Breeding pairs of homozygous female and hemizygous male B6.129P2-Fmr1^TM1Cgr/J mice (Fmr1-KO; #:003025) (The Dutch-Belgian Fragile X Consortium, 1994), and BTBR T⁺ Itpr3^tf/J mice (BTBR; #:002282) (Petkov et al., 2004; Timmermans et al., 2017) were purchased from Jackson Laboratories, Bar Harbor, ME, USA while C57BL/6JRj (B6) breeding pairs were purchased from Janvier Labs, Le Genest-Saint-Isle, France (# SC-C57J-F). All mice were further bred at the AAALAC-accredited animal facility of QPS Austria GmbH, Grambach, Austria. All animals were housed in single-sex groups of 3−5 mice per cage and kept in ventilated cages under a 12:12 h dark-light cycle and a temperature of 21 ± 1°C at 40 to 70% humidity. Dried pelleted standard rodent chow (Altromin^®, Lage, Germany) and water were provided ad libitum. All behavioral tests were performed during the light cycle phase (07:00 a.m.−4:00 p.m.). Each test was continuously performed at the same time of the day. Animals were allocated to experimental groups according to their strain and initial body weight. The use of animals was subject to the statutory provision under the Austrian Animal Welfare Act and was approved by the Styrian government, Austria (ABT13-67744/2020).

A total of 45 male BTBR and Fmr1-KO mice with a starting age of 4 weeks were allocated to receive either LP-211 (LP; 3 mg/kg, provided by M. Leopoldo, Bari, Italy. Purity > 98%), R-Baclofen (Bf; 1.5 mg/kg, Sigma Aldrich, St. Louis, MO, USA, G013) or vehicle (PEG400 20% v/v + Tween80 10% v/v + 0.9% saline). In addition, control groups of 15 male B6 animals received vehicle only. All animals were treated with an intraperitoneal injection 30 min before each behavioral test performed after age of 5 weeks as these strains are expected to exhibit relevant ASD behaviors mostly from this age (Leopoldo et al., 2011; Silverman et al., 2015).

Based on previous findings of these mouse models (Kazdoba et al., 2014; Silverman et al., 2015; Meyza and Blanchard, 2017; Costa et al., 2018; Zeidler et al., 2018), the behavioral test battery as well as compound dosing selection were designed as indicated in Figure 1. BTBR and Fmr1-KO mice are biologically and developmentally different, thus a different experimental design was applied for these animals. Each of these strains show ASD phenotypes such as modulated anxiety, at particular age stages, the testing timeline was therefore adjusted for each strain accordingly. Therefore, each analyzed parameter was scheduled based on phenotypic characteristics of BTBR and Fmr1-KO mice. All animals were weaned on postnatal day 21. Before each behavioral test, animals were habituated to the test room for 45 min.

Animal’s body weight was recorded every 7 days throughout the whole study. Veterinarian care was provided to animals that lost body weight by providing daily wet food pellets on the cage floor starting from day of weaning (postnatal day 21) until 6 weeks of age.

Data analysis was performed by GraphPad Prism™ 9.2 (GraphPad Software Inc., San Diego, CA, USA). All graphs present group means with standard error of the mean (SEM). The significance level was set at p < 0.05. Group means were compared using One-way or Two-way analysis of variance (ANOVA), with a subsequent post hoc; Dunn’s Multiple Comparison or Fischer’s LSD test. For comparison of two groups, Student’s t-test was performed. Significances were defined as *p < 0.05; ^**p < 0.01; ^***p < 0.001.

Pearson’s Correlation Coefficient (r) was used to compare animals’ body weight and latency to fall off the wire in the wire suspension test. Moreover, the results of the Pearson’s Correlation test of BTBR and Fmr1-KO mice were compared using Fischer’s Z transformation (Eid et al., 2011). To this aim, the online tool https://www.psychometrica.de/correlation.html#fisher was used. Z- and p-values are provided.

The exact group size and utilized statistical tests are mentioned in the figured legends.

All raw data of this study are provided in Supplementary Table 1.

