Deficits in Skilled Motor and Auditory Learning in a Rat Model of Rett Syndrome

doi:10.21203/rs.3.rs-31477/v2

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Deficits in Skilled Motor and Auditory Learning in a Rat Model of Rett Syndrome

https://doi.org/10.21203/rs.3.rs-31477/v2

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published 28 Sep, 2020

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Background

Rett Syndrome is an X-linked neurodevelopmental disorder caused by a mutation in the gene MECP2 . Individuals with Rett Syndrome display developmental regression at an early age, and develop a range of motor, auditory, cognitive and social impairments. Several studies have successfully modeled some aspects of dysfunction and Rett Syndrome-like phenotypes in transgenic mouse and rat models bearing mutations in the MECP2 gene. Here, we sought to extend these findings and characterize skilled learning, a more complex behavior known to be altered in Rett syndrome.

Methods

We evaluated the acquisition and performance of auditory and motor function on two complex tasks in heterozygous female Mecp2 rats. Animals were trained to perform a speech discrimination task or a skilled forelimb reaching task.

Results

Our results reveal that Mecp2 rats display slower acquisition and reduced performance on an auditory discrimination task than wild-type (WT) littermates. Similarly, Mecp2 rats exhibit impaired learning rates and worse performance on a skilled forelimb motor task compared to WT.

Conclusions

Together, these findings illustrate novel deficits in skilled learning consistent with clinical manifestation of Rett syndrome and provide a framework for development of therapeutic strategies to improve these complex behaviors.

Developmental Neuroscience

Rett syndrome

MeCP2

motor

auditory

learning

Rett syndrome is a rare neurodevelopmental disorder associated with a mutation in the X-linked gene MECP2. This disorder mostly affects females, who display normal early development followed by regression of already acquired skills (Chahrour & Zoghbi, 2007). Individuals with Rett syndrome exhibit a range of symptoms, including seizures, breathing abnormalities, motor impairments, changes in sensory responses, anxiety, and speech-language deficits (Chahrour & Zoghbi, 2007; Hagberg et al., 1986). The development of interventional strategies to improve function in these domains is of clear clinical importance.

To address this and provide a testbed for evaluation, transgenic mouse and rat models bearing mutations in the MECP2 gene have been developed (Goffin et al., 2011; Guy et al., 2001; Samaco et al., 2008; Veeraragavan et al., 2015). Some features of these models have been well-characterized and provide substantial insight into the pathophysiology of Rett syndrome. However, testing complex behaviors in Mecp2 rodents is in its early stages. Initial work successfully modeled psychomotor regression similarly seen in girls with Rett Syndrome by using a seed opening task in Mecp2 rats (Veeraragavan et al., 2015). Other studies using the same rat model have found abnormalities in gait, rotarod performance, and speech discrimination performance (Bhattacherjee et al., 2017; Engineer et al., 2015). It is likely that detailed characterization of other complex behaviors known to be altered in Rett syndrome, such as skilled learning, could expand the utility of these animal models. Here, we sought to characterize the acquisition and performance of skilled motor function, and to replicate the previous findings characterizing auditory discrimination performance.

To do so, we trained female heterozygous Mecp2 rats and wild-type (WT) littermate control rats to perform a speech discrimination task to assess auditory processing or a skilled forelimb reaching task to evaluate motor function. In both the complex auditory and motor tasks, Mecp2 rats exhibit acquisition of the task and improved performance with continued training. However, the rate of learning, as well as several metrics of performance, are reduced in Mecp2 rats compared to WT, indicative of a deficit in skilled learning. Impaired performance corresponds to dysregulated neural function in auditory and motor networks in transgenic Mecp2 models (Engineer et al., 2015; Morello et al., 2018). These results provide a novel characterization of skilled learning in Mecp2 rats and develop a framework for future studies to evaluate the effect of potential interventions on complex behavioral tasks.

