Social Interaction Deficits Emerge Alongside Anxiety-Like Behavior in TgF344-AD Rats

Social Interaction Deficits Emerge Alongside Anxiety-Like Behavior in TgF344-AD Rats | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 11 February 2025 V1 Latest version Share on Social Interaction Deficits Emerge Alongside Anxiety-Like Behavior in TgF344-AD Rats Authors : Daniela Černotová , Kristýna Malenínská , Anna Horáková , Ales Stuchlik , and Jan Svoboda 0000-0002-0343-5347 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173929882.27357606/v1 311 views 164 downloads Contents Abstract 1 Introduction 4 Discussion Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Alzheimer’s disease (AD) manifests by many behavioral changes, including anxiety and social withdrawal, which often precede and accompany cognitive decline. The TgF344-AD rat model recapitulates several AD-like pathological features and cognitive deficits, yet the progression of anxiety-like and social behavior impairments remains poorly characterized. In this study, we investigated anxiety-like and social behaviors in male and female TgF344-AD rats at 10 and 14 months of age using the open-field task, elevated plus-maze, and social interaction paradigms. While standard anxiety measures showed limited genotype effects, we observed subtle behavioral alterations in 10-month-old TgF344-AD rats, including decreased exploratory behavior and reduced locomotor activity in the elevated plus-maze. Notable social interaction deficits were identified in TgF344-AD rats, persisting across both age points. Our behavioral assessment provides an essential characterization of this model at intermediate disease stages when neuropathological changes and cognitive symptoms typically emerge. Most importantly, this work provides evidence for social behavior deficits in the TgF344-AD model and contributes to the understanding of non-cognitive changes in AD. Social Interaction Deficits Emerge Alongside Anxiety-Like Behavior in TgF344-AD Rats Daniela Černotová 1,2 , Kristýna Malenínská 1,3 , Anna Horáková 1 , Aleš Stuchlík 1 , Jan Svoboda* 1 1) Laboratory of Neurophysiology of Memory, Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, Prague 142 00, Czech Republic 2) Third Faculty of Medicine, Charles University, Ruská 87, Prague 100 00, Czech Republic 3) National Institute of Mental Health, Topolová 748, 250 67, Klecany, Czech Republic *Corresponding author Laboratory of Neurophysiology of Memory, Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, Prague 142 00, Czech Republic; E-mail:, tel.: [email protected] ; +420241062538 Abstract Alzheimer’s disease (AD) manifests by many behavioral changes, including anxiety and social withdrawal, which often precede and accompany cognitive decline. The TgF344-AD rat model recapitulates several AD-like pathological features and cognitive deficits, yet the progression of anxiety-like and social behavior impairments remains poorly characterized. In this study, we investigated anxiety-like and social behaviors in male and female TgF344-AD rats at 10 and 14 months of age using the open-field task, elevated plus-maze, and social interaction paradigms. While standard anxiety measures showed limited genotype effects, we observed subtle behavioral alterations in 10-month-old TgF344-AD rats, including decreased exploratory behavior and reduced locomotor activity in the elevated plus-maze. Notable social interaction deficits were identified in TgF344-AD rats, persisting across both age points. Our behavioral assessment provides an essential characterization of this model at intermediate disease stages when neuropathological changes and cognitive symptoms typically emerge. Most importantly, this work provides evidence for social behavior deficits in the TgF344-AD model and contributes to the understanding of non-cognitive changes in AD. Keywords: Alzheimer’s disease, TgF344-AD, social behavior, sociability, anxiety 1 Introduction Alzheimer‘s disease (AD) progressively disrupts cognitive functions and other mental abilities, including mood regulation or social behavior (Förstl & Kurz, 1999). Behavioral deficits emerge as a consequence of neuropathological changes, characterized mainly by the accumulation of amyloid-β plaques and neurofibrillary tangles, composed of hyperphosphorylated tau proteins. These pathological alterations result in neurodegeneration of specific brain areas, eventually leading to neuronal loss (Selkoe, 2001). However, many behavioral changes, such as mood dysregulation, anxiety, apathy, and social withdrawal, occur in the early phase and might be a prodromal sign of the disease (Palmer et al., 2007; Delrieu et al., 2014; Gallagher et al., 2011). Transgenic