The wire hanging test was performed as part of the general health check before treatment start of the efficacy study. Based on body weight results, mice were allocated to treatment groups, so that all groups would present similar body weight averages. The wire hanging test evaluating neuromuscular changes was performed at 4 weeks of age. The body weight of all animals was measured immediately thereafter and correlated with the wire hanging results. Results show that BTBR mice fell significantly earlier off the wire compared to B6 mice (Figure 2A; p ≤ 0.001). Body weights of BTBR mice was significantly higher compared to B6 mice (Figure 2B; p = 0.004). In addition, results of the wire hanging test were correlated with body weight data (Figures 2C, D). There was a significant negative correlation of body weight and the latency to fall in BTBR mice (Figure 2C; r = −0.6082; p ≤ 0.0001) but no correlation of these parameters in B6 mice (Figure 2D; r = 0.3872; p = 0.2137). Moreover, there was a significant difference between correlations of time hanging and body weight in BTBR mice and the B6 control group (z = −3.028; p = 0.001).

The Fmr1-KO mice, however, did not reveal any motor dysfunction by showing a comparable latency to fall and body weight levels compared to B6 control mice (Figures 2E, F). There was no significant correlation between body weight and the latency to fall in Fmr1-KO (Figure 2G; r = 0.1015; p = 0.5072) and B6 (Figure 2H; r = −0.05767; p = 0.8382). There was no significant difference between correlations of latency to fall off the wire and body weight in Fmr1-KO mice and the B6 control group (z = 0.488; p = 0.313).

Weekly body weight measurement of Fmr1-KO and BTBR mice showed that BTBR mice are heavier than B6 mice (Figure 3A; ANOVA: genotype, p < 0.001; age, p < 0.001; interaction, p < 0.001; post hoc test, p = 0.001). After one treatment with R-Baclofen at the age of 6 weeks and 2 treatments at the age of 10 weeks, BTBR mice lost about 10% of body weight at the age of 9 and 10 weeks (Figure 3A; p ≤ 0.03). Veterinarian care was provided to animals that lost body weight by providing daily wet food pellets on the cage floor until animals gained back the lost body weight. LP-211 treatment did not affect BTBR animals’ body weight.

Fmr1-KO mice showed a similar progression of body weight gain compared to B6 mice. Wet food pellets were provided to animals that lost body weight. Fmr1-KO mice showed higher body weights in week 5 and 6 than B6 mice but differences faded with age (Figure 3B; ANOVA: genotype, p = 0.42; age p < 0.001; interaction, p = 0.002; post hoc test, p = 0.04). In week 9 and 10, Fmr1-KO mice treated with LP-211 were heavier, as the vehicle-treated Fmr1-KO mice seem to stop gaining weight (Figure 3B; p = 0.04). R-Baclofen treatment did not affect the body weight of Fmr1-KO mice.

From this point onward, animals were treated with R-Baclofen, LP-211 or vehicle 30 min prior to each test.

Locomotor activity was measured during a 30 min trial in BTBR and B6 mice at the age of 6 weeks. Hyperactivity did not show a difference between BTBR and B6 mice (Figures 4A, B). Consistent with activity and hyperactivity levels, there were no differences observed in the moved distance between BTBR and B6 mice. Acute LP-211 and R-Baclofen treatment did not affect these parameters in BTBR mice (Figures 4A-C).

Fmr1-KO mice at 7 weeks of age, however, displayed a significantly higher activity and hyperactivity (Figures 4D, E; p = 0.004; p = 0.003, respectively) compared to B6 mice during the 30 min open field test. Fmr1-KO mice moved a longer distance (Figure 4F; p = 0.002) than B6 mice in the open field box.

Evaluation of parameters related to anxiety showed a significantly higher thigmotaxis (Figure 5A; p = 0.001) and shorter time spent in the center of the box (Figure 5B; p = 0.001) in BTBR mice compared to B6 mice. Anxiety in BTBR mice was further confirmed by the observed lower rearing number and rearing duration compared to B6 mice (Figures 5C, D; p ≤ 0.001). Acute LP-211 and R-Baclofen did not alleviate anxiety-like behavior observed in BTBR mice compared to vehicle-treated BTBR mice (Figures 5A-D).