Subjects

Twenty-four female Mecp2 heterozygous rats and 18 female wild-type littermates were used in this study. Rats were generated by breeding a Mecp2 heterozygous Sprague Dawley female with a wild-type Sprague Dawley male. Female breeders were obtained from Horizon Discovery (SAGE labs), using zinc-finger nuclease technology that generated a 71 base pair deletion in Exon 4 (Bhattacherjee et al., 2017; Engineer et al., 2015; Veeraragavan et al., 2015). Experimenters were blind to the genotype. Animals underwent behavioral assays and training between 4-8 months of age. Nine Mecp2 rats and 11 wild-type rats underwent auditory discrimination training. Fifteen Mecp2 rats and 13 wild-type rats underwent motor training. Following motor training, 7 of the Mecp2 rats and 9 wild-type littermates underwent intracortical microstimulation (ICMS). All animals were housed in a 12:12 hour reversed light-dark cycle, and were food deprived during training. A small subset of animals did not complete all tasks they were assigned to (Additional File 1). Similar to previous studies (Engineer et al., 2015), eighteen of the 24 Mecp2 rats had one or more seizures. When seizures occurred, rats were given a 30 minute break or removed from the session for the day. All protocols were approved by The University of Texas at Dallas Institutional Animal Care and Use Committee.

Auditory Behavioral Testing

Speech discrimination tasks and procedures were similar to previous studies (Engineer et al., 2008, 2014, 2015). Training sessions occurred twice a day for one hour each, five days a week. Rats were first trained to press a lever or nose poke for a food reward until they reached a criteria of 100 independent responses for two sessions. Once this criteria was met, rats began training on a sound detection task. Rats were trained to respond to the target speech sound ‘dad’ within 3 seconds of sound presentation until they reached the criteria of ≥ 75% correct for 5 sessions, or until they reached 50 sessions of detection. Rats received a 45 mg food pellet reward (45 mg dustless precision pellet, BioServ Frenchtown, NJ) for a correct response to the target sound ‘dad’, and received a 6 second time out for an incorrect response to silence catch trials (Fig. 1A). Following this detection task, rats were trained on a speech discrimination task, in which they were required to respond to the target sound ‘dad’, and ignore the non-target sounds ‘bad’, ‘sad’, ‘deed’, and ‘dood’ (Fig. 1D). Each rat was trained on the discrimination task for 3 weeks. Rats then began training on a speech in noise task in which they were tasked to respond to the target sound ‘dad’, and ignore non-target sounds (‘bad’, ‘sad’, ‘deed’, and ‘dood’) during various levels of background noise (0, 48, 54, 60, and 72 dB) with the speech sounds presented at 60 dB (Fig. 1G). Each rat was trained on the speech in noise task for 2 weeks.

All speech sounds were spoken by a female native English speaker, as in previous studies (Engineer et al., 2008, 2014, 2015). Sounds were presented so that the loudest 100 ms of the vowel was 60 dB. The STRAIGHT vocoder was used to frequency shift all speech sounds up by one octave into the rat hearing range, while leaving all temporal information intact (Kawahara, 1997).

Motor Behavioral Testing

Rats were trained on a skilled motor task, as in previous studies (Hays et al., 2013; Hulsey et al., 2016, 2019; Morrison et al., 2019). The task was an automated lever pressing task, in which the animal was required to learn to reach outside of a cage and press a lever twice in rapid succession. The behavior apparatus consisted of an acrylic cage, with a slot in the front right for access to a lever that was positioned -1 (inside) to 2 cm (outside) away from inside the edge of the chamber (Hays et al., 2013; Hulsey et al., 2016, 2019; Morrison et al., 2019) (Fig. 2A). Rewards pellets were delivered to a receptacle in the front left side of the apparatus. A potentiometer was affixed to the lever to record the angle of the lever relative to the horizontal. The lever allows for up to a 13° depression. A lever press was defined as a deflection greater than 9.5°, followed by a release to at least 4.75°. A spring supported the lever, providing 28 grams of resistance and allowing the lever to return to its horizontal resting position. An electronic controller board sampled the potentiometer position at 100 Hz and relayed the information to MotoTrak software the controlled the task criteria and collected data (MotoTrak, Vulintus, Louisville, CO).

