a-48ac-9a84-0bfa303dfa14\",\"order_by\":6,\"name\":\"Nadine Bernhardt\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"\",\"institution\":\"University Hospital Carl Gustav Carus, Technische Universität Dresden\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Nadine\",\"middleName\":\"\",\"lastName\":\"Bernhardt\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-02-11 09:53:24\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6006061/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6006061/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s12993-025-00284-z\",\"type\":\"published\",\"date\":\"2025-07-03T15:58:31+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":81822713,\"identity\":\"49f98eeb-d51d-4cda-96fb-ef4ee35225f9\",\"added_by\":\"auto\",\"created_at\":\"2025-05-02 11:39:38\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":134518,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBehavioral testing sequence\\u003c/strong\\u003e. All \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e and wt rats were subjected to a sequential behavioral test battery including the nestlet shredding test (NST), marble burying test (MBT), light-sound-confinement test (LSC) and forced swim test (FST) to reveal lower order repetitive behavior and the water-T-maze test (WTM) to test for higher order repetitive stereotypies. The sucrose consumption test (SCT) and social recognition test (SRT) were performed to exclude that anhedonic tendencies influence obtained results and confirm the context of social impairment in our model (control). Finally, an amphetamine-induced stereotypic behavior test (AMP) was performed, followed by tissue collection (TC) for post-mortem analysis. Behavioral testing started when animals of both genders were 12 weeks of age.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6006061/v1/e13515ad72bf957e346159f5.png\"},{\"id\":81822074,\"identity\":\"a3c4dbf4-f52e-40a9-90fb-9b0a808250a7\",\"added_by\":\"auto\",\"created_at\":\"2025-05-02 11:31:38\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":602780,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003eTsc2\\u003c/strong\\u003e\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e\\u003cstrong\\u003e+/-\\u003c/strong\\u003e\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e Eker rats display increased lower-order repetitive behavior without higher-order repetitive abnormalities. A\\u003c/strong\\u003e In the marble burying test, no significant change in the number of marbles buried was observed between genotypes (\\u003cem\\u003ep\\u003c/em\\u003e = 0.589) or sex (\\u003cem\\u003ep\\u003c/em\\u003e = 0.382). \\u003cstrong\\u003eB \\u003c/strong\\u003eIn the nestlet shredding test, \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e rats showed significantly increased shredding behavior compared to wt (\\u003cem\\u003ep\\u003c/em\\u003e = 0.001). \\u003cstrong\\u003eC, D, C’, D’ \\u003c/strong\\u003eData obtained from a light-sound confinement test. During the pre-test habituation, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e rats showed significantly increased grooming (\\u003cem\\u003ep\\u003c/em\\u003e = 0.001). In case of digging, a significant sex effect was observed, with females displaying more digging behavior than males (\\u003cem\\u003ep\\u003c/em\\u003e = 0.001). Further, a significant effect of genotype was revealed (\\u003cem\\u003ep\\u003c/em\\u003e = 0.002). In the test phase, both genotypes exhibited minimal digging and grooming behavior. During post-test recovery, a significant interaction between sex and genotype was observed in case of grooming (\\u003cem\\u003ep\\u003c/em\\u003e = 0.004). \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e female rats showed increased grooming behavior compared to wt and \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e males. Genotype and sex did not affect digging behavior in the post-test phase (\\u003cem\\u003ep\\u003c/em\\u003e = 0.578). C´ and D´ show pre- and post-test phase with individual values. \\u003cstrong\\u003eE\\u003c/strong\\u003e In the FST, no significant effect of genotype (\\u003cem\\u003ep\\u003c/em\\u003e = 0.497) and sex (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.961) was observed. \\u003cstrong\\u003eF\\u003c/strong\\u003e No effect of genotype (\\u003cem\\u003ep\\u003c/em\\u003e = 0.163) or sex (\\u003cem\\u003ep\\u003c/em\\u003e = 0.978) was detected in the reversal learning paradigm of the WTM test. Depicted are the total number of trials needed on day 2. \\u003cstrong\\u003eG, G´\\u003c/strong\\u003e After systemic administration of amphetamine, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e animals did not show increased susceptibility to repetitive behavior induction (\\u003cem\\u003ep\\u003c/em\\u003e = 0.846). \\u003cstrong\\u003eG\\u003c/strong\\u003e Depicted is the mean score of behavior categories following classification as described in the methods section. \\u003cstrong\\u003eG´\\u003c/strong\\u003e Shown are the proportions of scoring classifications per genotype. Animals of all genotypes (male (m), female (f)) showed predominant behavior of only two categories: (1) limited exploratory activity (sniffing, rearing, grooming) and (2) locomotion, but no resting (0) or stereotypic behavior (3).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6006061/v1/2862116be17e275f3ec06b93.png\"},{\"id\":81822073,\"identity\":\"7a9a5503-8b83-4e5f-8bcf-598d90bb538d\",\"added_by\":\"auto\",\"created_at\":\"2025-05-02 11:31:38\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":550902,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEker rats present with changes in social interaction. A \\u003c/strong\\u003eNo significant genotype effect was observed in the social recognition test (\\u003cem\\u003ep\\u003c/em\\u003e = 0.353). However, \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e rats displayed no habituation in subsequent sessions and no enhancement of exploration upon novel social stimulation. \\u003cstrong\\u003eA´\\u003c/strong\\u003e Comparison of social interaction in trial 2 and 5. In trial 2, a significant effect of genotype was observed (\\u003cem\\u003ep\\u003c/em\\u003e = 0.003) with \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e showing decreased social interaction time compared to wt. \\u003cstrong\\u003eB \\u003c/strong\\u003eExperimental Scheme of the water T-Maze test. \\u003cstrong\\u003eC-E\\u003c/strong\\u003e T-Maze test performance revealed no genotype effect on spatial memory in initial learning (acquisition phase, C, \\u003cem\\u003ep\\u003c/em\\u003e = 0.052), repetition phase (D, \\u003cem\\u003ep\\u003c/em\\u003e = 0.321), and reversal test (E, \\u003cem\\u003ep\\u003c/em\\u003e = 0.163). However, in the acquisition phase, a significant interaction of sex and genotype was observed (\\u003cem\\u003ep\\u003c/em\\u003e = 0.012), as male \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/-\\u003c/em\\u003e\\u003c/sup\\u003e rats required significantly fewer trials than male wt rats (\\u003cem\\u003ep\\u003c/em\\u003e = 0.001).\\u003cstrong\\u003e F \\u003c/strong\\u003eIn the sucrose consumption test, no significant effect of genotype was detected (\\u003cem\\u003ep\\u003c/em\\u003e = 0.441). \\u003cstrong\\u003eG \\u003c/strong\\u003eGraphical summary of altered ASD core domains in the Eker rat model.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6006061/v1/49eb16b7ad085e82f22dd36f.png\"},{\"id\":81822075,\"identity\":\"396cd058-40a0-4c0f-992c-19769ca32629\",\"added_by\":\"auto\",\"created_at\":\"2025-05-02 11:31:38\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":213218,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEker rats show altered dopamine metabolism in the PFC. A,B \\u003c/strong\\u003eHPLC analysis of dopamine (DA) levels in both striatum (CPu) and PFC. \\u003cstrong\\u003eA\\u003c/strong\\u003e DA levels in the CPu were not significantly altered in \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e rats compared to wt (\\u003cem\\u003ep\\u003c/em\\u003e = 0.484). \\u003cstrong\\u003eB\\u003c/strong\\u003e \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e rat PFC tissue showed significantly increased levels of DA (\\u003cem\\u003ep\\u003c/em\\u003e = 0.049). \\u003cstrong\\u003eC,D\\u003c/strong\\u003e DA turnover based on HPLC analysis of dopamine metabolites DOPAC and HVA in both CPu and PFC. \\u003cstrong\\u003eC\\u003c/strong\\u003e DA turnover in the CPU was not significantly altered in \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e rats compared to wt (\\u003cem\\u003ep\\u003c/em\\u003e = 0.61). \\u003cstrong\\u003eD\\u003c/strong\\u003e In PFC tissue a significant interaction of sex and genotype was observed, as male \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e rats showed significantly increased DA turnover compared to wt and female \\u003cem\\u003eTsc2 \\u003c/em\\u003e\\u003csup\\u003e+/-\\u003c/sup\\u003e animals (\\u003cem\\u003ep\\u003c/em\\u003e = 0.01). No outliers were detected in the dataset by applying a two-sided Grubbs´test (α = 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6006061/v1/535a51c889f19535d0df4c30.png\"},{\"id\":86179448,\"identity\":\"e5aade27-c8a5-4d12-9784-98d7c2af2ea4\",\"added_by\":\"auto\",\"created_at\":\"2025-07-07 16:17:39\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2454244,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6006061/v1/81e1e47d-2144-442d-9834-3ce43d558432.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Behavioral phenotyping identifies autism-like repetitive stereotypies in a Tsc2 haploinsufficient rat model\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eAutism Spectrum Disorder (ASD) is a neurodevelopmental disorder defined by deficits in two core behavioral domains: social interaction and repetitive behavior patterns (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). Despite a high global prevalence of approximately 1% (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e), efficacious drugs are missing, and early behavioral interventions remain the most promising treatment option (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). The biggest impeding factor for drug development remains ASD\\u0026acute;s complex and incompletely understood etiology. Disease pathogenesis arises from a multifaceted interaction between a range of genetic and environmental factors that together give rise to a wide symptom heterogeneity across ASD patients. Moreover, ASD is linked to over 100 candidate genes and a myriad of genetic risk factors like chromosomal rearrangements and copy number variations with mainly small to moderate effect sizes (\\u003cspan additionalcitationids=\\\"CR6 CR7 CR8\\\" citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e), thus posing additional challenge to generate disease models with high translational accuracy.\\u003c/p\\u003e \\u003cp\\u003eSimilarly, different transgenic ASD rodent models exhibit unique sets of behavioral alterations that fit the high aetiologic and symptomatic heterogeneity in humans (\\u003cspan additionalcitationids=\\\"CR11\\\" citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e). While social behavior is well-described in most ASD rodent models, characterizations of repetitive behavior profiles are less commonly reported. Repetitive behaviors in ASD patients include a range of motor patterns divided into a) \\u0026ldquo;lower order\\u0026rdquo; (stereotyped or repetitive motor movements), b) more complex \\u0026ldquo;higher order\\u0026rdquo; responses (compulsions, insistence on sameness, cognitive inflexibility and adherence to routine) and c) highly restricted interests such as unusual object fixation (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e). In rodent ASD models, strain-specific repetitive patterns can be revealed by applying combinations of behavioral tests that cover multiple response categories.