Fmr1-KO mice presented significantly less thigmotaxis compared to B6 mice (Figure 5E; p = 0.03). Moreover, low anxiety in Fmr1-KO mice was validated by the longer time animals spent in the center of the open field box compared to B6 mice (Figure 5F; p = 0.03). The rearing activity was comparable between Fmr1-KO and B6 mice. Acute treatment of LP-211 and R-Baclofen did not affect these parameters in Fmr1-KO mice, indicating no sedating effect of these drugs at the applied doses (Figures 5E-H).

Based on the observed results from the open field test, we aimed to further explore anxiety-like behavior by the widely used elevated plus maze (EPM) test in BTBR and Fmr1-KO mice at the age of 10 and 7 weeks, respectively. BTBR mice spent the same amount of time in open and closed arms and the center of the maze compared to B6 mice (Figures 6A–C). Fmr1-KO mice spent significantly more time in the open arms of the EPM compared to B6 mice (Figure 6D; p = 0.03). The cumulative duration in the closed arms, as well as central zone of the maze was similar between Fmr1-KO and B6 mice (Figures 6E, F; p = 0.06 and p = 0.68, respectively).

Examining the number of entries, BTBR mice entered the open arms as many times as B6 mice (Figure 7A). However, BTBR mice entered the closed arms more frequently than B6 mice (Figure 7B; p = 0.004). Furthermore, the BTBR mice entered the central zone of the maze more frequently than B6 mice (Figure 7C; p = 0.006). Acute LP-211 treatment of BTBR mice had neither significant effect on the number of entries to the closed arms nor the central zone of the EPM compared to the vehicle-treated BTBR (Figures 7B, C). There were no changes observed upon R-Baclofen injection in BTBR mice for the cumulative durations as well as frequencies to enter the open, closed, or central zone of the EPM compared to vehicle-treated BTBR mice. The frequency to enter the open arms was significantly higher in Fmr1-KO than B6 mice (Figure 7D; p = 0.003), while the frequency to enter the closed arms was similar between B6 and Fmr1-KO mice (Figure 7E; p = 0.07). None of the treatments was able to modulate this parameter in Fmr1-KO mice. Fmr1-KO mice entered the central zone of the EPM significantly more often compared to B6 mice (Figure 7F; p = 0.03). A decreased frequency to enter the open arms was observed in LP-211 treated Fmr1-KO mice compared to vehicle-treated Fmr1-KO mice (Figure 7D; p = 0.03). R-Baclofen treatment did not change the frequency of entries in open and closed arms and central zone of the EPM in Fmr1-KO mice compared to vehicle-treated Fmr1-KO mice.

Repetitive behavior was monitored by evaluating self-grooming in a 10 min session in BTBR and Fmr1-KO mice at the age of 10 and 7 weeks, respectively. BTBR mice spent significantly more time self-grooming compared to B6 mice (Figure 8A; p ≤ 0.001); however, there was no significant difference observed in the number of grooming episodes (Figure 8B). None of the compounds was able to reverse these parameters in BTBR mice.

Fmr1-KO mice spent a significantly longer time grooming with a higher number of grooming episodes during the 10 min of recording (Figures 8C, D; p = 0.002, p = 0.002, respectively). Acute R-Baclofen treatment significantly ameliorated the grooming duration as well as number of grooming episodes in Fmr1-KO mice (Figures 8C, D; p ≤ 0.001; p ≤ 0.001, respectively). Acute LP-211 treatment significantly reduced the grooming duration in Fmr1-KO mice (Figure 8C; p = 0.04).

Since R-Baclofen and LP-211 treatment only alleviated repetitive behavior in Fmr1-KO mice, we further studied the effect of these compounds on the social interest and communication test by measuring ultrasonic vocalization in these mice. This test was chosen to measure social communication as results of this test are less variable and more reliable compared to other tests such as the automated three-chamber social interaction test as less interventions to the test animal are needed to perform the test.

Male Fmr1-KO mice were exposed to fresh urine of estrous females and USV calls were recorded for 5 min at the age of 10 weeks. A significantly lower number of calls was observed in Fmr1-KO mice regardless of treatment compared to B6 mice (Figure 9A; p = 0.01). The latency to initiate the first call did not differ between Fmr1-KO and B6 mice and did not depend on the treatment (Figure 9B). R-Baclofen and LP-211 treatment did not affect these parameters in Fmr1-KO mice compared to vehicle-treated Fmr1-KO mice.