Training sessions were 30 minutes in duration and occurred twice per day separated by at least two hours, five days per week. Rats were first trained to press a lever that was positioned inside the apparatus. Throughout training, rats progressed through five stages, each requiring different criteria for trial success and consequent reward delivery, as detailed in Table 1. During the final stage, rats were rewarded for double pressing within a 500 ms window (Fig 2B). Once the final stage was completed, rats underwent intracortical microstimulation (ICMS).

Table 1

Motor behavioral training stage parameters.

Training Stage	Hit Time window (s)	Lever Location (cm)	Reward Criteria	Criterion for advancement to next stage
Stage 1	2.0	-1	Single lever press	30 pellets per session for 3 sessions
Stage 2	2.0	-1	Release of first lever press	30 pellets per session for 3 sessions
Stage 3	2.0	-1 à 2	Release of first lever press	30 pellets per session for 3 sessions
Stage 4	0.5 - 2.0	2	Double lever press	30 pellets per session for 3 sessions
Stage 5	0.5	2	Double lever press	100 pellets and 65% hit rate per session for 3 sessions

Intracortical Microstimulation

The day after the final day of motor training, rats underwent ICMS to evaluate left motor cortex organization contralateral to the trained paw. Rats were anesthetized with ketamine hydrochloride (70 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.), with supplementary doses given as needed to maintain anesthesia levels. Doxapram (20 mg/kg, i.p.) and glycopyrrolate (0.5 mg/kg, i.p.) were given to stabilize breathing and heart rate as needed. A small incision of the cistern magna was made to attenuate cortical swelling. A craniotomy and durotomy was performed to expose the left motor cortex. A tungsten electrode (0.1-1 MΩ) was inserted into the brain at a depth of 1.75 mm. Stimulation sites were then chosen at random on a grid with sites set 500 μm apart from each other. ICMS stimulation consisted of a 40 ms pulse train of 10 pulses.

ICMS procedures were conducted with 2 experimenters to ensure blinding to group and electrode location. The first experimenter placed the electrode and recorded data from each site. The second experimenter, blinded to the genotype of the rat and electrode position, delivered stimulations and classified movements. Stimulation was gradually increased from 20 μA to 250 μA, or until a movement was observed. The stimulation amplitude at which a movement was first seen was documented as the threshold. If no movement was seen at 250 μA, then that site was recorded as no response. Movements were classified as proximal forelimb, distal forelimb, head, or hindlimb. Cortical area was calculated by multiplying the number of sites eliciting a response by the area surrounding a site (0.25 mm²).

Social Behavior Tests

To assess social behavior, 9 Mecp2 rats and 9 WT littermates underwent a sociability and social novelty preference task. The behavioral arena was a rectangular box 70 cm in length x 30 cm wide x 50 cm depth. The apparatus was divided into three chambers. The two outside chambers each contained a wire cage. The experimental animal was placed inside the middle chamber, and allowed to freely explore for five minutes to habituate to the apparatus. To test sociability, an unfamiliar rat that had no prior contact was then placed into one of the wired cages, and the experimental rat freely explored for ten minutes. Sociability was defined as the amount of time the experimental rat spent in the chamber with the unfamiliar rat. The sociability index (time spent with the unfamiliar rat – time spent in the empty chamber) ÷ (time spent with the unfamiliar rat + time spent in the empty chamber) was used to indicate a preference to interact with or avoid the rat. To test social novelty, a second unfamiliar rat was placed in the wired cage in the chamber that had previously been empty. The experimental rat was allowed to freely roam for another 10 minutes. Social Novelty was defined as how much time was spent with the novel rat compared to the now familiar rat. The social novelty index (time spent with the novel rat – time spent with the familiar rat) ÷ (time spent with the novel rat + time spent with the familiar rat) was used to indicate a preference to interact with the novel rat over the familiar rat. The rats used during the sociability and social novelty tests were of the same age and sex as the experimental rats.