rodent models are widely used to reproduce behavioral and pathological features of AD. A double-transgenic rat model TgF344-AD shows several aspects of AD pathology, including expression of human amyloid precursor protein (APP) with the Swedish mutation, age-dependent amyloidosis, and tau hyperphosphorylation, leading to neurofibrillary tangle-like structures (Cohen et al., 2013). Also, TgF344-AD rats exhibit cognitive decline in several domains similar to patients with AD, such as working and reference memory, cognitive flexibility, aggravated anxiety, and depression-like behavior, starting as early as 6 months of age (Berkowitz et al. 2018; Morrone et al., 2020; Pentkowski et al., 2018; Saré et al., 2020). Whether the anxiety-like deficits persist or how they change with aging remains unknown. Moreover, some symptoms like social withdrawal, which is commonly seen in AD patients (Chung and Cummings, 2000), are still unexplored in the TgF344-AD model. In this study, we conducted a comprehensive behavioral assessment of TgF344-AD male and female rats (10 and 14 months of age), using the open-field task, elevated plus-maze, and social interaction paradigms. We hypothesized that anxiety-like behaviors would be exhibited, worsened with age, and coincide with the emergence of social deficits. The selected ages represent the mid-stage of the disease, where neuropathology is already present and cognitive symptoms start to develop (Bernaud et al., 2022; Fowler et al., 2022; Morrone et al., 2020, Tournier et al., 2021). Previously mentioned studies mostly focused on earlier (4, 6, and 9 months), middle (12 months), or later (16 and 18 months) time points, we bridge this gap in the behavioral phenotyping of TgF344-AD rats and provide evidence for social behavior deficits in this AD model. 2 Methods 2.1 Animals Male and female TgF344-AD (= AD) rats and wild-type Fisher 344 (= WT) rats were used for the experiments. In total, the experiments were performed with 34 male (WT = 11 and AD = 23) and 24 female (WT = 15, AD = 9) rats at the age of 10 months, and 21 male (WT = 12, AD = 9) and 18 female (WT = 7, AD = 10) rats at the age of 14 months. At the age of 3 weeks, a small tip of the tail was cut off for genotyping under isoflurane-induced general anesthesia. The animals were bred in the Animal Facility of the Institute of Physiology CAS, housed in pairs, and kept at standard conditions (22 °C, 50–60% humidity, 12 h light/dark cycle, water and food ad libitum ) until they reached the age of 10 or 14 months. Special attention was paid to the illumination not exceeding 50 lux to prevent retinal degeneration in albino rats (Rao, 1991). All experimental and housing conditions were approved by a resort Committee of Animal Welfare (Project of Experiments No. 80/2016) and complied with the European Community Council directive (2010/63/EC). 2.2 The Experimental Design The acclimatization period at the Institute of Physiology CAS was 10 days. Rats were handled for 5 days (5–10 min per day for each animal) before the experimental sessions. The battery of experiments was performed on 3 consecutive days in the following order: the open-field task, the elevated plus-maze task, and the social interaction task. The schematic representation is visualized in Fig. 1 . The apparatuses were cleaned before the first run of the day, between subjects, and after the last run using tap water and ethanol. On the day of the testing, rats were brought in their home cages into the experimental room 30 minutes before the testing. The level of illumination in the experiments was set at 5–10 lux. The locomotor activity of animals was assessed in a standard open-field task (proposed initially by Hall, 1934). The tested animal was placed in the center of the open-field apparatus and was allowed to explore freely for 5 minutes. One day after the open-field task, all animals were tested for anxiety-like behavior in the elevated plus-maze task. The protocol used was first introduced by Pellow et al. (1985). The tested rat was placed in the center of the elevated plus-maze facing 1 of the 2 open arms. Each animal was tested for 5 minutes. In the social interaction task, a partner animal was put in one corner of the apparatus facing the wall, and the very next moment, a tested animal was put in the opposite corner. The animals were allowed to freely interact throughout a 10-minute session. Before the experiments, the partner animals were marked on the back fur with a non-washable marker to be easily distinguished from the experimental animal. The partner animals had never interacted with the testing animals before. All experiments were recorded by a camera placed above the apparatus for