Overall, the results of the behavioral characterization and the efficacy study showed that male BTBR mice at the age of 4−10 weeks are a suitable model to study ASD symptoms, such as motor deficits, repetitive behavior, anxiety, and stress-like behavior. Acute R-Baclofen and LP-211 treatment could not ameliorate these alterations in BTBR mice. Fmr1-KO mice presented ASD-like behavior in terms of hyperactivity, repetitive behavior as well as communication impairments at the age of 4−10 weeks. Acute R-Baclofen and LP-211 treatment ameliorated repetitive behavior in Fmr1-KO mice.

Motor dysfunction in BTBR mice has been previously shown by Xiao et al. (2020) evaluating it from 2 weeks to 5 months of age. Researchers showed that abnormal cerebellar development could be an important factor related to motor dysfunction as well as dystonia-like behavior in these animals. Here we report motor abnormalities in BTBR mice at the age of 4 weeks as analyzed in the wire hanging test, that negatively correlated with body weights, suggesting that the body weight has a strong impact on motor abilities: only 4 out of 45 BTBR mice could hold the wire for more than 200 s. We thus conclude that motor disability in BTBR mice starts at an early age.

Approximately half of the children with ASD meet the diagnostic criteria of hyperactivity as well as attention-deficit disorder (Murray, 2010), thus evaluating locomotor activity in ASD animal models is an important parameter to evaluate their translatability. Here, when measuring the locomotor activity in the non-social context of the open field test, 6 weeks old BTBR mice presented no differences in activity and hyperactivity. A previous report of the same test performed with 22 days old BTBR mice showed that they traveled more than B6 controls in the first 15 min of the test session that was performed under red light (McFarlane et al., 2008). Furthermore, another study showed that 8 weeks old BTBR mice present hyperactivity compared to wild type control mice by an increased distance moved in all zones in the open field test (Xiao et al., 2020).

However, another study measured the distance moved in the open field test in 15 months old male BTBR mice for 30 min under room lightening, in which BTBR mice moved a comparable distance than B6 controls (Jasien et al., 2014). Differences in these results can be explained by different test settings like the age of animals, duration of recording, and room light condition. Overall, BTBR mice appear to present hyperactivity behavior at a young age until early adolescence but lose this phenotype during adulthood.

We further evaluated the anxiety level of BTBR mice by measuring anxiety parameters in the open field and EPM at the age of 6 and 10 weeks, respectively. BTBR mice presented a high anxiety profile both in open field and EPM test. When considering the total amount of the time spent in the open field box, which is a sum of thigmotaxis and time spent in the center, BTBR mice strongly exhibited anxious behavior by spending most of the time at the outer boundaries of the box and the least time in the center. Previous reports showed an anxiogenic profile of 14−17 weeks old BTBR mice by using the EPM as animals showed a significantly lower number of entries in the open arms. Authors mention that the lightening of the test room was a stressor to which BTBR mice show a strong response, manifesting high stress-like behavior in BTBR mice (Pobbe et al., 2011). Moreover, it is worth to mention that BTBR mice are shown to have impaired photoreceptor function and therefore present atypical visual perception which mimics some subset of ASD patients (Cheng et al., 2020). Another research group reported anxiety in 8−12 months old BTBR mice compared to wild type mice by the elevated open platform test, where BTBR mice spent more time at the edges and less time in the center of the open platform (Chao et al., 2018). Another study assessing anxiety-like behavior in aged (19−21 months old) BTBR mice showed a decreased anxiety-like behavior compared to existing results in younger mice by showing that animals spent less time in the closed arms of the EPM (O’Connor et al., 2021). We thus conclude that BTBR mice exhibit anxiety and stress-like behavior at adolescent age.

Furthermore, we confirmed repetitive behavior in the self-grooming test, as 10 weeks old BTBR mice spent a longer time self-grooming compared to controls. These results are consistent with previous reports testing BTBR mice for self-grooming from postnatal day 18 to 60 (McFarlane et al., 2008) as well as weeks 7 to 8 (Amodeo et al., 2014). Furthermore, O’Connor et al. (2021) showed higher repetitive behavior of self-grooming in 19−21 months old BTBR mice compared to wild type mice. We thus conclude that BTBR mice exhibit repetitive behavior at adolescent age, and, based on previous reports, maintain this behavior lifelong.