\\u003c/p\\u003e \\u003cp\\u003eCommonly used models are often based on single-gene disorders associated with ASD such as Tuberous Sclerosis (TSC) and Fragile X syndrome (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e), or high-effect risk genes like \\u003cem\\u003eSHANK3\\u003c/em\\u003e, \\u003cem\\u003eNLGN3\\u003c/em\\u003e, and \\u003cem\\u003eNLGN4\\u003c/em\\u003e, as they offer high construct validity and thereby increase relevance (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR17\\\" citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e). Among genetic aberrations reported in ASD, the \\u003cem\\u003eTSC2\\u003c/em\\u003e gene remains one of the major contributors (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). TSC is an early-onset, monogenic disorder caused by loss-of-function mutations in the \\u003cem\\u003eTsc1\\u003c/em\\u003e or \\u003cem\\u003eTsc2\\u003c/em\\u003e gene. Patients present with autism-like neurobehavioral phenotypes including social deficits and epileptic seizures (\\u003cspan additionalcitationids=\\\"CR21\\\" citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e), and are co-diagnosed with ASD in up to 69% of cases, suggesting genetic linkage of ASD and TSC (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). While the connection between genetic and anatomical alterations is not yet fully understood, molecularly, \\u003cem\\u003eTsc1\\u003c/em\\u003e and \\u003cem\\u003eTsc2\\u003c/em\\u003e mutations are known to result in overactive mTORC1 (mammalian target of rapamycin complex 1), thereby interfering with metabolic processes including cell growth, proliferation, differentiation, and protein synthesis (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e). Hyperactive mTOR signaling is associated with impaired neurodevelopment, synaptic plasticity, and signaling, and thus strongly correlates to neurodevelopmental diseases like epilepsy and ASD (\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rat model carries a spontaneous germ line mutation of the \\u003cem\\u003eTsc2\\u003c/em\\u003e gene (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e), making it a valuable tool for studying TSC etiology and its implications in ASD (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR30\\\" citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e). Reportedly, Eker rats recapitulate brain abnormalities and cellular pathology present in human TSC (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR33\\\" citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e), show altered synaptic plasticity (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e), and present with mild but consistent autism-like social impairments (\\u003cspan additionalcitationids=\\\"CR36\\\" citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). The observed social deficits were found to improve upon selective inhibition of mTORC1 in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals, supporting hyperactive mTORC1 signaling as a potential driver of behavioral manifestations in ASD (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). However, along with concerns about absent spontaneous seizure activity, the lack of repetitive behavior characterization sparks debate about the suitability of the Eker rat as an ASD model (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTherefore, the present study aimed to provide an extended characterization of ASD-like repetitive behavior in Tsc2+/- Eker rats by exposing animals to a test battery addressing both lower and higher order repetitive phenotypes to strengthen the model\\u0026rsquo;s translational value in ASD research.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimals\\u003c/h2\\u003e \\u003cp\\u003eAll experiments were performed in the heterozygous Eker rat strain with \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e+/\\u0026minus;\\u003c/sup\\u003e mutation on a Long-Evans background, RRID:RGD_625624 and their wild-type (wt, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/+\\u003c/em\\u003e\\u003c/sup\\u003e) littermates which were used as control. Animals were bred at and obtained from the National Institute of Mental Health in Klecany, Czechia at 8 weeks of age. Acclimatization and daily handling were performed for 4 weeks prior to behavioral experiments. The animals were housed in pairs of 2\\u0026ndash;3 in standard housing cages (Makrolon\\u0026reg;, Type IV-S, Tecniplast Deutschland GmbH, Hohenpei\\u0026szlig;enberg, Germany) in a controlled environment of 21\\u0026ndash;24\\u0026deg;C and an average humidity of 55% under an automated 12 h/12 h day-night cycle. Food and water were provided \\u003cem\\u003ead libitum\\u003c/em\\u003e. Experiments took place during the late-day phase and animals\\u0026rsquo; weight was controlled twice a week.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eBehavioral testing\\u003c/h3\\u003e\\n\\u003cp\\u003eBehavioral experiments started when animals were 12 weeks of age, in accordance with relevant literature on social behavior of \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e). A total of 11 \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e and 11 wt rats was used and subjected to the behavioral testing sequence and following post-mortem analysis. Sample-size was calculated a-priori using G*Power. Genotype groups included both male (\\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;6) and female (\\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;5) animals, respectively. Experiments were conducted in two separate birth cohorts, with sex and gender being counterbalanced across cohorts. Experimental testing and analysis were performed by trained and blinded experimenters. A broad behavioral test battery assessing both lower (marble burying test, nestlet shredding test, light-sound-confinement test, forced swim test) and higher order (water-T-maze test) repetitive behavior was used. Additional readouts for social behavior (social recognition test), reward function (sucrose consumption test) and cognition (water-T-maze test) were collected to report repetitive behavior alterations in the context of already known abnormalities. Behavioral tests were performed in a fixed sequence depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, allowing for a minimum of 24 h resting period between tests to limit stress and potential carryover effects.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eNestlet shredding test\\u003c/h3\\u003e\\n\\u003cp\\u003eThe nestlet shredding test was used to detect lower-order repetitive behavior in non-stressful conditions (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). Animals were placed individually in a standard cage containing bedding material, fresh nestlet material, and a square tissue paper (5 cm \\u0026times; 5 cm, weighing 2.5 g) in the opposite corner. The test was conducted for 30 minutes without food or water supply before the animals were returned to their home cage. The remaining unshredded nestlet material was then weighed (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003eMarble burying test\\u003c/h3\\u003e\\n\\u003cp\\u003eThe marble burying test was used to detect lower-order motor stereotypy in form of repetitive digging behavior in non-stressful conditions (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e). Animals were placed individually in a standard cage with bedding material filled up to 5 cm height, in which 9 clean, commercially available glass marbles (approx. 15 mm diameter) were evenly distributed on half of the cage\\u0026acute;s surface. Animals were allowed to explore for 30 minutes without food and water supply, after which they returned to their home cage and the number of buried marbles that were at least 2/3 covered in bedding material was counted (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003eLight-sound-confinement test\\u003c/h3\\u003e\\n\\u003cp\\u003eA light-sound-confinement test (LSC) was performed to assess lower-order repetitive digging and grooming behavior in stressful conditions through the application of combined auditory and visual stress. The animals were placed in a plexiglass tube (30 cm height and 12 cm diameter) (Stoeltus Co., Ireland) fixed on a standard cage and the tube was fitted with a commercially available loudspeaker (SoundCore Select 2 Bluetooth Loudspeaker, Anker, Germany) and a light source (KL 1600 LED, Schott AG, Germany). Behavior of the rats was filmed from two sides using ManyCam (Visicom Media Inc.) for 30 min in total. For the first 10 min, activity of the animals was recorded without audiogenic or visual stressors (pre-test phase). During the following 10 min test phase, animals were exposed to both visual and auditory stressors. Light was applied at a maximum of 1400 lux combined with white noise bursts (100 dB SPL, 50 ms) (programmed with Audacity Version 2.3.0 and Matlab Version R2019a), played with random interstimulus intervals ranging between 10\\u0026ndash;20 s. After removal of both stressors, the activity of the animals was recorded for another 10 min (post-test phase). Total grooming time and digging time were analyzed for each phase (\\u003cspan additionalcitationids=\\\"CR42\\\" citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eForced swim test\\u003c/h2\\u003e \\u003cp\\u003eThe forced swim test (FST) was conducted to measure active coping strategies in stress response (\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e). Further, response behavior was assessed for repetitive patterns. For habituation, animals were placed in a glass cylinder (60 cm height, 24 cm diameter) filled with 23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C water to 30 cm height for 15 min individually. Afterward, animals were dried under a red-light source (SIL-6 red light lamp Beurer, Hans Dinslage GmbH, Germany) and placed back in their home cage. After 24 h, animals were tested in a water height of 40 cm and activity was recorded for 5 min. The total time of immobility per animal was determined using the software EthoVision\\u0026reg; XT (Noldus Information Technology, Netherlands) (\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e). Additionally, potential repetitive climbing patterns were manually assessed by a trained experimenter and potential uninterrupted repeated circular swimming was evaluated in EthoVision\\u0026acute;s heat map feature.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eWater-T-maze\\u003c/h3\\u003e\\n\\u003cp\\u003eThe water-T-maze (WTM) test was conducted to detect higher-order repetitive behavior as well as assess learning and memory (\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e). The experiment was conducted in a T-shaped pool (Maze Engineers, Conduct Science\\u0026copy;, USA) filled with 23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C opaque water (XSL titanium white, Kremer pigments, Germany), which consisted of a long stem (L 70 cm, W 15.5 cm, H 46 cm) that bifurcates into two arms (L 121 cm \\u0026times; W 15.5 cm \\u0026times; H 46 cm). A transparent platform (15.5 cm x 15.5 cm, 24 cm height) was randomly placed in one of the two arms of the T-maze and was submerged 1 cm below water level. On day one (acquisition phase), animals were trained to find the platform position in a fixed arm. Animals were placed in the starting arm and allowed to choose either direction after reaching the end of the stem. Once an animal reached either arm within a test period of 60 s, the arm was closed with a plexiglass door (47 cm \\u0026times; 20 cm), confining the animal for 5 s before removal to a holding cage for 10 s and subsequent test repetition. The training was repeated until the animal chose the correct arm, and escaped onto the platform, in five consecutive trials. On day two, 24 h later, animals were retrained by repeating the previous day\\u0026acute;s test procedure (repetition phase). Once position learning was completed, the platform was switched to the opposite arm of the T-maze and the test procedure was repeated as described (reversal test). During each holding period outside of the maze, animals were dried under a red-light source (SIL-6 red light lamp Beurer, Hans Dinslage GmbH, Germany). The number of attempts required by each animal to make five consecutive correct decisions was recorded; the maximum number of trials was limited to 25 per day.