subsequent offline analysis. Fig. 1: Experimental design. The WT and AD rats (males and females), aged 10 or 14 months, were handled for five days and then underwent the open-field task (5 min), elevated plus-maze task (5 min), and social interaction task (10 min) on three consecutive days. The experiments were performed in the same room at dimmed light (5–10 lux). Created in BioRender.com. 2.3 The Open-Field and Elevated Plus-Maze Apparatuses The open-field apparatus was used for the open-field and social interaction tasks. The apparatus was a white wooden box (70 x 70 x 40 cm) with a black floor. The elevated plus-maze was used to assess anxiety-like behavior. It consisted of 4 black cross-shaped Plexiglas arms (11 cm wide, 50 cm long), elevated 60 cm above the floor. An 11 x 11 cm square black platform joined each arm at the center. Two opposite arms remained without walls (‘open arms’), while the other two contained 30 cm tall orange opaque sides (‘closed arms’). 2.4 Data Analysis In the open-field task, the evaluated parameters were the following: the time spent in the center sector (defined as 50 x 50 cm), the total distance traveled, and the total times of rearing and self-grooming. In the elevated plus-maze, the time (duration) spent in open arms was measured for each subject, and the ratio between the time in the open arm and total time was calculated. Furthermore, the duration of peeking out (from closed arms) and head dipping (downward from open arms) was measured for each subject. In the social interaction task, the time of socialization of the experimental rat toward the partner rat was analyzed. Specific parameters, such as the total time spent interacting with the other rat, time spent by anogenital (nose of the experimental animal-to-anogenital of the partner animal) exploration, non-anogenital (nose-to-nose) exploration, and non-social parameters such as rearing of the animal and its time spent by self-grooming were analyzed. Time spent in specific sectors in the open-field arena and elevated plus-maze were analyzed using the automatic video analysis in the EthoVision XT 17.5 (Noldus). Other behavioral data were tagged manually by the experimenter blinded to the experimental group in Boris software v. 7.9.7 (Friard & Gamba, 2016). The statistical analysis was made in GraphPad Prism 10.4.1. A two-way ANOVA with the main factors of sex and genotype was conducted for both 10- and 14-month-old groups separately, followed by a Tukey’s multiple comparisons post-hoc test in case of significant interaction effect. When necessary, data were logarithmically transformed to improve conformity with the assumptions of homogeneity and normal distribution (log(y) for open-field distance, time in center, peeking from the elevated plus-maze, rearing in the social interaction task; log(y+0.01) for open arm/total time in the elevated plus-maze; log(y+1) for head dipping in the elevated plus-maze). The graphs were created in GraphPad Prism 10.4.1 software. The statistical significance was set at p < 0.05. All data are presented as a group mean ± standard error of the mean (SEM). All two-way ANOVA results are provided in Supp. Tab. 1 ; only significant results are described in the result section 3. 3 Results 3.1 Open-Field Task Two-way ANOVA analysis in 10-month-old rats revealed a significant effect of sex on locomotor activity (F 1, 54 = 20.53, p < 0.0001), with males traveling shorter distances than females ( Fig. 2A ). Similarly, 10-month-old males showed significantly less rearing compared to females (F 1, 54 = 17.39, p = 0.0001) ( Fig. 2C ). In contrast, no significant differences were observed in distance traveled or rearing behavior in 14-month-old rats ( Fig. 2A+C ). Grooming analysis revealed no significant difference in either age group, although a trend towards significance was observed for sex in 10-month-old rats (F1, 54 = 3,777, p = 0.0572) ( Fig. 2D ). Additionally, no significant differences were found in time spent in the center for either age group ( Fig. 2B ). Fig. 2: Effects of sex and genotype in the open-field task. (A) The significant sex effect was revealed in the traveled distance (F 1, 54 = 20.53, p < 0.0001); females had higher locomotor activity than males. (B) No difference was revealed in time spent in the center in either age or group. (C) Analysis showed a significant sex difference in rearing in 10-month-old rats (F 1, 54 = 17.39, p = 0.0001), with males showing less rearing than females. (D) No difference was observed in grooming behavior, although a trend towards significance was observed for sex in 10-month-old rats (F1, 54 = 3,777, p = 0.0572). (E) Representative trajectories of 10-month-old (left) and 14-month-old (right) rats. Data in the graphs show mean ± SEM; the dots represent individual values. Two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 3.2 Elevated Plus-Maze Task Three AD male rats from the 10-month-old group fell down the EPM apparatus and were not included in the data analysis. Additionally, the distance traveled in one AD male rat from the 10-month-old group was not analyzed due to technical issues during the analysis. Analysis of elevated plus-maze behavior did not reveal differences in time spent in the open arm in 10- and 14-month-old rats ( Fig. 3A ). A significant genotype effect was observed in the traveled distance at 10 months (F 1, 50 = 4.761, p = 0.0338), and significant sex effect at 14 months (F 1, 34 = 7.026, p = 0.0121) ( Fig. 3B ). At 10 months, two-way ANOVA showed a significant genotype effect (F 1, 51 = 10.55, p = 0.0021) and a trend toward significant sex effect (F 1, 51 = 4.017, p = 0.0504) in peeking behavior. These results demonstrate that AD rats exhibited reduced peeking compared to their WT counterparts ( Fig. 3C ). Head dipping did not differ significantly in this age group ( Fig. 3D ). At 14 months, neither peeking out nor head dipping differed between groups ( Fig. 3C+D ). Fig. 3: Effects of sex and genotype in the elevated plus-maze task. (A) The rats spent similar time in the open arms, as no statistical difference was observed. (B) A significant genotype effect was observed in the traveled distance at 10 months (F 1, 50 = 4.761, p = 0.0338), and a significant sex effect at 14 months (F 1, 34 = 7.026, p = 0.0121). (C) A significant genotype effect was revealed (F 1, 51 = 10.55, p = 0.0021) and a trend toward the sex effect (F 1, 51 = 4.017, p = 0.0504) in peeking out from the closed arms at 10 months. Both AD males and females exhibited reduced peeking compared to their WT counterparts. (D) Furthermore, head dipping from the open arm was not significantly different at 10 or 14 months of age. (E) Representative trajectories of 10-month-old (left) and 14-month-old (right) rats. Data in the graphs show mean ± SEM; the dots represent individual values. Two-way ANOVA, * p < 0.05. 3.3 Social Interaction Task Three rats from the 10-month-old group (one WT female and two WT males) were not included in the analysis as they showed minimal activity in this testing session and did not interact with partner rats. No aggressive behavior was observed between rats. Two-way ANOVA showed a significant main effect of genotype at 10 (F 1, 51 = 4.799, p = 0.0331) and 14 months (F 1, 34 = 8.383, p = 0.0066) ( Fig. 4A ). Analysis of specific social interaction subtypes revealed that nose-to-nose investigation times mirrored the total social investigation duration pattern, with male AD rats showing significantly reduced anogenital exploration compared to all other groups at 14 months. These findings are described in detail in the Supp. Fig. 1 . As for the nonsocial parameters, the analysis of rearing behavior showed significant effects in both age groups. Two-way ANOVA revealed an effect of genotype at 10 months (F 1,51 = 7.966, p = 0.0068) and 14 months (F 1,34 = 7.242, p = 0.011), with a trend towards sex significance (F1, 34 = 3,790, p = 0.0599) at 14 months ( Fig. 4B ). For grooming behavior, 10-month-old rats showed only a significant effect of sex (F 1,51 = 4.056, p = 0.0493) ( Fig. 4C ). At 14 months, the analysis revealed both a significant interaction (F 1,34 = 4.867, p = 0.0342) and a main effect of sex (F 1,34 = 8.148, p = 0.0073). Post-hoc comparisons demonstrated that AD males groomed significantly more than AD females (p = 0.0047) ( Fig. 4C ). Fig. 4: Social interaction task results. (A) The analysis of behavior during social interactions showed prominent deficits in the AD group. Specifically, a significant genotype effect was confirmed in the total interaction time of both 10-month-old (F 1, 51 = 4.799, p = 0.0331) and 14-month-old rats (F 1, 34 = 8.383, p = 0.0066). Similarly, total rearing time showed a significant genotype effect in both 10-month-old (F 1,51 = 7.966, p = 0.0068) and 14-month-old (F 1,34 = 7.242, p = 0.011) rats, and a trend toward sex significance (F1, 34 = 3,790, p = 0.0599) at 14 months. On the other hand, grooming in both age groups showed significant effects of sex (F 1,51 = 4.056, p = 0.0493 for 10-month-old, F 1,34 = 8.148, p = 0.0073 for 14-month-old) and sex x group interaction effect in 14-month-old (F 1,34 = 4.867, p = 0.0342) rats. Post-hoc tests showed that AD male rats spent more time grooming themselves than AD female rats (p = 0.0047). (B) Representative examples of social interactions (anogenital above, non-anogenital in the middle), rearing, and grooming (below). Data in the graphs show mean ± SEM; the dots represent individual values. Two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. 