Based on a study by Pietropaolo et al. (2011), the B6 background is more suitable to study core autism symptoms of social impairments, repetitive behaviors, as well as hyperactivity compared to the FVB background. Here, Fmr1-KO mice on a B6 background were thus used to evaluate ASD-like behaviors. The results obtained from the behavioral characterization of Fmr1-KO mice are consistent with previous reports (Michalon et al., 2012; Kramvis et al., 2013; Hébert et al., 2014; Kazdoba et al., 2014; Gantois et al., 2017; Gaudissard et al., 2017). In summary, Fmr1-KO mice did not show any motor dysfunction and presented comparable body weights as B6 mice. Although a micro-computerized tomography analysis performed by Leboucher et al. (2019) showed that the deficiency in the Fmr1 gene impacts skeleton and bone morphology in 16 weeks old Fmr1-KO mice, it does not influence the animal’s body weight and motor performance. Moreover, in Fmr1-KO mice the body weight does not correlate with the latency to fall off the wire in the wire suspension test. Thus, a heavier mouse does not necessarily fall off earlier compared to a lighter mouse.

The observed differences in the latency to fall off the wire in control animals of BTBR and Fmr1-KO studies could be caused by the different breeding pairs that were used for each study.

Results of the behavioral characterization of Fmr1-KO mice in the open field and EPM test indicate hyperactivity as well as lower anxiety in this mouse model consistent with previous reports performed in 2−5 months old Fmr1-KO mice using the same tests (Mineur et al., 2002; Qin et al., 2002; Saré et al., 2016). Studies evaluating overlapping anxiety and hyperactivity in humans show that around 50% of adults and up to 30% of children with attention deficit hyperactivity disorder (ADHS) meet both diagnostic criteria (Pliszka, 2000; Michelini et al., 2015; Mulraney et al., 2018; van der Meer et al., 2018). Though it must be pointed out that the complexity of neuropsychiatric disorder symptoms such as hyperactivity such as excessive activity, decreased sleep need, disturbed behavior, etc., challenges the interpretation of data from animal studies (Nestler and Hyman, 2010; Yen et al., 2013). Hence, the lower anxiety-like behavior observed in Fmr1-KO mice detected by a higher frequency to enter and more time spent in the open arms of the EPM test could be affected by the higher activity and hyperactivity observed in these mice. Although hyperactivity might influence the results of anxiety analyses, evaluation of Fmr1-KO mice in the EPM and open field test definitively demonstrates that these animals have a reduced anxiety.

Repetitive behaviors are considered a key characteristic of ASD (Caldwell-Harris, 2021), thus reproducing them in animal models is crucial for the use of these models for further ASD research. Fmr1-KO mice showed repetitive behaviors by spending more time as well as more episodes of self-grooming compared to controls. These data are consistent with previous reports about the grooming behavior of 3 months old Fmr1-KO mice on a B6 background (Mines et al., 2010; Pietropaolo et al., 2011).

Results from the ultrasonic vocalization recording of 10 weeks old Fmr1-KO mice revealed impaired sociability and communication deficits in this mouse model. Some studies showed a similar average quantity of calls from adult Fmr1-KO compared to wild type mice. One example is the study where Fmr1-KO mice were bred on an FVB background and USV was recorded while exposing mice to fresh urine collected from estrus female mice for 5 min (Hodges et al., 2017). Moreover, another study reports Fmr1-KO on B6 background at the age of 2−3 months while directly exposed to receptive female mice for 2 min (Belagodu et al., 2016). Another study reported a reduced rate of ultrasonic calls during mating for 10 min by 2−3 months old Fmr1-KO mice on an FVB background compared to wild type mice and thus on a different background than the one used here (Rotschafer et al., 2012). We can conclude that not only age but also the genetic background as well as the test setting, e. g., female mice vs. urine of female mice (Hoffmann et al., 2009) as well as exposure time can affect the outcome of the ultrasonic vocalization test of adult male Fmr1-KO mice (Premoli et al., 2021).