\\u003c/p\\u003e\\n\\u003ch3\\u003eSucrose consumption test\\u003c/h3\\u003e\\n\\u003cp\\u003eThe sucrose consumption test was performed to assess reward function and depressive episodes as reported previously (\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e). Animals were habituated to a standard bottle of sweetened condensed milk (Nestl\\u0026eacute;, Milchm\\u0026auml;dchen, (1:3)) in their home cage for 30 min. After 24 h, animals were habituated to the single housing standard cage type III (Makrolon\\u0026reg;, Tecniplast Deutschland GmbH, Germany) for 30 min and subsequently food restricted for 21 h (15 g per animal). On the day of testing, animals were exposed to the bottle containing sucrose solution for 10 min. Bottle weight was recorded before and after the test phase. Animals were weighed on each test day as well as the day after sucrose consumption (\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSocial recognition test\\u003c/h2\\u003e \\u003cp\\u003eThe social recognition test was used to evaluate sociability towards an unknown partner rat. Animals were kept in isolation for three consecutive days and were familiarized with the test box (45 cm \\u0026times; 45 cm, LE802S Panlab square arena, S.L.U. Panlab, Spain) for 1 h every day. On the test day, each experimental animal was presented with an unfamiliar sex- and age-matched conspecific of the same strain and placed in a locked startle box (Grid Rod Animal Holder, OCB Systems Ltd., United Kingdom) with openings for nose-to-nose contact. Experimental animals were allowed to explore or interact for a test period of 60 s in a total of four trials with the same social-partner animal in intervals of 10 min. In a fifth trial, experimental animals were presented with a novel social partner animal. Following each trial, the social-partner animal was returned to their home cage for the 10 min interval time, and the startle box was disinfected. All trials were videotaped and the examination time spent in close proximity was manually determined (\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAmphetamine-induced stereotypic behavior test\\u003c/h2\\u003e \\u003cp\\u003eAn amphetamine-induction test was conducted to characterize the effect of amphetamine on repetitive behavior stereotypies. Animals were injected intraperitoneally with 2.0 mg amphetamine (Lipomed AG, Switzerland) per kg body weight, based on the emergence of amphetamine-induced stereotypy in wt animals above doses of 2 mg/kg (\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e). Rats were placed in a testing box (46.5 \\u0026times; 46.5 cm \\u0026times; 44 cm) and video-recorded for 2 h. Behavior following amphetamine application was analyzed in 5 min intervals, and the most prominent behavior was scored for each interval. Scoring was performed based on an adapted protocol described in Kelly et al. (1975) (\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e) by dividing behavior in (0) no locomotor activity (sleeping, residing without apparent sniffing) (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e) limited exploratory activity (discontinuous sniffing, rearing or grooming) (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e) locomotion (frequent rearing, sniffing) and (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e) stereotypic behavior (repetitive grooming, rearing and licking). Scoring was performed manually by a single trained and blinded experimenter. Blinding was achieved by number-coding animals for video-analysis to eliminate genotype-bias.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePost-mortem High-Performance Liquid Chromatography\\u003c/h2\\u003e \\u003cp\\u003eAnimals were intraperitoneally anesthetized using pentobarbital (60 mg/kg) and decapitated. Whole brains were extracted and frozen in methylbutane for 2 min at -20 to -40\\u0026deg;C and stored at -80\\u0026deg;C. Bilateral micro-punches (\\u0026Oslash; 1 mm) of prefrontal cortex (PFC) and striatum (caudate putamen, CPu) tissue were homogenized in 500 \\u0026micro;l 0.1 M perchloric acid by ultrasonication that was applied for 3 \\u0026times; 10 s on ice. After protein quantification (PierceTM 660 nm Protein Assay; Thermo Fisher Scientific Inc., USA), homogenates were centrifuged for 15 min at 13,000 g at 4\\u0026deg;C. The supernatant was separated via HPLC (1260 Infinity II LC System, OpenLab LC ChemStation Software, Agilent Technologies, USA) on a PRONTOSIL 120-5-C18SH (VDS Optilab, Germany) analytical column followed by electrochemical detection (Coulochem III, Thermo Fisher Scientific Inc., USA) of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels as \\u0026micro;g/g protein. Dopamine turnover was calculated from concentration levels, adjusted to the protein content of the sample, as neurotransmitter to metabolite ratio (DA/(DOPAC\\u0026thinsp;+\\u0026thinsp;HVA)).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eStatistical analysis was performed using GraphPad Prism 9.5.1. All behavioral tests as well as HPLC measures were analyzed using a two-way analysis of variance (ANOVA) for the main effect of genotype (wt, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e+/\\u0026minus;\\u003c/sup\\u003e Eker) and sex (male, female). In consideration of our small dataset we did not test for normality but relied on the visual inspection of qq-plot for residuals and the robustness of ANOVA, to violations of normality, especially when the sample sizes are small and equal across groups (\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e). Tukey\\u0026rsquo;s HSD was used to adjust for multiple post hoc comparisons. No animals were excluded from analysis. Statistical significance was set at \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eTsc2\\u003c/b\\u003e \\u003cb\\u003ehaploinsufficiency increases lower-order repetitive behavior patterns\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo determine if the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e genotype induces ASD-associated behavior in rats, a set of behavioral tests was performed on the Eker model to reveal repetitive behavior patterns.\\u003c/p\\u003e \\u003cp\\u003eFirst, lower-order restricted behavior was observed under non-stressful conditions using the nestlet shredding and marble burying test. Both behavioral tests are commonly used to reveal stereotypic repetitive behavior (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e). While no differences in marble-burying behavior were observed between wild-type (wt) and \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA), \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats displayed significantly increased nestlet shredding behavior (\\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;16.39, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB), indicating lower-order repetitive behavior in Eker rats.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eSince repetitive behavior represents a strategy to deal with stress and anxiety, next, stereotyped movements were assessed under stressful conditions. The Light-Sound-Confinement test was utilized to investigate repetitive digging and grooming behavior under the application of multiple stressors. First, animals were subjected to the pre-test phase, in which they adjusted to the novel test environment in the absence of additional stressors. \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats showed significantly increased grooming behavior compared to wt (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, C\\u0026acute;; \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;23.47, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.001), further suggesting repetitive behavioral abnormalities in the Eker rat model. Conversely, digging time was significantly reduced in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats compared to wt in the pre-test phase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, D\\u0026acute;; \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;13.43, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.002). Notably, a significant sex effect was observed in the case of digging in the pre-test phase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, D\\u0026acute;; \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;47.92, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.001), as female rats displayed higher levels of digging behavior than males.\\u003c/p\\u003e \\u003cp\\u003eNext, animals were subjected to the test phase, where unpredictable visual and acoustic stressors were introduced to elevate stress levels. Neither genotype demonstrated grooming behavior nor digging behavior during stressor application (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, D). Interestingly, a sex and genotype interaction for grooming was observed during recovery in the post-test phase as female \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats displayed significantly increased grooming behavior compared to male \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e and wt animals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, C\\u0026acute;; \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;11.22, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.004). In contrast, digging behavior did not significantly differ between genotypes or sex during recovery in the post-test phase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, D\\u0026acute;). However, animals might need more time to recover from the stress phase, thus the occurrence of a genotype effect at later timepoints cannot be excluded.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, while \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals were found to display lower-order repetitive behavior in the form of increased grooming in non-stressful conditions, high-stress application in the Light-Sound-Confinement (LSC) test did not exacerbate repetitive behavior patterns in the Eker rat. However, female \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats might recover faster following high-stress levels and showed higher compulsive grooming behavior compared to wt. No repetitive digging behavior was found in the Eker rat model.\\u003c/p\\u003e \\u003cp\\u003eTo test whether \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats show altered mobility as a stress response, animals were subjected to the forced swim test (FST). The total time of immobility did not differ significantly between \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e and wt animals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE), suggesting that stress-coping responses involving mobility are not altered in the Eker rat model. Additionally, no repetitive behavior that could present as repetitive circling or climbing was detected in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats.\\u003c/p\\u003e \\u003cp\\u003eThe presence of higher-order repetitive behavior was investigated using the water T-maze test (WTM), as its reversal learning paradigm reveals an animal\\u0026acute;s ability to switch behavioral strategies. Higher order repetitive behavior would have presented as perseverative behavior by not successfully completing the task criterion, a consistent choice of novel platform location, thus, animals would reach the maximum number of trials on day 2 of the WTM. Instead, both wt and \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats completed the task successfully with no significant difference (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF). Both recalling of position habit learning (repetition phase) and finding an unknown platform position (reversal phase) showed no significant difference in attempts needed to fulfill criterion (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, E). Thus, unaltered performance in the WTM suggests that the Eker rat does not demonstrate a significant lack of behavioral response variability and therefore does not display higher-order repetitive behavior.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eStress-induced repetitive behavior is thought to act as anxiety relief by activating the dopaminergic reward system, thereby representing an important strategy for emotional regulation in ASD and obsessive-compulsive disorder models (\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e). In fact, abnormal lower-order repetitive stereotypies are associated with overactivation of the dopaminergic cortical basal ganglia circuitry (\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e). Accordingly, treatment with the indirect dopamine agonist amphetamine exacerbates repetitive motor stereotypies and can reveal underlying changes in the dopamine (DA) system in rodent models (\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e). Therefore, we opted to investigate whether administering amphetamine at a dosage of 2 mg/kg would reveal signs of repetitive behavior in Eker rats. To determine if the Eker rat model presents with altered DA function and connected occurrence of repetitive behavior, an amphetamine challenge protocol was applied. Behavioral categorization using an adapted scoring protocol from Kelly et al. (1975) (\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e) revealed that both wt and \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals mainly presented with limited (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e) and frequent exploratory behavior (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e) (rearing, sniffing, grooming) but did not exhibit repetitive stereotypic behavior (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e) (repetitive rearing and grooming) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG). A mean behavioral categorization score was calculated to show the overall locomotive activity of sex and genotype. Repeated ANOVA measures did not reveal significant effects of sex on behavior categories, so data from males and females were combined. No significant effect on the extent and type of locomotion was found between wt and \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals after amphetamine injection (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG, G\\u0026acute;). Thus, the application of amphetamine provoked unaltered responses in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals and did not induce repetitive behavior.