4 Discussion Anxiety, depression, and social withdrawal often emerge as early manifestations of AD and may precede cognitive decline (Palmer et al., 2007; Delrieu et al., 2014; Gallagher et al., 2011). Numerous studies have demonstrated anxiety-like behavior and reduced social exploration across various rodent AD models (Pentkowski et al., 2021, Pietropaolo et al., 2012; Filali et al., 2011; Locci et al., 2021). In our study, we investigated these behaviors and social interactions in the TgF344-AD rat model at 10 and 14 months of age – a critical period when β-amyloid plaques accumulate in the brain and cognitive symptoms emerge. We observed anxiety-like traits in the elevated plus-maze together with social interaction deficits in the AD genotype. The social deficits increased with age and showed more pronounced effects in males. In the open-field task, our findings diverged from previous studies (Lopez et al., 2024, Pentkowski et al., 2018; Wu et al., 2020), showing no genotype differences in center exploration. However, we found that 10-month-old males, regardless of genotype, exhibited reduced locomotory activity and rearing time – parameters of horizontal and vertical exploratory activity, respectively – compared to females ( Fig. 2A–C ). Our results contrast with earlier work by Cohen et al. (2013), who reported increased vertical exploratory activity (more frequent rearing) in 15-month-old AD rats, although more recent studies documented decreased horizontal exploration (distance traveled, time spent in the center or frequency of center visits) in both male and female AD rats between 6–12 months of age (Saré et al., 2020, Srivastava et al., 2023; Wu et al., 2020). This variability likely reflects methodological differences across studies – lighting during experiments, length of the testing session, explored parameters, or varying definitions of the center zone for analysis. Our elevated plus-maze findings also showed some divergence from previous research that reported reduced open-arm exploration in TgF344-AD rats (Lopez et al., 2023; Pentkowski et al., 2018; Wu et al., 2020). While traditional measures showed no genotype differences in open-arm exploration – similar to Srivastava et al. (2023) at rats’ 9 and 12 months – our analysis of additional exploratory behaviors revealed that 10-month-old AD rats of both sexes exhibited significantly reduced peeking from the closed arms ( Fig. 3B ). These differences disappeared at 14 months, with no differences in open arm time or exploratory behaviors ( Fig. 3A+C ). The locomotor patterns in the elevated plus-maze partially aligned with our open-field results: males walked less than females, but only at 14 months, not 10 months. Instead, at 10 months, locomotion was primarily affected by the AD genotype. These age-specific findings align with other studies showing behavioral deficits at particular ages, such as Saré et al. (2020) reporting hypoactivity in female AD rats at 6 and 12 months but not at 18 months, and Srivastava et al. (2023) observing anxiety-like deficits in females at 9 months but not at 12 months. A key finding of our study was the marked reduction in social investigation ( Fig. 4A ) and less rearing ( Fig. 4B ) during the social interaction task in both 10- and 14-month-old AD rats compared to WT rats. The results further showed that nose-to-nose interactions were affected sooner than anogenital interactions ( Supp. Fig. 1 ). The social deficits were particularly pronounced in male AD rats at 14 months; they showed significantly decreased anogenital interaction compared to WT males ( Supp. Fig. 1 ). Our findings complement existing research on social behavior in AD models. Reduced social interest has been reported in 6-month-old APP/PS1 male and female mice with the same genetic mutations (Pietropaolo et al., 2012; Filali et al., 2011). Although Locci et al. (2021) found no differences later at 10 months, the authors observed reduced sociability in APP NLGF and 5xFAD mice, other AD models of amyloidosis. In TgF344-AD rats specifically, Chaney et al. (2021) reported reduced social exploration at 9 months, though these differences weren’t present at 15 and 18 months and the sex of the animals was not specified in this study. The differences in rearing behavior during the social interaction task indicate variations between WT and AD rats. Specifically, WT rats displayed an increase in rearing during the social interaction task compared to their open-field task performance, whereas AD rats did not exhibit a similar increase. However, this variation could