Standard Behavioral Assessments

To assess repetitive behaviors, 9 Mecp2 rats and 9 WT littermates underwent a marble burying test. Rats were habituated to the novel bedding (BioFresh nitrocellulose comfort bedding). Rats were then placed into a new cage with 15 marbles for 10 minutes. The number of marbles buried was recorded.

9 Mecp2 rats and 9 WT littermates also underwent a spontaneous alternation task. Rats were placed in a plus maze and were allowed to freely explore for 10 min. A spontaneous alternation occurred when a rat entered all arms within five consecutive entries. The percentage of spontaneous alternation was calculated by dividing the number of alternations by the number of possible alternations (number of alternations) ÷ (number of total arm entries – 4) x 100.

Statistical Analysis

Statistical analysis was performed with MATLAB software. Speech discrimination performance was measured as percent correct, determined by the average of correct responses to the target speech sound and correct rejections to the non-target speech sounds. Performance on the speech in noise task was taken from the first week of training. The average reaction time to respond to the target sound was also assessed. Two-way repeated measure ANOVAs were used to analyze discrimination performance between experimental groups as well as discrimination performance over time. Post hoc unpaired t-tests were used to determine statistical significance. Behavioral analysis of motor function included average training stage progression, number of trials per session, and lever inter-press interval (IPI). Significant differences for average stage progression were determined using the Wilcoxon rank-sum test. Mean IPI was calculated from the fourth and fifth stages. Significant differences in behavior measures were determined using unpaired t-tests. The chi-square test was used to determine significance in the portion of rats that completed the study. To assess ICMS data, an unpaired t-test was used to determine significance in movement representation size, total map size, and average thresholds. Measures for the behavioral assays include the number of marbles buried, sociability, social novelty, and percent alternations. Unpaired t-tests were used to determine group differences in behavioral assays. All data is reported as mean +/- standard error of the mean (SEM).

A number of studies document alterations in auditory processing in individuals with Rett syndrome (Bader et al., 1989; Foxe et al., 2016; Kálmánchey, 1990; Stach et al., 1994; Stauder et al., 2006) and animal models (Engineer et al., 2015; Goffin et al., 2011; Liao et al., 2012). We sought to evaluate the acquisition and performance on a speech discrimination task in Mecp2 rats to confirm previous findings (Engineer et al., 2015). Mecp2 and WT rats were initially trained to respond to presentation of the speech sound ‘dad’. Detection of the speech sound, calculated as the percentage of trials in which rats correctly responded to the target sound, did not significantly improve in either group over the course of five weeks of training (Fig. 1B; Two-way Repeated Measures ANOVA, F(4, 28) = 1.3288, p = 0.238). No differences in performance were observed in between groups on this simple task (Fig. 1B; Two-way Repeated Measures ANOVA, Effect of Group; F(1, 7) = 0.025, p = 0.878). A significantly slower reaction time was observed in the Mecp2 group compared to the WT control group (Fig. 1C; Mecp2: 2.45 ± 0.07 s, WT: 2.13 ± 0.1 s, Unpaired t-test, p = 0.029). These findings are consistent with previous studies and suggest that gross auditory processing is generally intact in Mecp2 rodents (Engineer et al., 2015; Goffin et al., 2011; Liao et al., 2012).

