Communication‐related assessments in an Angelman syndrome mouse model

Abstract Introduction Angelman syndrome (AS) is a neurodevelopmental disorder characterized by motor deficits, seizures, some autistic‐like behaviors, and severe impairment of speech. A dysfunction of the maternally imprinted UBE3A gene, coupled with a functional yet silenced paternal copy, results in AS. Although studies of transgenic mouse models have revealed a great deal about neural populations and rescue timeframes for specific features of AS, these studies have largely failed to examine intermediate phenotypes that contribute to the profound communicative disabilities associated with AS. Methods Here, we use a variety of tasks, including assessments of rapid auditory processing and social communication. Expressive vocalizations were directly assessed and correlated against other core behavioral measures (motor, social, acoustic perception) to model putative influences on communication. Results AS mice displayed the characteristic phenotypes associated with Angelman syndrome (i.e., social and motor deficits), as well as marginal enhancements in rapid auditory processing ability. Our characterization of adult ultrasonic vocalizations further showed that AS mice produce fewer vocalizations and vocalized for a shorter amount of time when compared to controls. Additionally, a strong correlation between motor indices and ultrasonic vocalization output was shown, suggesting that the motor impairments in AS may contribute heavily to communication impairments. Conclusion In summary, the combination of motor deficits, social impairment, marginal rapid auditory enhancements, and altered ultrasonic vocalizations reported in a mouse model of AS clearly parallel the human symptoms of the disorder. This mouse model offers a novel route to interrogate the underlying genetic, physiologic, and behavioral influences on the under‐studied topic of impaired communication in AS.