R-Baclofen, a GABA_B receptor agonist, has been already used in various clinical and preclinical studies to ameliorate ASD phenotypes (Cryan et al., 2004; Erickson et al., 2014; Qin et al., 2015; Berry-Kravis et al., 2017; Veenstra-VanderWeele et al., 2017; Stoppel et al., 2018; Möhrle et al., 2021). The applied drug concentration is critical, especially in studies measuring behavioral changes where deleterious side effects such as sedation or hyperactivity need to be avoided. A study showed a paradoxical effect of chronic R-Baclofen not only in the Fmr1-KO mouse model but also in B6 control mice by increasing activity (Zeidler et al., 2018). Another study reported the sedative effects of R-Baclofen in higher doses such as 3−5 mg/kg intraperitoneal treatment in the BTBR and C58 ASD mouse models (Silverman et al., 2015). Therefore, in the current study, the concentration of 1.5 mg/kg was carefully selected to induce disease ameliorating effects while preventing any side effects. Concentrations less than 3 mg/kg have been reported to be effectless on locomotor activity, repetitive behavior, as well as sociability in B6 mice (Silverman et al., 2015), therefore, no B6 mice were treated with R-Baclofen in the current study.

In 2005, Silverman et al. (2015) evaluated the efficacy of R-Baclofen on both sexes of BTBR mice at the age of 10−12 weeks with a single dose of 1 and 3 mg/kg 60 min prior to testing. Both doses improved sociability in BTBR mice but only the 3 mg/kg dose could improve the repetitive behavior of these mice. The same research group showed in an earlier study that 1 mg/kg of R-Baclofen did not reverse the social impairment of male-female interaction in BTBR mice (Silverman et al., 2015). In the present study, BTBR vehicle-treated mice showed higher anxiety and higher repetitive grooming behavior. R-Baclofen treatment did not improve these parameters. Thus, it might be possible that each behavioral assay requires a different optimal dose of R-Baclofen to be effective. Chronic treatment with R-Baclofen might be more useful, not only because ASD is a lifelong disorder and would require constant treatment, but it would also blunt the observed side effects when using higher concentrations. As discussed by Silverman et al. (2015), comprehensive studies conducting both acute and chronic treatments for each behavioral assay would be the most effective way of assessing compound efficacy.

In Fmr1-KO mice, there were no improvements in hyperactivity, anxiety level, and communication detected after R-Baclofen treatment. However, acute R-Baclofen injection could significantly reduce the repetitive grooming behavior in Fmr1-KO mice. While both BTBR and Fmr1-KO are used in ASD research, (Silverman et al., 2015) they are genetically and behaviorally very different (Nestler and Hyman, 2010; Meyza and Blanchard, 2017; Nikvarz et al., 2017; Niu et al., 2017). Therefore, the different effects of R-Baclofen on BTBR and Fmr1-KO mice’ behavior are not unexpected. Nonetheless, as previously mentioned, a more efficient way to study compound effects, is to study it separately for each behavioral assay considering both acute and chronic administrations.

The serotonin 5-HT₇ receptor agonist LP-211 has already been used to alleviate object recognition memory and stereotypic behavior by an acute administration of 3 mg/kg in 3–4 months old Fmr1-KO mice but did not affect wild type mice (Costa et al., 2018). In addition, Costa et al. (2015, 2018) showed that activation of the 5-HT₇ receptor by LP-211 reversed mGluR-LTD in the hippocampal region of Fmr1-KO mice and prevented internalization of AMPA receptors by stimulating the adenylate cyclase and protein kinase A. LP-211 not only reversed mGluR-LTD in Fmr1-KO mice but also in wild type animals, exhibiting the potential of this compound to modulate synaptic plasticity impairments in Fmr1-KO mice (Costa et al., 2015). In 2020, Armstrong et al. (2020) showed that improvement of pharmacological properties and pharmacokinetics of a serotonergic compound, a combination of 5-HT₁A, 5-HT₂C, and 5-HT₇ receptor agonists, can ameliorate social deficits, prevent audiogenic seizures, and decrease repetitive behavior in male and female Fmr1-KO mice on a FVB background. Chronic treatment with a low dose of LP-211 was further shown to improve Rett-syndrome-related phenotypes such as memory deficits and anxiety (De Filippis et al., 2014, 2015). Since LP-211 was already shown to penetrate the brain within 30 min after intraperitoneal injection (Hedlund et al., 2010; Costa et al., 2018), we used acute injections of 3 mg/kg of LP-211 30 min before each testing in Fmr1-KO mice resulting in significantly reduced anxious behavior in Fmr1-KO mice.