We next assessed performance in these rats on a more challenging auditory task that requires discrimination of the target sound from a number of similar distractor sounds, either in a quiet environment or in the presence of background noise. Mecp2 rats demonstrated significantly impaired performance compared to WT in the absence of background noise over the course of three weeks of testing (Fig. 1E; Two-way Repeated Measures ANOVA, Effect of Group; F(1, 18) = 5.99, p = 0.025). Post-hoc analysis revealed that Mecp2 rats performed significantly worse in week 2 of discrimination training, and a trend towards worse performance on weeks 1 and 3 (unpaired t-test, week 1: p = 0.054, t = 2.05; week 2: p = 0.0172, t = 2.62; week 3: p = 0.055, t = 2.04). The presence of background noise significantly degraded discrimination performance in both groups (Fig. 1H; Mixed Design ANOVA, Effect of Noise Level; F(4, 14) = 50.66, p = 3.55x10^-8). However, Mecp2 rats performed significantly worse than WT in multiple levels of background noise (Fig. 1H; Mixed Design ANOVA, Effect of Noise Level*Group; F(4, 14) = 3.392, p = 0.039). Post-hoc analysis revealed that Mecp2 rats performed significantly worse when 0-60 dB background noise was present (pairwise comparisons, 0 dB: F(1, 17) = 9.57, p = 0.007; 48 dB: F(1, 17) = 6.97, p = 0.017; 54 dB: F(1, 17) = 7.82, p = 0.012; 60 dB: F(1, 17) = 16.68, p = 0.001; 72 dB: F(1, 17) = 1.30, p = 0.268). Consistent with reduced performance, Mecp2 rats exhibited significantly slower reaction times (Fig. 1F, 1I; Unpaired t-test, discrimination: Mecp2: 1.82 ± 0.11 s, WT: 1.52 ± 0.04 s, p = 0.013; speech in noise: Mecp2: 1.99 ± 0.08 s, WT: 1.69 ± 0.07 s, p = 0.012). These findings indicate that Mecp2 rats exhibit deficits on a complex auditory task that are not apparent in a simple task.

Previous studies report modest deficits in motor function in Mecp2 heterozygous or KO animal models (Bhattacherjee et al., 2017; Guy et al., 2001; Patterson et al., 2016; Samaco et al., 2013; Stearns et al., 2007; Veeraragavan et al., 2015; Wu et al., 2016), but these deficits largely fail to replicate the substantial motor dysfunction observed in Rett patients. Given the appearance of deficits on a challenging auditory task, we next sought to characterize acquisition and performance of Mecp2 rats on a skilled forelimb motor task. Mecp2 and WT rats underwent training on a task that requires the rats to reach their forelimb through a narrow slot in the cage and press a lever twice in rapid succession to receive a food reward. The training was staged to become progressively more challenging as performance improved, such that longer reaches and faster presses were required (Table 1). Mecp2 rats demonstrated significantly slower progression through training stages than WT animals over training, indicative of an impairment in skilled motor learning (Fig. 2C; Wilcoxon rank-sum test, weeks 2-12, p < 0.05). Consequently, a smaller proportion of Mecp2 rats completed the final stage of training within 12 weeks compared to WT (Fig. 2D; Mecp2: 3 of 15 rats, WT: 9 of 13 rats, χ² test, χ² = 6.89, p = 0.009). Mecp2 rats displayed a significantly slower interval between successive lever presses than WT (Fig. 2E; Mecp2: 0.86 ± 0.1 s, WT: 0.57 ± 0.04 s, Unpaired t-test, p = 0.013). Additionally, Mecp2 rats performed fewer trials during behavioral training sessions, consistent with previous reports of motor hypoactivity (Bhattacherjee et al., 2017; Guy et al., 2001; Patterson et al., 2016; Samaco et al., 2013; Stearns et al., 2007; Veeraragavan et al., 2015; Wu et al., 2016) (Fig. 2F; Mecp2: 114.29 ± 11.27, WT: 144.19 ± 8.61, Unpaired t-test, p = 0.049). Together, these results indicate an impairment in skilled motor function in Mecp2 rats.