| INTRODUC TI ON
Angelman Syndrome (AS) affects 1 in 15,000 live births (Mertz et al., 2013;Õiglane-Shlik et al., 2006) and is characterized by developmental delays, epilepsy, difficulties with motor control, microcephaly, abnormal laughter, and social behavior, and altered sleep patterns (Mabb et al., 2011;Williams et al., 2006). AS symptoms are present during the first year of life (Fryburg et al., 1991), and therefore AS is classified as a neurodevelopmental disorder.
AS is further characterized by atypical social communication, which is also a core symptom of autism spectrum disorders (ASD; Williams et al., 2006). Less is known about the social communication symptom domain of AS compared with other symptoms, particularly as it relates to the genetic underpinnings of the disorder.
Identification of genetic influences on core behavioral systems that influence social communication in AS is the focus of the current study.
AS is caused by a genetic mutation or deletion in the maternally inherited UBE3A allele (15q11.2; Kishino et al., 1997;Margolis et al., 2015). Since the paternal copy of the gene is silenced in neurons, mutation or deletion of the maternal allele results in little to no UBE3A gene product in neurons. The UBE3A gene encodes a protein product also known as E6-associated protein (E6AP), which is a HECT domain-containing E3 ubiquitin ligase Mabb & Ehlers, 2010). This protein first received attention for its role in the ubiquitination and degradation of the tumor repressor p53 . Mutations in the HECT domain that primarily affect the ubiquitin ligase activity of UBE3A cause AS (Cooper et al., 2004;Nawaz et al., 1999), strongly suggesting that loss of ubiquitin ligase function from UBE3A has a serious impact on neurodevelopment. Importance of UBE3A in neurodevelopment is further demonstrated by a de novo missense mutation that disrupts a PKA phosphorylation site on the E3 ligase, upregulating UBE3A protein, with enhanced enzyme activity and increased dendritic spine growth. This increase in UBE3A activity and altered synaptic function may contribute to the pathogenesis of ASD (Jason et al., 2015).
Mouse models of AS have been developed to further understand the neuroscience of the disorder. In a seminal original paper on the model, Jiang et al., (1998) reported that mice with a heterozygous maternal knockout of the rodent homolog Ube3a (denoted as Ube3a m−/p+ or more simply, Ube3a AS) displayed characteristic symptoms of AS, including motor dysfunction and seizures, context-dependent learning impairments, deficits in hippocampal longterm potentiation, and increased cytoplasmic p53 in Purkinje cells and hippocampal pyramidal neurons. Subsequently, Yashiro et al. (2009) and Sato & Stryker (2011) used the model to show effects on experience-dependent cortical plasticity during a developmental time period in which sensory input greatly impacts synaptic connectivity. Recent work from Judson et al., (2016) used conditional Ube3a mouse models to show that GABAergic UBE3A loss is the driving factor underlying increased circuit excitability, contributing in turn to the seizure and epileptic phenotype seen in individuals with AS.
The current study was designed to further explore the role of UBE3A in the language-and socio-communicative-related impairments of AS using a Ube3a mutant mouse model. Social communication is a core and highly impactful feature of AS, yet it has been particularly resistant to rodent study, and hence basic research approaches. This study has two primary components: first, the study replicated patterns of atypical behavior in AS mice on motor-related tasks (Sonzogni et al., 2018) and social-related tasks (Stoppel & Anderson, 2017). Secondly, it included novel communication-specific measures of complex auditory processing and expressive communicative vocalization. These latter tasks were selected for their ability to detect subtle rapid changes in complex spectrotemporal acoustic stimuli (e.g., speech), a skill fundamental to language development (Benasich & Tallal, 2002;Farmer & Klein, 1995;Fitch et al., 2001;Hari & Kiesilä, 1996;Kraus et al., 1996). Indeed, other language-impaired populations show difficulties with processing rapidly changing auditory information, regardless of whether it is speech-or non-speech-based (Cohen-Mimran & Sapir, 2007;Tallal et al., 1993;Tallal & Newcombe, 1978;Tallal & Piercy, 1973;Vandermosten et al., 2011). Moreover, infant acoustic processing indices accurately predict long-term language outcomes, thus emphasizing the importance of intact auditory processing for typical language development (Benasich et al., 2006;Benasich & Tallal, 2002). We also looked to extend evidence of anomalous communicative vocalizations in AS mice (Jiang et al., 2010;Mandel-Brehm et al., 2015;Stoppel & Anderson, 2017), specifically by examining relationships between ultrasonic vocalizations (USVs) produced by adult AS model mice, and other core behavioral measures. Thus, we assessed whether general motor deficits, social behavioral anomalies, and/or low-level acoustic processing anomalies may be statistically related within-subjects to anomalous USV vocalization production. This statistical modeling could shed light on the neurologic underpinnings of communicative deficits in AS. The novel results of the current study point strongly to motor anomalies (rather than atypical social or receptive-acoustic processing systems) as a major functional correlate of communicative impairments in AS.
Philpot at UNC-Chapel Hill (Chapel Hill, NC)  and were rederived on a C57BL/6J background strain at the Center for Mouse Genome Modification (CMGM) at UConn Health (Farmington, CT)-a C57 background strain was chosen due to reports of audiogenic seizures for Ube3a knockout mice on a 129 background strain. Audiogenic seizures did not occur in AS C57 mice at the age of 3 months (Born et al., 2017;Mandel-Brehm et al., 2015). Ube3a expression in the Ube3a FLOX/p+ mice have been extensively characterized by its developers using a variety of Cre-drivers (Berrios et al., 2016;Bruinsma et al., 2015;Judson et al., 2016;McCoy et al., 2017;Sidorov et al., 2018). For this study, CMV-Cre male mice (B6.C-Tg(CMV-cre)1Cgn/J; JAX stock #006054) were obtained from The Jackson Laboratory (Bar Harbor, ME) and crossed with female Ube3a FLOX/p+ mice to generate an F2 generation, which consisted of 15 litters averaging 5 mice per litter. This resulted in female Ube3a m−/p+ (AS) mice and female Ube3a m+/p+ / Cre + (Control) mice, as well as Ube3a FLOX/p+ and wild-type males. Subjects were genotyped using the protocols outlined in Judson et al., 2016. Twenty-two female AS mice and 17 female Control mice (C57BL/6J background; littermates) were used for experimentationonly females were selected for experimentation due to previous work assessing communication in an adult AS mouse model (Stoppel & Anderson, 2017) as an effort to replicate findings. Subjects were given food and water ad lib and single-housed after postnatal day (P) 30 in standard Plexiglass mouse cages. All mice were kept on a 12 hr:12 hr light/dark cycle with experimentation occurring during their light cycle. The subject's testing order was randomized prior to the start of experimentation-experimenters remained blind to genotype. All testing procedures occurred in compliance with National Institutes of Health and approved by the University of Connecticut's Institutional Animal Care and Use Committee (IACUC).