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, the Eker rat model was found to display lower-order repetitive behavior alterations under low-stress conditions in the form of excessive nestlet shredding and grooming. Surprisingly, the application of stressors did not exacerbate repetitive behavior patterns. Furthermore, no higher-order repetitive behavior was detected, indicating sufficient behavioral flexibility in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats. Lastly, the Eker model seems to display wt-like behavioral responses following manipulation of the dopamine system through amphetamine administration, suggesting no major alterations of the DA system to be present.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eRepetitive behavior alterations in the Eker rat are observed in a context of impaired social behavior\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eApart from repetitive stereotypies, ASD presents with deficits in social behavior and communication, as well as cognitive impairment and behavioral inflexibility (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e). While autistic-like social behavior has been reported in Eker rats, learning and memory performance was found to be unaltered, suggesting normal cognitive function (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTo validate that \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats show previously reported deficits in social interaction, first, animals were challenged with a social recognition test in which exploration time with an unfamiliar rat was assessed. Eker rats showed overall less interaction duration compared to wt during three out of five trials (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), with a significantly decreased interaction time during the second trial (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA\\u0026acute;, \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;12.47, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.003), confirming the previously reported general decrease in social interest. Wt animals expectedly displayed variety in social exploratory behavior, with a gradual decrease in interaction during the first four trials and an increase in interaction upon the introduction of a novel actor rat in trial 5. In contrast, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals were found to maintain similar levels of interaction time across all trials, independent of actor rat familiarity or novelty (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), suggesting restricted social interest in Eker rats.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eTsc2\\u003c/em\\u003e haploinsufficiency was found to cause changes in activity-dependent hippocampal synaptic plasticity (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e). To assess whether Eker rats show signs of impaired spatial memory, which is dependent on hippocampal learning, animals were challenged with the water T maze test (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB-E). During the acquisition phase, a significant interaction of sex and genotype was observed (\\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;4.353, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.012) as male \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats needed significantly fewer trials than wt males to learn the correct arm position (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC; \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.001). However, no negative effect of the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u003c/em\\u003e\\u003c/sup\\u003e genotype on learning speed was observed, suggesting that learning is not impaired in Eker rats. In the repetition test, 24 hours later, wt and \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals found the correct arm position equally well (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). Similarly, no significant differences in number of trials needed to criterion were detected between genotypes in the reversal test (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). These results indicate that spatial memory formation is not affected in Eker rats and are in line with previous reports (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTo test if alterations in reward function might influence behavioral test results, a sucrose preference test was performed. No difference between genotypes nor interaction effects have been observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF), indicating that \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals show intact reward function and do not develop anhedonic tendencies.\\u003c/p\\u003e \\u003cp\\u003eIn summary, Eker rats show signs of social behavior impairment in the form of abnormal social interest. On the contrary, spatial learning and memory as well as reward function remained unaffected by \\u003cem\\u003eTsc2\\u003c/em\\u003e haploinsufficiency. Herein, we provide evidence that the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animal model of ASD presents with an overt repetitive behavior pattern that occurs alongside social behavior impairments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG, G\\u0026acute;).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEker rats show evidence for impaired PFC-dependent dopaminergic signaling\\u003c/h2\\u003e \\u003cp\\u003eNext, we opted to explore neurochemical alterations in the brain of \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats by determining DA levels in post-mortem brain tissue. Despite absent changes in amphetamine response after systemic administration, the dopaminergic pathway remains a promising target due to its strong connection to repetitive behavior formation and general movement modulation (\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e). An important DA target is the cortico-striatal-thalamo-cortical (CSTC) pathway, a neuronal circuit that is crucial for movement selection and initiation (\\u003cspan additionalcitationids=\\\"CR58\\\" citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e) and is implicated in influencing stereotyped behavior such as repetitive self-grooming and digging in rodents (\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e). Thus, we analyzed neurotransmitter levels in the striatum (CPu) and prefrontal cortex (PFC), as both are key areas regulating and modulating CSTC activity and function and are linked to stereotyped behavior initiation (\\u003cspan additionalcitationids=\\\"CR63\\\" citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e). Therefore, region-specific HPLC for dopamine (DA), and downstream DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was performed. While no significant alteration of DA level was found in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rat CPu tissue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA), significantly increased levels of DA were present within the PFC of \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals compared to wt (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB; \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;4.478, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.0485). Similarly, no changes in DA turnover were observed in CPu tissue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). In the PFC, a significant sex x genotype interaction revealed a significantly decreased DA/(DOPAC\\u0026thinsp;+\\u0026thinsp;HVA) ratio in male \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats compared to female \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e and wt rats (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD; \\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;8.5, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.01), suggesting significantly increased DA turnover in male \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals. Overall, a significant effect of genotype was found, revealing an increased DA turnover rate in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats compared to wt (\\u003cem\\u003eF\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e)\\u0026thinsp;=\\u0026thinsp;15, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.001). Thus, alterations in PFC-specific dopamine metabolism might contribute to social and repetitive behavioral alterations in the \\u003cem\\u003eTsc2\\u003c/em\\u003e haploinsufficient Eker rat.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eRestricted repetitive behavior is one of the core pathological features of ASD and presents with high phenotypic symptom heterogeneity. Animal models of ASD are expected to offer construct validity, e.g., in the form of associated genetic mutations, and recapitulate characteristic key manifestations. Here, using a series of behavioral tests accounting for a spectrum of stereotyped behavior, we sought to characterize lower-order and higher-order repetitive behavior of the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e haploinsufficient Eker rat. Importantly, we show that \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats demonstrate a moderate phenotype of ASD-like behavior in the repetitive behavior domain involving increased lower-order motor stereotypies but no higher-order repetitive behavior, which was accompanied by a PFC-specific increase in DA levels.\\u003c/p\\u003e \\u003cp\\u003eWhile the Eker rat strain has been postulated as a model for ASD and ASD-like behavior (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e), to our knowledge, this is the first study to evaluate the repetitive behavior pattern in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. Since the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker strain offers high construct validity and was indicated to present with social deficits, we hypothesized that Eker rats additionally develop repetitive stereotypies that are typically exacerbated under stress. Reported deficits include reduced novel object recognition and environmental exploration in the open field paradigm, as well as decreased social exploration in the form of non-anogenital (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e) and anogenital exploration (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). Interestingly, Petrasek et al. (2021) (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e) further reported alterations in ultrasonic vocalization in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e pups, suggesting phases of impaired social communication which is consistent with ASD characteristics in humans. A study on hippocampal plasticity reported a significant decrease in synaptic modification capacity in the form of reduced LTP and LTD amounts (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e). While this finding implies an impact on cognitive function, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals were found to display unimpaired learning abilities in a Morris water maze paradigm (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e), suggesting that molecular and functional changes are subtle and do not elicit a major behavioral impact (\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). Other TSC rodent models have been more thoroughly assessed regarding ASD-like behavior. \\u003cem\\u003eTsc1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e mice showed deficits in social interaction and impaired hippocampal learning revealed through the Morris water maze task and a fear conditioning test (\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e). In a mouse model with Purkinje cell-specific \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e heterozygous genotype, repetitive digging behavior (marble burying test) and decreased social exploration, but no alterations in learning and memory were detected (\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e). Interestingly, ASD symptom manifestations overall seem to be highly strain-dependent, as several heterozygous TSC rodent models present with specific combinations of social deficits, cognitive impairment and repetitive behavior. However, while social interaction and learning are commonly assessed domains in TSC rodent models, repetitive behavior analyses remain less frequently reported.