We were able to reproduce the positive effect of an acute LP-211 administration on repetitive behavior in Fmr1-KO mice even at a younger age than previously used (Costa et al., 2018). Additionally, Lacivita et al. (2020) synthesized and evaluated a variety of long-chain arylpiperazine compounds with biased selectivity to 5-HT₇R that showed higher drug-like properties and a high affinity to distinctive 5-HT₇ receptors resulting in a significantly improved repetitive behavior in 12–16 weeks old Fmr1-KO mice.

Altogether, previous reports conclude that LP-211 corrects synaptic plasticity, abnormal intracellular signaling, as well as memory and repetitive behavior. From our current report, we can further conclude that LP-211 rescues some of the behavioral abnormalities including repetitive behaviors in Fmr1-KO mice and it seems that LP-211 can also be used to ameliorate anxiety (Leopoldo et al., 2011; Costa et al., 2015, 2018; Lacivita et al., 2017).

We did not detect a positive effect of LP-211 on behavioral abnormalities in BTBR mice. Although, previous reports showed serotoninergic axon density reduction in the hippocampus (Guo and Commons, 2017) and a rescue of repetitive behavior in BTBR mice after acute treatment with a 5-HT₆ receptor blocker (Amodeo et al., 2021). Finally, we can conclude that repetitive behavior seemed to be linked to the serotoninergic system as reported previously and that serotoninergic compounds could improve this behavior in ASD mouse models such as Fmr1-KO mice (Lewis and Kim, 2009; Muller et al., 2016). Further research is needed to improve the pharmacological properties and study the effect of serotoninergic compounds such as LP-211 in other ASD mouse models.

So far, no FDA-approved compound exists to improve core symptoms of ASD. Animal studies are critical and valuable tools to investigate the treatment effects of already existing pharmacological compounds and to develop new drug candidates. Our preclinical data from the characterization of BTBR and Fmr1-KO mice by using a similar but strain-specific behavioral test battery supports the use of these mouse models for ASD research. Moreover, evaluating the efficacy of two existing pharmacological tools showed the value of these strains for future novel therapeutics. We could show some of the core and secondary symptoms in these models, and also point out how precisely mouse models can mimic the ASD phenotypic spectrum with a wide diversity in abnormality severity of abnormality. We observed strong hyperactivity in Fmr1-KO mice, but a moderate level in BTBR mice. Measurements of anxiety-like behavior revealed high levels of this behavior in BTBR mice, but lower anxious behavior in Fmr1-KO mice which could be explained as a side effect of the observed hyperactivity. Repetitive behavior was detected in both models. In addition, Fmr1-KO mice showed impaired communication confirmed by ultrasonic vocalization measurements.

Altogether these observations support the comorbidity of ASD core symptoms with motor dysfunction (Bougeard et al., 2021; Posserud et al., 2021), hyperactivity, and anxiety in ASD individuals (Avni et al., 2018; Nimmo-Smith et al., 2020). Our data support the idea that the GABAergic and serotoninergic systems play an important role for ASD phenotypic symptoms. We could improve the repetitive behavior, one of the core symptoms of ASD, observed in Fmr1-KO mice with only a single injection of a low dose of R-Baclofen or LP-211.

Although ASD symptoms of BTBR and Fmr1-KO mice and the impact of R-Baclofen and LP-211 have been studied previously, only a fraction of the age-related ASD phenotype of these models are understood so far. Moreover, as observed in the current study, each compound mostly shows a slight effect on the behavioral abnormalities of BTBR and Fmr1-KO mice. A combined treatment of R-Baclofen and LP-211 should be considered as a strategy for future ASD studies.