We next sought to explore whether large scale changes in cortical circuits could explain motor performance deficits in Mecp2 rats. To do so, we conducted ICMS mapping to document movement representations in motor cortex after the conclusion of motor training. No differences in overall area of the motor cortex or the size of individual movement representations were observed between Mecp2 and WT rats (Fig. 3A, Unpaired t-test, overall area: Mecp2: 15.82 ± 2.09 mm², WT: 17.92 ± 1.25 mm², p = 0.38; Fig. 3B, distal forelimb area: Mecp2: 5.39 ± 0.44 mm², WT: 4.97 ± 0.45 mm², p = 0.53; proximal forelimb area: Mecp2: 1.32 ± 0.27 mm², WT: 2.11 ± 0.68 mm², p = 0.34; hindlimb area: Mecp2: 2.43 ± 0.4 mm², WT: 2.53 ± 0.45 mm², p = 0.88; head area: Mecp2: 6.46 ± 1.43 mm², WT: 8.06 ± 0.94 mm², p = 0.35). Additionally, the stimulation threshold to evoke a movement was similar between groups (Fig. 3C; Mecp2: 104.52 ± 5.47 µA, WT: 94.19 ± 7.38 µA, Unpaired t-test, p = 0.3). The absence of gross changes in cortical movement representations suggests that motor dysfunction may arise from changes in synaptic function in motor networks, rather than large scale changes in corticospinal connectivity.

Previous studies have documented social and cognitive deficits in Mecp2 rodents, so we sought to corroborate those findings (De Filippis et al., 2014; Gandaglia et al., 2019; Samaco et al., 2013; Ure et al., 2016; Veeraragavan et al., 2015; Wu et al., 2016). Mecp2 and WT rats underwent a social interaction task in which we measured their sociability and social novelty. No difference in sociability or social novelty was observed between groups (Fig. 4D, 4E; Unpaired t-test, sociability index: Mecp2: 0.47 ± 0.08, WT: 0.36 ± 0.08, p = 0.4; social novelty index: Mecp2: 0.2 ± 0.1, WT: 0.17 ± 0.04, p = 0.72). Mecp2 and WT rats also exhibited comparable behavior on a marble burying task (Fig. 4A; Mecp2: 8 ± 0.8 marbles, WT: 7.89 ± 1.37 marbles, Unpaired t-test, p = 0.94). In a spontaneous alternation task, Mecp2 rats have significantly less spontaneous alternations compared to WT controls (Fig. 4B; Mecp2: 55% ± 3.77, WT: 69% ± 3.31, unpaired t-test, p=0.012). Spontaneous alternation is also used to measure anxiety or locomotor activity, however the number of entries to each arm was not different (Fig. 4C; Mecp2: 27.44 ± 2.36 entries, WT: 29 ± 1.52 entries, unpaired t-test, p=0.59), suggesting that locomotor activity or anxiety did not drive any spontaneous alternation behavior.

The present study provides a novel characterization of complex skill learning in Mecp2 rats. We report that heterozygous Mecp2 rats display slower acquisition and reduced performance on an auditory discrimination task than WT littermates. Similarly, Mecp2 rats exhibit impaired learning rates and worse performance on a skilled forelimb motor task compared to WT. Together, these findings illustrate skilled performance deficits that are novel and consistent with clinical manifestation.

We find that Mecp2 rats were impaired during a more challenging auditory task, corroborating a previous report (Engineer et al., 2015). These findings are consistent with clinical evidence that while individuals with Rett Syndrome generally have normal gross auditory processing, higher levels of auditory processing are often degraded (Bader et al., 1989; Engineer et al., 2015; Goffin et al., 2011; Kálmánchey, 1990; Liao et al., 2012; Stach et al., 1994; Stauder et al., 2006). This is also supported by evidence of higher hearing thresholds and decreased response strength to low frequency sounds in the primary auditory cortex of Mecp2 rats (Engineer et al., 2015). Additionally, we found that Mecp2 rats were slower at responding to the target sound on all speech discrimination tasks. Both individuals with Rett Syndrome and Mecp2 animal models exhibit delayed cortical responses to auditory stimuli (Bader et al., 1989; Engineer et al., 2015; Foxe et al., 2016; Goffin et al., 2011; Liao et al., 2012). This degraded auditory processing likely contributes to the slower reaction times to auditory stimuli observed in the current study.