also be attributed to factors such as repeated arena exposure and the extended test duration (10 mins vs. 5 mins in the open-field task), which might have enhanced WT rats’ comfort and exploration in a familiar environment. Social odors and ultrasonic vocalizations play crucial roles in social behaviors. Previous studies have documented hyposmia in AD males across 6-, 12- and 18-month age groups, though these findings were based on a small sample size (Saré et al., 2020). Furthermore, early vocal deficits were revealed in AD males at 6 and 9 months (Rudisch et al., 2024). While we did not directly measure vocalizations, these factors may contribute to the social interaction deficits we observed. Although we did not observe prominent differences in standard measures from the open-field or elevated plus-maze tasks, several factors suggest an anxiety-like phenotype in AD rats. Notably, many male AD rats completely avoided the open arms despite maintaining activity in closed arms and both sexes of AD rats peeked less from the closed arms. The sensitivity of the elevated plus-maze test and the discrepancy between other study results may have been affected due to prior experience with other tests and differing handling procedures (described in Blokland et al., 2012; Schmitt & Hiemke, 1998). The decrease in social exploration, lower rearing, and concurrent increase in self-grooming behavior, especially evident in 14-month-old male AD rats, may indicate an anxiety-like response to social novelty ( Fig. 4C ). However, additional data are needed to confirm this hypothesis, as we did not distinguish between specific grooming patterns, which are more suitable for evaluating stressful and comfort conditions (Kalueff & Tuohimaa, 2004). An important methodological consideration is that TgF344-AD rats, generated on the Fisher background strain, develop retinal degeneration and vision problems with age (Pena et al., 2021; Tsai et al., 2014). As albinos, they are sensitive to prolonged exposure to standard light intensities (Rao, 1991). We maintained housing illumination below 50 lux and conducted experiments under dimmed light (5–10 lux). Most previous studies using TgF344-AD rats do not specify housing conditions or experimental lighting (Chaney et al., 2021, Lopez et al., 2023, Pena et al., 2021, Pentkowski et al., 2018; Rudisch et al., 2024, Saré et al., 2020, Srivastava et al., 2023, Wu et al., 2020), which could potentially confound behavioral data and exacerbate vision loss in AD rats. Sex differences in disease progression are notable in TgF344-AD rats, with certain symptoms being more prominent in males and others in females (Lopez et al., 2024; Saré et al., 2020; Srivastava et al., 2023). Cognitive decline in male TgF344-AD rats appears earlier than in females. Male rats show learning and memory impairments in the Morris water maze (MWM) at both 9 and 12 months, while female rats only show these impairments at 12 months (Srivastava et al., 2023). Interestingly, these cognitive changes coincide with distinct metabolic profiles – males show enhanced glucose metabolism at 12 months while females display reduced glucose metabolism (Srivastava et al., 2023) and more advanced β-amyloid pathology (Chaudry et al., 2022). Female TgF344-AD rats also tend to exhibit more prominent anxiety-like behaviors and hypoactivity in open-field tasks at earlier time points (Lopez et al., 2024; Saré et al., 2020; Srivastava et al., 2023). These sex-dependent manifestations underscore the importance of considering sex as a biological variable in AD research. The temporal progression of anxiety-related behavior, followed by social deficits, may reflect the sequential impact of AD pathology on distinct neural circuits. Our work provides new insights into anxiety-like behaviors during the mid-stage of the disease and demonstrates reduced social engagement across the two tested ages (10 and 14 months). Importantly, these findings parallel clinical observations of increased social anxiety and withdrawal in AD patients, suggesting that the TgF344-AD model recapitulates important aspects of the social behavioral alterations associated with AD pathology in humans. Limitations Several limitations should be considered when interpreting our findings. First, we did not control for potential confounding factors such as animal motivation or depression-like behaviors, which could have influenced our behavioral observations. This is particularly relevant for our social interaction findings, where we observed the most pronounced differences. Future studies should incorporate specific controls for these variables to better isolate disease-specific effects on social behavior. Second, our