\\u003c/p\\u003e \\u003cp\\u003eASD-related rodent studies have focused on fixated action patterns such as obsessive self-grooming, digging, and nestlet shredding to quantify lower-order repetitive behavior (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e). Here, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats were found to present with lower-order stereotypies in the form of increased nestlet shredding and self-grooming behavior. Conversely, no excessive digging was revealed in the marble burying test or occurred spontaneously. Instead, Eker rats spent significantly less time digging than wt rats. This contradictory finding fits the highly heterogeneous behavioral profiles of both human patients and rodent ASD models (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e). Some do not manifest repetitive behavior at all (\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e), others show anomalies only in specific behavioral tests. \\u003cem\\u003eShank3\\u003c/em\\u003e knockout mice, as an established ASD model, manifest repetitive behavior in the form of increased grooming and nestlet shredding but show decreased digging and marble-burying behavior (\\u003cspan additionalcitationids=\\\"CR71\\\" citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e). Similar patterns can be found in \\u003cem\\u003eShank2\\u003c/em\\u003e mutant lines (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e). Genetic factors exert great influence on innate digging behavior, thus, strain-variations in digging responses are common (\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e74\\u003c/span\\u003e). Although excessive digging is commonly used as a measure for repetitive stereotypies, it can also be interpreted as an indicator of exploratory drive, depending on the motivational background (\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e). Thus, reduced digging, especially in the habituation setting of this study, may correspond to a decrease in explorative behavior, which has been previously reported in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAcute stress is known to play a role in altering repetitive behavior patterns, such as increasing grooming behavior (\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR76\\\" citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e75\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR77\\\" class=\\\"CitationRef\\\"\\u003e77\\u003c/span\\u003e). Thus, repetitive behavior was evaluated under stressful conditions in a here-described LSC test and the FST. Interestingly, the application of stress did not exacerbate repetitive behavior patterns in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. Further, we report the absence of higher-order repetitive abnormalities in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. Higher-order repetitive behaviors are often modeled using reversal learning paradigms such as the water T-maze or Morris water maze test (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e). Animals are trained to prefer correct-rewarded over incorrect-unrewarded choices, after which the choice and reward relation is reversed. The number of trials needed to switch to the new correct-reward association is meant to reflect cognitive flexibility and insistence on sameness (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e). Together, Eker rats were found to present with mild changes in stereotypic behavior but seem to lack a higher-order repetitive behavior domain. Several ASD rodent models (C58 and BTBR T\\u0026thinsp;+\\u0026thinsp;tf/J mice) do not manifest with apparent cognitive inflexibility as they show unaltered responses in classical reversal learning paradigms. However, only when using a probabilistic reversal learning test approach with a reward rate of 80% instead of 100%, which is commonly used to model cognitive inflexibility in ASD patients, significant differences became apparent (\\u003cspan additionalcitationids=\\\"CR79\\\" citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e78\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e80\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eImportantly, we found that alterations in lower-order repetitive behavior in the Eker rat occur alongside previously reported social and cognitive behavioral patterns. Here, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals showed mild impairments in the social recognition test, confirming deficits in the social domain of ASD manifestations (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). Interestingly, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats maintained similar interaction time throughout all trials, while wt rats expectedly decreased the interaction time during the first four trials with the same partner and increased interaction time upon the introduction of a novel partner. Changes in the social recognition test can arise for several reasons, such as olfactory system impairments or locomotion deficits (\\u003cspan additionalcitationids=\\\"CR82\\\" citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR83\\\" class=\\\"CitationRef\\\"\\u003e83\\u003c/span\\u003e). Additionally, decreased social interaction could be attributed to generalized alterations in exploratory drive that need to be specifically addressed. Potential cross-target effects should be excluded to increase the robustness of social deficit findings in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. The social recognition test is thought to reflect both social interest and short-term recognition memory in rodent studies (\\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e). Thus, our results can be interpreted as both impaired social interest and target discrimination in Eker rats, as well as altered short-term recognition memory suggesting an impact on cognitive function. Here, we show that the Eker model does not present significant changes in spatial learning and memory, as assessed in the WTM test. Our results fit previous reports showing typical learning and spatial memory in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals in the Morris water maze, radial maze, and conditioned taste aversion test (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e). Contrary, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals surprisingly show increased performance compared to wt upon modification to a delayed matching-to-place task meant to model episodic-like memory by adding a 2 h interval between trials (\\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e). However, improved performance in an episodic memory task can also be interpreted as a cognitive disadvantage; that is, Eker rats might perform better in finding novel platform locations due to a lack of memory formation of previous platform locations (\\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e). The entirety of observed brain malformations, cellular aberrations, and impairments of neuroplasticity suggest cognitive deficits to be present in the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rat (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). However, behavioral assessments so far have failed to show robust impairments in cognition. As discussed previously, applying higher task difficulty in the form of a probabilistic reversal learning test might be more sufficient to reveal higher-order repetitive behavior and cognitive inflexibility.\\u003c/p\\u003e \\u003cp\\u003eGiven the growing understanding of sex discrepancies in ASD symptom manifestation (\\u003cspan citationid=\\\"CR85\\\" class=\\\"CitationRef\\\"\\u003e85\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR86\\\" class=\\\"CitationRef\\\"\\u003e86\\u003c/span\\u003e), sex effects are important to consider in ASD models. Thus, repetitive behavior alterations were assessed in both male and female \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. While little is known about sex-specific differences in ASD models, available data remains inconsistent with variance between animal strains and specific tasks (\\u003cspan citationid=\\\"CR87\\\" class=\\\"CitationRef\\\"\\u003e87\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR88\\\" class=\\\"CitationRef\\\"\\u003e88\\u003c/span\\u003e). For example, the BTBR ASD model was found to present with repetitive grooming and marble burying in male but not female mice (\\u003cspan citationid=\\\"CR89\\\" class=\\\"CitationRef\\\"\\u003e89\\u003c/span\\u003e), while repetitive grooming in Nlgn4 ASD mice appears in female animals exclusively (\\u003cspan citationid=\\\"CR90\\\" class=\\\"CitationRef\\\"\\u003e90\\u003c/span\\u003e). Here, we report a significant effect of sex before stress application in the LSC test, with increased digging behavior in female compared to male animals. Additionally, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e females were found to show increased grooming behavior compared to both wt and males. In a \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e mouse model, female animals exhibited deficits in habituation response and following decreases in anxiety (\\u003cspan citationid=\\\"CR91\\\" class=\\\"CitationRef\\\"\\u003e91\\u003c/span\\u003e), suggesting that observed differences in repetitive behavior during the LSC test may arise from an impaired habituation response to the novel environment in female \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats. Further, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e males presented with improved performance in the acquisition phase of the WTM. In line with our finding, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e males are reported to show increased performance in an episodic memory task (\\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e), suggesting altered memory formation in the Eker rat. Interestingly, this phenotype does not seem to occur in female Eker rats, suggesting sex differences in cognition; however, a confirmation and extension to other cognitive domains needs to be made before drawing meaningful conclusions. The here reported sex effects should be interpreted carefully, as male and female animal numbers are too small to draw meaningful conclusions. Overall, the discussed sex effects are considered to be of small relevance regarding the main concern of genotype effects in the Eker ASD model.\\u003c/p\\u003e \\u003cp\\u003eWhile the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker model presents with both social impairment and repetitive behavior, symptom expression remains mild. Deviations from human pathology in the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rat model have been proposed to originate from its genetic basis. Homozygous deletion is embryonically lethal in both mice and rats, thus heterozygous TSC models are used (\\u003cspan citationid=\\\"CR92\\\" class=\\\"CitationRef\\\"\\u003e92\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR93\\\" class=\\\"CitationRef\\\"\\u003e93\\u003c/span\\u003e). However, heterozygous animal models do not always accurately mirror typical neuropathy. In case of TSC, spontaneous seizure development as a hallmark manifestation cannot be recapitulated in animal models so far (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). Often, \\u0026ldquo;second hit paradigms\\u0026rdquo; are therefore applied to model neurodevelopmental diseases with complex gene-environmental etiology, as they are thought to recapitulate human disease development more closely (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e) and have been reported to induce ASD-like phenotypes (\\u003cspan citationid=\\\"CR94\\\" class=\\\"CitationRef\\\"\\u003e94\\u003c/span\\u003e). Chemical \\u0026ldquo;second hit\\u0026rdquo; induction has been used to generate epileptic seizures in the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rat model and was found to increase ASD-associated behavior in mutant animals (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). Thus, introducing a \\u0026ldquo;second hit\\u0026rdquo; paradigm may exacerbate behavioral alteration in the Eker rat model also in respect to its repetitive behavior profile. Finally, pharmacological testing of mTOR inhibitors or dopamine receptor antagonists and their effect on repetitive phenotypes as well as lifespan analysis of stereotypies, especially considering early symptom onset in ASD, may be applied in the Eker model to challenge its translational accuracy and support predictive validity.