Given that Mecp2 rats were impaired during a more challenging auditory task, we sought to characterize performance on a skilled forelimb motor task. Overall, Mecp2 rats had a significant impairment in skilled motor function compared to WT controls. In the current study, the reduced forelimb movement speed, as assessed by longer latencies between presses on the task, are consistent with deficits in motor performance in Mecp2 rats (De Filippis et al., 2015; Veeraragavan et al., 2015). Additionally, Mecp2 rats initiated fewer trials during behavioral testing sessions, consistent with motor hypoactivity previously documented with the open field and rotarod tasks (Bhattacherjee et al., 2017; Guy et al., 2001; Patterson et al., 2016; Samaco et al., 2013; Stearns et al., 2007; Veeraragavan et al., 2015; Wu et al., 2016). The impairments in motor learning and skilled motor function reported here are consistent with clinical observations. Moreover, these findings expand the utility of the transgenic rat model and suggest that the model may be useful as a framework to evaluate interventions aimed at improving motor function.

Studies have documented impaired motor cortex plasticity in both Mecp2 rodent models (Morello et al., 2018; Moretti et al., 2006) and Rett Syndrome patients (Bernardo et al., 2020), which may contribute to motor dysfunction. Based on this data and our observation of deficits in skilled motor function, we sought to explore whether large scale changes in cortical circuits could explain poor motor performance in Mecp2 rats. However, no differences in the cortical movement representation area or stimulation threshold to evoke movements were observed. The technique used to evaluate motor function in this study, intracortical microstimulation, largely provides a coarse evaluation of direct corticospinally-evoked movements. Similar to the auditory system, our findings indicate that motor circuits are largely preserved in Mecp2 heterozygous rats, and the motor dysfunction likely reflects changes in synaptic plasticity or dysfunction in higher processing areas. Future studies directed at examining finer-scale synaptic changes in motor networks may provide more insight into skilled motor dysfunction.

Along with severe auditory and motor dysfunction, Rett Syndrome patients exhibit cognitive and social deficits. This has been modeled in various rodent models of Rett Syndrome (De Filippis et al., 2014; Gandaglia et al., 2019; Samaco et al., 2013; Ure et al., 2016; Veeraragavan et al., 2015; Wu et al., 2016). We sought to corroborate some of these findings in our Mecp2 female rat model. To do so, Mecp2 and WT rats underwent a social interaction task, a marble burying task, and a spontaneous alternation task to assess sociability and social preference, repetitive behaviors, and working memory, respectively. While we did not observe differences in social or repetitive behaviors, we did observe a working memory impairment, indicated by a reduction in percent alternation in the spontaneous alternation task. This finding is consistent with a previous study that observed working memory impairments in a novel Mecp2 male mouse model (Gandaglia et al., 2019). Conversely, we did not observe abnormal social behavior or repetitive movements. This difference may arise from the fact that female Mecp2 rodents used in the present study exhibit a less severe phenotype compared to male rodents (Patterson et al., 2016; Samaco et al., 2013). Additionally, some studies have suggested that social behavior may improve over time in Rett Syndrome patients (Zappella et al., 2003), which may explain the lack of abnormal social behavior in the current study. A previous study documented abnormal social behavior in juvenile female Mecp2 rats (Veeraragavan et al., 2015), while the current study used adult Mecp2 rats. Future studies evaluating social behavior at several timepoints may provide more insight to the manifestation and time course of abnormal social behavior in Mecp2 rats.

Several studies have successfully modeled MECP2 dysfunction and Rett Syndrome-like phenotypes in Mecp2 mice (Goffin et al., 2011; Guy et al., 2001; Liao et al., 2012; Morello et al., 2018; Moretti et al., 2006; Samaco et al., 2013; Stearns et al., 2007). The Mecp2 rat model used in the present study provides the opportunity for more complex behavioral analysis that may offer more insight into the pathology of Rett Syndrome. Additionally, our experiments were conducted in adult Mecp2 heterozygous females, which is more analogous to heterozygous mutations found in girls with Rett syndrome. A recent preceding study highlights the utility of the female Mecp2 rat model, using the model to identify psychomotor regression similarly seen in Rett Syndrome girls (Veeraragavan et al., 2015).