| Rotarod (P50 -53)
Testing began at P58 with the Rotarod task-a task used to assess sensorimotor ability and motor learning. Subjects were placed on a cylindrical drum (6 cm in diameter; 7.5 cm in width) that rotated at an accelerating rate-4 rotations per minute (RPM) to 40 RPM over the span of 2 min. Over the course of three consecutive days, each subject completed four trials with an intertrial interval of 15 min.
The latency to fall from the rotating cylindrical drum (distance to fall = 20 cm) was recorded and averaged across trials per day.

| Modified Three-Chamber Social Preference Task (P62)
Subjects were assessed on a modified Three-Chamber Social task to evaluate general sociability by exploiting a mouse's typical preference for a conspecific (i.e., social stimulus) over an object (i.e., nonsocial stimulus). See Yang et al., (2011) for original methods. After completing a five-minute habituation period, subjects were placed in the center of a three-chambered testing apparatus (overall dimensions: 40.5 cm × 62 cm × 23 cm; each chamber: 40.5 cm × 20 cm × 23 cm) and allowed to freely explore all chambers for ten minutes. During this testing session, one chamber contained a caged, same-sex Control mouse ("Novel Mouse"), F I G U R E 1 Syllable Repertoire generated by MUPET. Generated via MUPET analysis, forty unique syllables were assigned to one of ten possible categories (Heckman et al., 2016). Eight of the ten possible categories were observed in the syllable repertoire (Short (expected frequency range: 70 kHz; example: syllable #1), Flat (expected frequency range: 70 kHz; example, syllable #2), Down-FM (expected frequency range: 80 -60 kHz; example, syllable #9), Up-FM (expected frequency range: 60 -80 kHz example: syllable #20), Chevron (expected frequency range: 70 -80 kHz; example: syllable #32), 2-Frequency Step (expected frequency range: 60 -80 kHz, example: syllable #35), Noisy (expected frequency range: 40 -80 kHz example: syllable #36), and Complex (expected frequency range: >50 kHz example: syllable #39). The number located in the bottom-left corner of each syllable indicates the number of times that syllable was produced across all subjects
A single female subject was placed in a Plexiglass mouse tub (28 cm × 16.5 cm × 12 cm) and allowed to freely explore the chamber for twenty minutes. Following this habituation period, a second, same-genotype female (i.e., "newcomer mouse") was introduced and the two subjects were able to freely interact with one another.
Under these conditions, the original female mouse will vocalize in the presence of the newcomer female mouse (Ferhat et al., 2016;Maggio and Whitney, 1985). Importantly, any vocalizations made by the newcomer mouse were balanced across genotype, so that total calls recorded came from with a Control or AS pair. During the tenminute interaction, a Brüel & Kjaer Type 4954-B microphone (Brüel & Kjaer, Naerum, Denmark), recording at 192,000 Hz and connected to an RME Fireface UC audio interface (RME Audio, Haimhausen, Germany), was placed 5 cm above the top of the Plexiglass tub. tion & characteristics of each syllable category). Any USV that did not fall within this frequency or duration range, in addition to USVs that occurred less than 5 ms following a previous USV, were excluded from analyses. Following this inclusion criteria, a syllable repertoire was generated for all mice in the study that identified 40 unique syllables (not all mice made all syllables during their session; Figure 1).