\\u003c/p\\u003e \\u003cp\\u003eVarious brain networks and dopamine-dependent signaling pathways have been linked to repetitive behavior formation (\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e). Here, we report alterations in PFC but not striatal DA levels of \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals, both of which are components of the CSTC pathway that regulates motor activity (\\u003cspan additionalcitationids=\\\"CR58\\\" citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR95\\\" class=\\\"CitationRef\\\"\\u003e95\\u003c/span\\u003e). The PFC has been shown to mediate repetitive behavior patterns (\\u003cspan citationid=\\\"CR96\\\" class=\\\"CitationRef\\\"\\u003e96\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR97\\\" class=\\\"CitationRef\\\"\\u003e97\\u003c/span\\u003e), is thought to be involved in goal-directed reinforcement learning (\\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e80\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR98\\\" class=\\\"CitationRef\\\"\\u003e98\\u003c/span\\u003e) and shows disrupted function in various animal models of ASD-linked risk genes and ASD patients (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e). Projections from the PFC into the substantia nigra pars compacta regulate the majority of DA release in the striatum. Striatal overactivation is further implicated in repetitive behavior development (\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e). The \\u003cem\\u003eScn1a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e ASD mouse model, for example, exhibits hyperactivity and excessive self-grooming upon increased excitation in the PFC (\\u003cspan citationid=\\\"CR99\\\" class=\\\"CitationRef\\\"\\u003e99\\u003c/span\\u003e). Observed increases in PFC DA levels occurring alongside increases in DA turnover, thus, fit with our observation of repetitive behavior formation in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. Interestingly, DA/(DOPAC/HVA) rates showed differences between sexes in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals, suggesting that PFC-dependent DA increase causes compensatory changes through elevation of DA turnover in male \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats. While observed alterations in PFC-DA turnover may partially explain sex differences in repetitive behavior, extended mechanistic analysis of DA receptor and enzyme function are necessary to build this hypothesis and connect repetitive phenotypes to dysfunction of the dopaminergic PFC system. Considering that we did not find changes in homeostatic CPu DA levels, both regions possibly exert different effects on specific types of repetitive behavior, as the striatum for example is involved in maintaining and flexibly changing choice patterns, thereby influencing reversal learning and behavioral flexibility (\\u003cspan citationid=\\\"CR100\\\" class=\\\"CitationRef\\\"\\u003e100\\u003c/span\\u003e). Further, we recognize that other brain regions have been implicated in repetitive behavior formation, including the hippocampus, amygdala, and ventral tegmental area (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e), which may be addressed to characterize region-specific alterations of DA in the Eker rat. Additionally, the usage of animals for HPLC analysis that have undergone behavioral assessment to reduce animal numbers and comply with ethical standards raises the possibility of interference. To minimize this, a single low dose of amphetamine, established to not generate behavioral stereotypies in wt animals, was used for behavioral testin,g and tissue was sampled after a one-day rest, circumventing acute DA alterations as well as long-term effects which manifest after 1\\u0026ndash;3 weeks (\\u003cspan citationid=\\\"CR101\\\" class=\\\"CitationRef\\\"\\u003e101\\u003c/span\\u003e) or after repeated treatment (\\u003cspan citationid=\\\"CR102\\\" class=\\\"CitationRef\\\"\\u003e102\\u003c/span\\u003e). Lastly, alterations in dopaminergic signaling in the PFC are further known to result in changed social behavior and social recognition in ASD-associated animal models (\\u003cspan citationid=\\\"CR103\\\" class=\\\"CitationRef\\\"\\u003e103\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR104\\\" class=\\\"CitationRef\\\"\\u003e104\\u003c/span\\u003e). This study lacks experimental evidence to specifically connect alterations in DA level to behavioral alterations; however, we demonstrate that changes in PFC-dependent DA levels occur simultaneously with ASD-like alterations in the social and repetitive behavior domain in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eFor the first time, we characterized the repetitive behavior profile of the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rat model. In summary, we report the presence of lower-order repetitive behaviors that occurred alongside decreased social interaction in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats. Importantly, we showed that DA homeostasis is altered in the PFC of \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats, suggesting a contribution to ASD-like repetitive and social behavior manifestation. We propose that our findings add translational value to the usage of the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rat model in preclinical ASD research.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eASD\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eautism spectrum disorder\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eCPu\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003estriatum (caudate putamen)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eCSTC\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ecortico-striatal-thalamo-cortical\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eDA\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003edopamine\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eDOPAC\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003e3,4-dihydroxyphenylacetic acid\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eFST\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eforced swim test\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eHVA\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ehomovanillic acid\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eLSC\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003elight-sound-confinement test\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eLTD\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003elong-term depression\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eLTP\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003elong-term potentiation\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003emTORC1\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003emammalian target of rapamycin complex 1\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003ePFC\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eprefrontal cortex\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eTSC\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003etuberous sclerosis complex\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003ewt\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ewild-type\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003e\\u003cem\\u003eWTM\\u003c/em\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ewater-T-maze\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\" \\u003cp\\u003e \\u003cstrong\\u003eEthics approval\\u003c/strong\\u003e \\u003cp\\u003e All experiments were conducted in accordance with the European Union guidelines on care and use of laboratory animals (EU directive 2010/63/EU) and were approved by the local ethics committees (Landesdirektion Sachsen, No. 10/2018, Institutional Animal Care and Use Committee No. 66/2016).\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThe work reported in this study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project-ID 518530049, Project-ID 521379614 \\u0026ndash; SFB/TRR 393 and Project-ID 454245598 \\u0026ndash; GRK2773/1.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eM.Z. behavioral experiments. B.H. Audacity/Matlab programming, HPLC analysis. T.P., I.V., breeding, genotyping, and provided materials. N.B., T.P., R.W. study conceptualization and design. N.B. data and statistical analysis. A.R., N.B manuscript draft. N.B., A.R., figure design. N.B. funding acquisition. All authors contributed to revisions of the manuscript draft.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eWe acknowledge the valuable technical assistance provided by Kristin Wogan.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eAll data is provided within the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAmerican Psychiatric Association. Diagnostic and statistical manual of mental disorders: DSM-5. 5th edition. American Psychiatric Association. 2013. Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1176/appi.books.9780890425596\\u003c/span\\u003e\\u003cspan address=\\\"10.1176/appi.books.9780890425596\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChristensen DL, Bilder DA, Zahorodny W, Pettygrove S, Durkin MS, Fitzgerald RT, et al. Prevalence and Characteristics of Autism Spectrum Disorder Among 4-Year-Old Children in the Autism and Developmental Disabilities Monitoring Network. J Dev Behav Pediatr. 2016;37(1):1\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZeidan J, Fombonne E, Scorah J, Ibrahim A, Durkin MS, Saxena S, et al. Global prevalence of autism: A systematic review update. Autism Res. 2022;15(5):778\\u0026ndash;90.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMcCracken JT, Anagnostou E, Arango C, Dawson G, Farchione T, Mantua V, et al. Drug development for Autism Spectrum Disorder (ASD): Progress, challenges, and future directions. Eur Neuropsychopharmacol. 2021;48:3\\u0026ndash;31.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGogate A, Kaur K, Khalil R, Bashtawi M, Morris MA, Goodspeed K, et al. The genetic landscape of autism spectrum disorder in an ancestrally diverse cohort. npj Genom Med. 2024;9(1):1\\u0026ndash;22.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWillsey HR, Willsey AJ, Wang B, State MW. Genomics, convergent neuroscience and progress in understanding autism spectrum disorder. Nat Rev Neurosci. 2022;23(6):323\\u0026ndash;41.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSatterstrom FK, Kosmicki JA, Wang J, Breen MS, De Rubeis S, An JY, et al. Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism. Cell. 2020;180(3):568\\u0026ndash;e58423.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGaugler T, Klei L, Sanders SJ, Bodea CA, Goldberg AP, Lee AB, et al. Most genetic risk for autism resides with common variation. Nat Genet. 2014;46(8):881\\u0026ndash;5.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGeschwind DH. Genetics of Autism Spectrum Disorders. Trends Cogn Sci. 2011;15(9):409\\u0026ndash;16.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKazdoba TM, Leach PT, Crawley JN. Behavioral phenotypes of genetic mouse models of autism. Genes Brain Behav. 2016;15(1):7\\u0026ndash;26.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eM\\u0026ouml;hrle D, Fern\\u0026aacute;ndez M, Pe\\u0026ntilde;agarikano O, Frick A, Allman B, Schmid S. What we can learn from a genetic rodent model about autism. Neurosci Biobehavioral Reviews. 2020;109:29\\u0026ndash;53.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKas MJ, Glennon JC, Buitelaar J, Ey E, Biemans B, Crawley J, et al. Assessing behavioural and cognitive domains of autism spectrum disorders in rodents: current status and future perspectives. Psychopharmacology. 2014;231(6):1125\\u0026ndash;46.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWhitehouse CM, Lewis MH. Repetitive Behavior in Neurodevelopmental Disorders: Clinical and Translational Findings. Behav Anal. 2015;38(2):163\\u0026ndash;78.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSundberg M, Sahin M. Cerebellar Development and Autism Spectrum Disorder in Tuberous Sclerosis Complex. J Child Neurol. 2015;30(14):1954\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNiu M, Han Y, Dy ABC, Du J, Jin H, Qin J, et al. Autism Symptoms in Fragile X Syndrome. J Child Neurol. 2017;32(10):903\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJamain S, Radyushkin K, Hammerschmidt K, Granon S, Boretius S, Varoqueaux F, et al. Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc Natl Acad Sci U S A. 2008;105(5):1710\\u0026ndash;5.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBetancur C, Buxbaum JD. SHANK3 haploinsufficiency: a common but underdiagnosed highly penetrant monogenic cause of autism spectrum disorders. Mol Autism. 2013;4:17.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRadyushkin K, Hammerschmidt K, Boretius S, Varoqueaux F, El-Kordi A, Ronnenberg A, et al. Neuroligin-3-deficient mice: model of a monogenic heritable form of autism with an olfactory deficit. Genes Brain Behav. 2009;8(4):416\\u0026ndash;25.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSpecchio N, Pietrafusa N, Trivisano M, Moavero R, De Palma L, Ferretti A, et al. Autism and Epilepsy in Patients With Tuberous Sclerosis Complex. Front Neurol. 2020;11:639.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCrino PB, Nathanson KL, Henske EP. The New England Journal of Medicine. Massachusetts Medical Society; 2006 [cited 2025 Jan 21]. The Tuberous Sclerosis Complex. Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.nejm.org/doi/full/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.nejm.org/doi/full/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1056/NEJMra055323\\u003c/span\\u003e\\u003cspan address=\\\"10.1056/NEJMra055323\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eK\\u0026uacute;tna V, O\\u0026rsquo;Leary VB, Newman E, Hoschl C, Ovsepian SV. Revisiting Brain Tuberous Sclerosis Complex in Rat and Human: Shared Molecular and Cellular Pathology Leads to Distinct Neurophysiological and Behavioral Phenotypes. Neurotherapeutics. 2021;18(2):845\\u0026ndash;58.