behavioral analysis was not comprehensive across all testing paradigms. For instance, grooming and rearing behaviors were not evaluated in the elevated plus maze, limiting our ability to draw conclusions about how different environmental contexts affect rats’ behavior. Additionally, our behavioral assessments focused exclusively on later disease stages when pathology was already established, potentially missing important early behavioral changes. To address these limitations, future investigations should examine social interactions during the prodromal phase of the disease. Such studies would benefit from incorporating advanced methodologies, including deep learning-based behavioral analysis tools to detect subtle changes in social dynamics and ultrasonic vocalization recordings to provide a more complete characterization of social interactions. These approaches could help identify early behavioral biomarkers of the disease. Conclusion In this study, we conducted the open-field, elevated plus-maze, and social interaction paradigms in TgF344-AD male and female rats (10 and 14 months of age). While traditional measures of anxiety-like behavior in the open-field and elevated plus-maze tasks showed limited genotype effects, we identified subtle sex-dependent behavioral changes, including reduced exploratory behaviors and locomotor activity, particularly in males. Furthermore, AD rats showed particularly pronounced social interaction deficits that persisted across both age points, accompanied by increased self-grooming behavior in males. Our findings of anxiety-like behaviors and social deficits parallel clinical observations in AD patients, supporting the validity of the TgF344-AD rat model for studying these early non-cognitive symptoms of AD. Future studies should continue to consider sex as a biological variable and carefully control for methodological factors, particularly lighting conditions, which may significantly impact behavioral outcomes in this light-sensitive strain. CRediT authorship contribution statement DČ – formal analysis, investigation, writing - original draft, writing - review and editing, visualization KM – conceptualization, methodology, formal analysis, writing - review & editing AH – investigation, formal analysis AS – formal analysis, writing - review and editing, supervision, funding acquisition JS – conceptualization, formal analysis, writing - review & editing, project administration, funding acquisition Declaration of competing interest None. Data availability The data generated and/or analyzed in this study are available from the corresponding authors upon reasonable request. Funding sources This research was supported by the Czech Science Foundation (GACR) grant 25-16227S and by the project National Institute for Neurology Research (Programme EXCELES, ID Project No. LX22NPO5107)—Funded by the European Union–Next Generation EU. Declaration of generative AI and AI-assisted technologies in the writing process The authors used Claude 3.5 Sonnet to improve the readability and language of the manuscript. The authors reviewed and edited the content as needed and take full responsibility for the content of the published article. References Berkowitz, L. E., Harvey, R. E., Drake, E., Thompson, S. M., & Clark, B. J. (2018). Progressive impairment of directional and spatially precise trajectories by TgF344-Alzheimer’s disease rats in the Morris Water Task. 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Medicine and Science in Sports and Exercise, 52(7), 1456–1469. https://doi.org/10.1249/MSS.0000000000002294 Supplementary Material File (fig3.eps) Download 17.78 MB Information & Authors Information Version history V1 Version 1 11 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords alzheimer’s disease anxiety sociability social behavior tgf344-ad Authors Affiliations Daniela Černotová Institute of Physiology Czech Academy of Sciences View all articles by this author Kristýna Malenínská Institute of Physiology Czech Academy of Sciences View all articles by this author Anna Horáková Institute of Physiology Czech Academy of Sciences View all articles by this author Ales Stuchlik Institute of Physiology Czech Academy of Sciences View all articles by this author Jan Svoboda 0000-0002-0343-5347 [email protected] Institute of Physiology Czech Academy of Sciences View all articles by this author Metrics & Citations Metrics Article Usage 311 views 164 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Daniela Černotová, Kristýna Malenínská, Anna Horáková, et al. Social Interaction Deficits Emerge Alongside Anxiety-Like Behavior in TgF344-AD Rats. Authorea . 11 February 2025. 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