Subjects
Twenty 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). Animals underwent behavioral assays and training between 4-8 months of age. Ten 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 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. Of the Mecp2 rats that were trained, 6 were excluded from the study due to health complications, including malocclusion. 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. 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.
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. 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).