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJeste SS, Varcin KJ, Hellemann GS, Gulsrud AC, Bhatt R, Kasari C, et al. Symptom profiles of autism spectrum disorder in tuberous sclerosis complex. Neurology. 2016;87(8):766\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGadad BS, Hewitson L, Young KA, German DC. Neuropathology and Animal Models of Autism: Genetic and Environmental Factors. Autism Res Treat. 2013;2013:731935.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePalavra F, Robalo C, Reis F. Recent Advances and Challenges of mTOR Inhibitors Use in the Treatment of Patients with Tuberous Sclerosis Complex. Oxidative Med Cell Longev. 2017;2017(1):9820181.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSwiton K, Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J. Molecular neurobiology of mTOR. Neuroscience. 2017;341:112\\u0026ndash;53.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLipton JO, Sahin M. The Neurology of mTOR. Neuron. 2014;84(2):275\\u0026ndash;91.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEker R, Mossige J. A Dominant Gene for Renal Adenomas in the Rat. Nature. 1961;189(4767):858\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYeung RS, Xiao GH, Jin F, Lee WC, Testa JR, Knudson AG. Predisposition to Renal Carcinoma in the Eker Rat is Determined by Germ-Line Mutation of the Tuberous Sclerosis 2 (TSC2) Gene. Proc Natl Acad Sci USA. 1994;91(24):11413\\u0026ndash;6.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGranak S, Tuckova K, Kutna V, Vojtechova I, Bajkova L, Petrasek T, et al. Developmental effects of constitutive mTORC1 hyperactivity and environmental enrichment on structural synaptic plasticity and behaviour in a rat model of autism spectrum disorder. Eur J Neurosci. 2023;57(1):17\\u0026ndash;31.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChi OZ, Wu CC, Liu X, Rah KH, Jacinto E, Weiss HR. Restoration of Normal Cerebral Oxygen Consumption with Rapamycin Treatment in a Rat Model of Autism\\u0026ndash;Tuberous Sclerosis. Neuromolecular Med. 2015;17(3):305\\u0026ndash;13.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChi OZ, Liu X, Fortus H, Werlen G, Jacinto E, Weiss HR. Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis. Neuromolecular Med. 2024;26(1):10.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eK\\u0026uacute;tna V, Uttl L, Waltereit R, Krištofikov\\u0026aacute; Z, Kaping D, Petr\\u0026aacute;sek T, et al. Tuberous Sclerosis (tsc2+/-) Model Eker Rats Reveals Extensive Neuronal Loss with Microglial Invasion and Vascular Remodeling Related to Brain Neoplasia. Neurotherapeutics. 2020;17(1):329\\u0026ndash;39.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVon Der Brelie C, Waltereit R, Zhang L, Beck H, Kirschstein T. Impaired synaptic plasticity in a rat model of tuberous sclerosis. Eur J Neurosci. 2006;23(3):686\\u0026ndash;92.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTakahashi DK, Dinday MT, Barbaro NM, Baraban SC. Abnormal Cortical Cells and Astrocytomas in the Eker Rat Model of Tuberous Sclerosis Complex. Epilepsia. 2004;45(12):1525\\u0026ndash;30.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWaltereit R, Japs B, Schneider M, de Vries PJ, Bartsch D. Epilepsy and Tsc2 Haploinsufficiency Lead to Autistic-Like Social Deficit Behaviors in Rats. Behav Genet. 2011;41(3):364\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchneider M, de Vries PJ, Sch\\u0026ouml;nig K, R\\u0026ouml;\\u0026szlig;ner V, Waltereit R. mTOR inhibitor reverses autistic-like social deficit behaviours in adult rats with both Tsc2 haploinsufficiency and developmental status epilepticus. Eur Arch Psychiatry Clin Neurosci. 2017;267(5):455\\u0026ndash;63.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePetrasek T, Vojtechova I, Klovrza O, Tuckova K, Vejmola C, Rak J, et al. mTOR inhibitor improves autistic-like behaviors related to Tsc2 haploinsufficiency but not following developmental status epilepticus. J Neurodev Disord. 2021;13:14.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAngoa-P\\u0026eacute;rez M, Kane MJ, Briggs DI, Francescutti DM, Kuhn DM. Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice. J Vis Exp. 2013;(82):50978.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKozlova AA, Rubets E, Vareltzoglou MR, Jarzebska N, Ragavan VN, Chen Y, et al. Knock-out of the critical nitric oxide synthase regulator DDAH1 in mice impacts amphetamine sensitivity and dopamine metabolism. J Neural Transm (Vienna). 2023;130(9):1097\\u0026ndash;112.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eThomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology. 2009;204(2):361\\u0026ndash;73.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCampeau S. Apparatus and General Methods for Exposing Rats to Audiogenic Stress. Bio Protoc. 2016;6(21):e1994.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eValsamis B, Schmid S. Habituation and Prepulse Inhibition of Acoustic Startle in Rodents. J Vis Exp. 2011;(55):3446.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBouwknecht JA, Spiga F, Staub DR, Hale MW, Shekhar A, Lowry CA. Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors; relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus. Brain Res Bull. 2007;72(1):32\\u0026ndash;43.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCommons KG, Cholanians AB, Babb JA, Ehlinger DG. The Rodent Forced Swim Test Measures Stress-Coping Strategy, Not Depression-like Behavior. ACS Chem Neurosci. 2017;8(5):955\\u0026ndash;60.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePorsolt RD, Anton G, Blavet N, Jalfre M. Behavioural despair in rats: A new model sensitive to antidepressant treatments. Eur J Pharmacol. 1978;47(4):379\\u0026ndash;91.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSlattery DA, Cryan JF. Using the rat forced swim test to assess antidepressant-like activity in rodents. Nat Protoc. 2012;7(6):1009\\u0026ndash;14.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGuariglia SR, Chadman KK. Water T-maze: A useful assay for determination of repetitive behaviors in mice. J Neurosci Methods. 2013;220(1):24\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMeyerolbersleben L, Winter C, Bernhardt N. Dissociation of wanting and liking in the sucrose preference test in dopamine transporter overexpressing rats. Behav Brain Res. 2020;378:112244.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHadar R, Edemann-Callesen H, Reinel C, Wieske F, Voget M, Popova E, et al. Rats overexpressing the dopamine transporter display behavioral and neurobiological abnormalities with relevance to repetitive disorders. Sci Rep. 2016;6(1):39145.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWinslow JT, Camacho F. Cholinergic modulation of a decrement in social investigation following repeated contacts between mice. Psychopharmacology. 1995;121(2):164\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKelly PH, Seviour PW, Iversen SD. Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Res. 1975;94(3):507\\u0026ndash;22.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBlanca MJ, Alarc\\u0026oacute;n R, Arnau J, Bono R, Bendayan R. Non-normal data: Is ANOVA still a valid option? Psicothema. 2017;29(4):552\\u0026ndash;7.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSun J, Yuan Y, Wu X, Liu A, Wang J, Yang S, et al. Excitatory SST neurons in the medial paralemniscal nucleus control repetitive self-grooming and encode reward. Neuron. 2022;110(20):3356\\u0026ndash;e33738.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMuehlmann AM, Lewis MH. Abnormal repetitive behaviours: shared phenomenology and pathophysiology. J Intellect Disabil Res. 2012;56(5):427\\u0026ndash;40.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMoy SS, Riddick NV, Nikolova VD, Teng BL, Agster KL, Nonneman RJ, et al. Repetitive behavior profile and supersensitivity to amphetamine in the C58/J mouse model of autism. Behav Brain Res. 2014;259. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1016/j.bbr.2013.10.052\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.bbr.2013.10.052\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKim H, Lim CS, Kaang BK. Neuronal mechanisms and circuits underlying repetitive behaviors in mouse models of autism spectrum disorder. Behav Brain Funct. 2016;12(1):3.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGandhi T, Lee CC. Neural Mechanisms Underlying Repetitive Behaviors in Rodent Models of Autism Spectrum Disorders. Front Cell Neurosci. 2021;14:592710.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eParr-Brownlie LC, Hyland BI. Bradykinesia Induced by Dopamine D2 Receptor Blockade Is Associated with Reduced Motor Cortex Activity in the Rat. J Neurosci. 2005;25(24):5700\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKalueff AV, Stewart AM, Song C, Berridge KC, Graybiel AM, Fentress JC. Neurobiology of rodent self-grooming and its value for translational neuroscience. Nat Rev Neurosci. 2016;17(1):45\\u0026ndash;59.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ede Brouwer G, Fick A, Harvey BH, Wolmarans DW. A critical inquiry into marble-burying as a preclinical screening paradigm of relevance for anxiety and obsessive-compulsive disorder: Mapping the way forward. Cogn Affect Behav Neurosci. 2019;19(1):1\\u0026ndash;39.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTaylor GT, Lerch S, Chourbaji S. Marble burying as compulsive behaviors in male and female mice. Acta Neurobiol Exp (Wars). 2017;77(3):254\\u0026ndash;60.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVaccarino F, Franklin KBJ. Self-stimulation and circling reveal functional differences between medial and lateral substantia nigra. Behav Brain Res. 1982;5(3):281\\u0026ndash;95.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAbbott AE, Linke AC, Nair A, Jahedi A, Alba LA, Keown CL, et al. Repetitive behaviors in autism are linked to imbalance of corticostriatal connectivity: a functional connectivity MRI study. Soc Cogn Affect Neurosci. 2018;13(1):32\\u0026ndash;42.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKim IH, Rossi MA, Aryal DK, Racz B, Kim N, Uezu A, et al. Spine Pruning Drives Antipsychotic-sensitive Locomotion via Circuit Control of Striatal Dopamine. Nat Neurosci. 2015;18(6):883\\u0026ndash;91.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChi OZ, Liu X, Fortus H, Werlen G, Jacinto E, Weiss HR. Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis. Neuromolecular Med. 2024;26(1):10.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGoorden SMI, van Woerden GM, van der Weerd L, Cheadle JP, Elgersma Y. Cognitive deficits in Tsc1+/\\u0026ndash;mice in the absence of cerebral lesions and seizures. Ann Neurol. 2007;62(6):648\\u0026ndash;55.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eReith RM, McKenna J, Wu H, Hashmi SS, Cho SH, Dash PK, et al. Loss of \\u003cem\\u003eTsc2\\u003c/em\\u003e in Purkinje cells is associated with autistic-like behavior in a mouse model of tuberous sclerosis complex. Neurobiol Dis. 2013;51:93\\u0026ndash;103.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePearson B, Pobbe R, Defensor E, Oasay L, Bolivar V, Blanchard D, et al. Motor and cognitive stereotypies in the BTBR T\\u0026thinsp;+\\u0026thinsp;tf/J mouse model of autism. Genes Brain Behav. 2011;10(2):228\\u0026ndash;35.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eShin W, Kweon H, Kang R, Kim D, Kim K, Kang M, et al. Scn2a Haploinsufficiency in Mice Suppresses Hippocampal Neuronal Excitability, Excitatory Synaptic Drive, and Long-Term Potentiation, and Spatial Learning and Memory. Front Mol Neurosci. 2019;12:145.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBauer HF, Delling JP, Bockmann J, Boeckers TM, Sch\\u0026ouml;n M. Development of sex- and genotype-specific behavioral phenotypes in a Shank3 mouse model for neurodevelopmental disorders. Front Behav Neurosci. 2023;16:1051175.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYoo T, Cho H, Lee J, Park H, Yoo YE, Yang E et al. GABA Neuronal Deletion of Shank3 Exons 14\\u0026ndash;16 in Mice Suppresses Striatal Excitatory Synaptic Input and Induces Social and Locomotor Abnormalities. Front Cell Neurosci [Internet]. 2018 Oct 9 [cited 2025 Jan 29];12. Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.frontiersin.org/journals/cellular-neuroscience/articles/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.frontiersin.org/journals/cellular-neuroscience/articles/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.3389/fncel.2018.00341/full\\u003c/span\\u003e\\u003cspan address=\\\"10.3389/fncel.2018.00341/full\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePe\\u0026ccedil;a J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature. 2011;472(7344):437\\u0026ndash;42.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJung S, Park M. Shank postsynaptic scaffolding proteins in autism spectrum disorder: Mouse models and their dysfunctions in behaviors, synapses, and molecules. Pharmacol Res. 2022;182:106340.