A number of limitations of this study merit consideration. While the presented data provides initial evidence of skilled learning deficits in auditory and motor tasks in Mecp2 rats, rodent models fail to capture the complexity of skilled learning in humans. Future studies that expand evaluation to a broader range of tasks will provide a more comprehensive assessment of skilled learning in this model. In the current study, we observed significantly slower reaction times in Mecp2 rats, consistent with reduced performance on the speech discrimination task. Based on the present data, we cannot exclude the possibility that slower reaction time arise from abnormal motor function. However, given that Mecp2 rodents have generally intact motor function but abnormal auditory cortical responses (Engineer et al., 2015; Goffin et al., 2011; Liao et al., 2012), we believe that the speech discrimination impairment is due to atypical cortical auditory processing. Future studies should incorporate recording of neural responses in brainstem and auditory cortex in conjunction with behavioral discrimination to examine the contribution of auditory processing. One of the central findings of the present study is the observation of impaired motor learning and performance on a skilled task. The current study also found that Mecp2 rats performed fewer trials during motor behavioral training sessions, which is consistent with previous reports of motor hypoactivity with the open field and rotarod tasks (Bhattacherjee et al., 2017; Guy et al., 2001; Patterson et al., 2016; Samaco et al., 2013; Stearns et al., 2007; Veeraragavan et al., 2015; Wu et al., 2016). It is possible that impaired motor skills could arise from lack of motivation or general muscle weakness. Preliminary results from a previous study indicate potential muscle weakness in Mecp2 females, but the magnitude of this effect is unclear (Patterson et al., 2016). Moreover, recent evidence found that impairments in motor cortex plasticity correlates with motor deficit in individuals with Rett syndrome, suggesting that central changes contribute to motor dysfunction (Bernardo et al., 2020). Future studies are needed to delineate the contributions of central and peripheral mechanisms on motor dysfunction.

The development of interventional strategies to improve the quality of life of individuals with Rett Syndrome is of clinical importance. Characterization of more complex behaviors known to be altered in Rett syndrome could expand the utility of animal models. The current study characterizes skilled motor learning, replicates previous work evaluating auditory discrimination performance in female Mecp2 rats and establishes a framework for future studies to evaluate the effect of potential interventions on complex behavioral tasks.

Methyl CpG binding protein 2 (Mecp2), wild-type (WT), intracortical microstimulation (ICMS)

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by The International Rett Syndrome Foundation, a 2018 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation (CTE), and the NIH National Institute of Neurological Disorders and Stroke R01 NS094384 (SAH) and National Institute on Deafness and Other Communication Disorders R01 DC017480 (CTE).

Authors’ contributions

KSA, MPK, SAH, and CTE were all involved in designing the experiment. KSA, AEB, RAM, and AA were responsible for data collection and data management. KSA and CTE were responsible for data analysis. KSA, CTE and SAH were involved in writing and editing the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We would like to thank Helena Zhang, Aaron Kuo, Joy Mong, Mai Shedd, Joanna John, Naser Asfoor, Yun-Yee Tsang, Prathima Kandukuri, Ayushi Bisaria, Peyton Demetrovich, Stephanie Abe, Eric Bellinghausen, Archana Iyer, Rachel Herd, Manolo Rios, Gabby Spurlin, Lauren McLeod, Nikitha Vicas, Parker Lachowsky, Josh Currie, Asad Mehkri, Krishna Patel, Merelyn Thomas, Anima Gnawali, Armin Tutnjic, Daanish Khazi-Syed, Mahitha Roy, Angela Abraham, and Ajay Tulsi for help with motor and auditory behavioral training. We would also like to thank Daniel Hulsey, Sadmaan Sarker, Nikitha Vicas, Ruby Solorzano, and Peyton Demetrovich for ICMS support and David Pruitt for analysis help.

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Deficits in Skilled Motor and Auditory Learning in a Rat Model of Rett Syndrome

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