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eThomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology. 2009;204(2):361\\u0026ndash;73.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGarc\\u0026iacute;a-Villamisar D, Rojahn J. Comorbid psychopathology and stress mediate the relationship between autistic traits and repetitive behaviours in adults with autism. J Intellect Disabil Res. 2015;59(2):116\\u0026ndash;24.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKatz RJ, Roth KA. Stress induced grooming in the rat\\u0026ndash;an endorphin mediated syndrome. Neurosci Lett. 1979;13(2):209\\u0026ndash;12.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHomberg JR, Van Den Akker M, Raas\\u0026oslash; HS, Wardeh G, Binnekade R, Schoffelmeer ANM, et al. Enhanced motivation to self-administer cocaine is predicted by self-grooming behaviour and relates to dopamine release in the rat medial prefrontal cortex and amygdala. Eur J Neurosci. 2002;15(9):1542\\u0026ndash;50.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAmodeo DA, Jones JH, Sweeney JA, Ragozzino ME. Differences in BTBR T\\u0026thinsp;+\\u0026thinsp;tf/J and C57BL/6J mice on probabilistic reversal learning and stereotyped behaviors. Behav Brain Res. 2012;227(1):64\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWhitehouse CM, Curry-Pochy LS, Shafer R, Rudy J, Lewis MH. Reversal learning in C58 mice: Modeling higher order repetitive behavior. Behav Brain Res. 2017;332:372\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSolomon M, Smith AC, Frank MJ, Ly S, Carter CS. Probabilistic Reinforcement Learning in Adults with Autism Spectrum Disorders. Autism Res. 2011;4(2):109\\u0026ndash;20.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMin JY, Park S, Cho J, Huh Y. The anterior insular cortex processes social recognition memory. Sci Rep. 2023;13(1):10853.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCholeris E, Clipperton-Allen AE, Phan A, Valsecchi P, Kavaliers M. Estrogenic involvement in social learning, social recognition and pathogen avoidance. Front Neuroendocr. 2012;33(2):140\\u0026ndash;59.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKim SH, An K, Namkung H, Saito A, Rannals MD, Moore JR, et al. Anterior Insula\\u0026ndash;Associated Social Novelty Recognition: Pivotal Roles of a Local Retinoic Acid Cascade and Oxytocin Signaling. AJP. 2023;180(4):305\\u0026ndash;17.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWaltereit R, Welzl H, Dichgans J, Lipp HP, Schmidt WJ, Weller M. Enhanced episodic-like memory and kindling epilepsy in a rat model of tuberous sclerosis. J Neurochem. 2006;96(2):407\\u0026ndash;13.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWerling DM, Geschwind DH. Sex differences in autism spectrum disorders. Curr Opin Neurol. 2013;26(2):146\\u0026ndash;53.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLeow KQ, Tonta MA, Lu J, Coleman HA, Parkington HC. Towards understanding sex differences in autism spectrum disorders. Brain Res. 2024;1833:148877.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJeon SJ, Gonzales EL, Mabunga DFN, Valencia ST, Kim DG, Kim Y, et al. Sex-specific Behavioral Features of Rodent Models of Autism Spectrum Disorder. Exp Neurobiol. 2018;27(5):321\\u0026ndash;43.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMurta V, Seiffe A, Depino AM. Sex Differences in Mouse Models of Autism Spectrum Disorders: Their Potential to Uncover the Impact of Brain Sexual Differentiation on Gender Bias. Sexes. 2023;4(3):358\\u0026ndash;91.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBove M, Sikora V, Santoro M, Agosti LP, Palmieri MA, Dimonte S, et al. Sex differences in the BTBR idiopathic mouse model of autism spectrum disorders: Behavioural and redox-related hippocampal alterations. Neuropharmacology. 2024;260:110134.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEl-Kordi A, Winkler D, Hammerschmidt K, K\\u0026auml;stner A, Krueger D, Ronnenberg A, et al. Development of an autism severity score for mice using Nlgn4 null mutants as a construct-valid model of heritable monogenic autism. Behav Brain Res. 2013;251:41\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSar\\u0026eacute; RM, Lemons A, Figueroa C, Song A, Levine M, Beebe Smith C. Sex-Selective Effects on Behavior in a Mouse Model of Tuberous Sclerosis Complex. eNeuro. 2020;7(2):ENEURO.0379-19.2020.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOnda H, Lueck A, Marks PW, Warren HB, Kwiatkowski DJ. Tsc2+/\\u0026ndash; mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J Clin Invest. 1999;104(6):687\\u0026ndash;95.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRennebeck G, Kleymenova EV, Anderson R, Yeung RS, Artzt K, Walker CL. Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development. Proc Natl Acad Sci U S A. 1998;95(26):15629\\u0026ndash;34.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZheng W, Wang M, Cui Y, Xu Q, Chen Y, Xian P, et al. Establishment of a two-hit mouse model of environmental factor induced autism spectrum disorder. Heliyon. 2024;10(9):e30617.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi W, Pozzo-Miller L. Dysfunction of the corticostriatal pathway in autism spectrum disorders. J Neurosci Res. 2020;98(11):2130\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMorency MA, Stewart RJ, Beninger RJ. Effects of unilateral microinjections of sulpiride into the medial prefrontal cortex on circling behavior of rats. Prog Neuropsychopharmacol Biol Psychiatry. 1985;9(5):735\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKelly E, Meng F, Fujita H, Morgado F, Kazemi Y, Rice LC, et al. Regulation of autism-relevant behaviors by cerebellar\\u0026ndash;prefrontal cortical circuits. Nat Neurosci. 2020;23(9):1102\\u0026ndash;10.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDoll BB, Jacobs WJ, Sanfey AG, Frank MJ. Instructional control of reinforcement learning: A behavioral and neurocomputational investigation. Brain Res. 2009;1299:74\\u0026ndash;94.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHan S, Tai C, Jones CJ, Scheuer T, Catterall WA. Enhancement of Inhibitory Neurotransmission by GABAA Receptors Having α2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Model of Autism. Neuron. 2014;81(6):1282\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eO\\u0026rsquo;Neill M, Brown VJ. The effect of striatal dopamine depletion and the adenosine A2A antagonist KW-6002 on reversal learning in rats. Neurobiol Learn Mem. 2007;88(1):75\\u0026ndash;81.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVanderschuren LJ, Schmidt ED, De Vries TJ, Van Moorsel CA, Tilders FJ, Schoffelmeer AN. A single exposure to amphetamine is sufficient to induce long-term behavioral, neuroendocrine, and neurochemical sensitization in rats. J Neurosci. 1999;19(21):9579\\u0026ndash;86.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTaracha E, Czarna M, Turzyńska D, Maciejak P. Amphetamine-induced prolonged disturbances in tissue levels of dopamine and serotonin in the rat brain. Pharmacol Rep. 2023;75(3):596\\u0026ndash;608.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBlum K, Bowirrat A, Sunder K, Thanos PK, Hanna C, Gold MS, et al. Dopamine Dysregulation in Reward and Autism Spectrum Disorder. Brain Sci. 2024;14(7):733.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSato M, Nakai N, Fujima S, Choe KY, Takumi T. Social circuits and their dysfunction in autism spectrum disorder. Mol Psychiatry. 2023;28(8):3194\\u0026ndash;206.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"behavioral-and-brain-functions\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"babf\",\"sideBox\":\"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)\",\"snPcode\":\"12993\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12993/3\",\"title\":\"Behavioral and Brain Functions\",\"twitterHandle\":\"@BBF_Journal\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Autism spectrum disorder, tuberous sclerosis complex, repetitive behavior, Eker rat\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6006061/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6006061/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBesides deficits in social communication and interaction, repetitive behavior patterns are core manifestations of autism spectrum disorder (ASD). Phenotypes are heterogeneous and can range from simple lower-order motor stereotypies to more complex higher-order cognitive inflexibility and fixated interests. Due to ASD\\u0026rsquo;s multifaceted etiology, animal models are often generated from monogenic diseases associated with ASD, such as Tuberous Sclerosis Complex (TSC), and are expected to copy behavioral core deficits to increase the model\\u0026acute;s translational value for ASD disease research and novel treatment development. The global haploinsufficient \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rat model has been shown to display ASD core symptoms in the social domain. However, the presence and extent of aberrant repetitive behavior patterns in the Eker rat remain to be investigated. Thus, the present study applied a set of behavioral tests to determine the repetitive behavioral profile in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rats and used brain-region-specific neurotransmitter analysis to support findings on a molecular level. \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e animals demonstrated lower-order repetitive behavior in the form of excessive self-grooming and nestlet shredding under non-stressful conditions that co-occurred alongside social interaction deficits. However, no higher-order repetitive behavior was detected in \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats. Interestingly, \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e rats exhibited increased levels of homeostatic dopamine in the prefrontal cortex, supporting the link between aberrant cortical dopaminergic transmission and the appearance of lower-order repetitive phenotypes. Together, our results support the \\u003cem\\u003eTsc2\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e+/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e Eker rat as a model of ASD-like behavior for further investigation of ASD-related development and neurobiology.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Behavioral phenotyping identifies autism-like repetitive stereotypies in a Tsc2 haploinsufficient rat model\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-05-02 11:31:33\",\"doi\":\"10.21203/rs.3.rs-6006061/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-05-09T21:13:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-05-08T21:48:58+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-05-07T18:38:22+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-05-06T11:46:17+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"85623290308450979018385685822617366466\",\"date\":\"2025-05-01T20:22:40+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"223502206151335924569477569378949135815\",\"date\":\"2025-05-01T17:12:31+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"293743581002634137695180036482313543136\",\"date\":\"2025-04-28T19:13:06+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"133282416273114247176736083684575452873\",\"date\":\"2025-04-28T17:11:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"316155819817595277187536274867175828931\",\"date\":\"2025-04-26T17:11:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-04-26T17:08:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-04-24T06:17:47+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Behavioral and Brain Functions\",\"date\":\"2025-04-23T12:11:07+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"behavioral-and-brain-functions\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"babf\",\"sideBox\":\"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)\",\"snPcode\":\"12993\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12993/3\",\"title\":\"Behavioral and Brain Functions\",\"twitterHandle\":\"@BBF_Journal\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"362f8a11-ba31-4109-9ae4-0b1f3665fa82\",\"owner\":[],\"postedDate\":\"May 2nd, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-07-07T16:07:41+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-6006061\",\"link\":\"https://doi.org/10.1186/s12993-025-00284-z\",\"journal\":{\"identity\":\"behavioral-and-brain-functions\",\"isVorOnly\":false,\"title\":\"Behavioral and Brain Functions\"},\"publishedOn\":\"2025-07-03 15:58:31\",\"publishedOnDateReadable\":\"July 3rd, 2025\"},\"versionCreatedAt\":\"2025-05-02 11:31:33\",\"video\":\"\",\"vorDoi\":\"10.1186/s12993-025-00284-z\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12993-025-00284-z\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6006061\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6006061\",\"identity\":\"rs-6006061\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}