| Modified prepulse inhibition paradigm
Subjects completed testing with a rapid auditory processing task that utilizes a modified prepulse inhibition paradigm (see Fitch with an open and opaque Plexiglass chamber (to prevent escape; 20.5 cm × 21.5 cm × 30.5 cm), and presented with auditory stimuli generated by RPvdsEx software and a RZ6 multifunction processor (Tucker Davis Technologies, Alachua, FL). The load-cell platforms recorded the subject's motor reflex response to a 105dB, 50ms in duration, white noise burst ranging from 1,00Hz, -10,000Hz [known as the startle-eliciting stimulus (SES)]. Information from the platforms was processed and recorded with a Biopac MP150 acquisition system and Acqknowledge 4.1 software (Biopac Systems, Goleta, CA).
The modified prepulse inhibition paradigm is used to assess the differences in ASR when the SES is presented with or without a preceding acoustic cue. The subject's cue detection/discrimination ability can be measured by analyzing differences in ASR between cued and uncued trials. In cued trials, an auditory stimulus (i.e., the cue) is presented 50ms before the SES and if the subject can detect the cue, their ASR will be reduced (attenuated) relative their ASR when the cue was not presented (i.e., uncued trial) or the cue was not detected. This phenomenon can be quantified by calculating an "attenuation score," which compares the mean amplitude of the cued ASR to the mean amplitude of the uncued ASR:

| Normal single tone (NST) (P70, P81)
Normal Single Tone (NST) was used to assess subject's baseline prepulse inhibition and general hearing ability. Against a silent background, subjects were asked to detect a 50ms auditory cue that consisted of a simple, 8,000Hz, pure tone (70dB) (NST 8kHz). 104 trials were used during the testing session, and on half of the trials, the cue was presented 50ms before the 105dB SES (i.e., cued trial) [52 cued trials and 52 uncued trials; pseudorandomized, even trial distribution]. Attenuation scores calculated from this task were used a covariate on subsequent auditory processing task as a measure to eliminate individualized differences in hearing ability and prepulse inhibition.
An ultrasonic version of this task was conducted following the completion of all nonultrasonic auditory tasks. The number of trials, intertrial interval durations, distribution of cued and uncued trials, and volumes for the cue and the SES were similar to that of NST 8kHz; however, the cue frequency was changed to 40,000Hz (NST 40kHz).

| Embedded tone 100 (P73 -78)
The Embedded Tone (EBT) task tested the subject's the ability to detect a change in frequency within a constant, pure tone background (75 dB; 10,500 Hz). Ube3a AS and Control mice were subjected to AttenuationScore = meancuedASR meanuncuedASR x100 300 pseudorandom trials with the intertrial interval varying between 16 s -24 s to ensure the subject was not able to predict the onset of the next trial. Cued trials contained a 5,600 Hz cue that occurred 100 ms before the 105 dB SES and varied in duration, ranging from 2 ms -100 ms. Uncued trials contained a cue that occurred "0 ms" before the SES. EBT 100 was administered for five consecutive days.

| Single Arbitrary Waveform: Chevron (P82)
Subjects were required to detect a mouse ultrasonic vocalizationspecifically, a chevron syllable from a wild-type c57 mouse (44 ms in duration; amplitude ranged from 72,000 Hz -85,000 Hz). Similar to NST, the cue was presented on half of the 104 trials 50 ms before the SES (pseudorandomly and evenly distributed) with ITIs ranging from 16s -24s. Single Arbitrary Waveform (SAW): Chevron demands a higher level of processing and introduces a social-context component to the discrimination requirement.

| Statistical analysis
Behavioral data were subjected to statistical analysis by Task post hoc analyses were conducted to confirm appropriate Genotype size using G*Power 3.1 software (Faul et al., 2007) with α = 0.05 and power (1-β) = 0.80.

| RE SULTS
Power analyses confirmed the Genotype size used was adequate to detect significance on tasks with moderate to large effect sizes.
Future studies with a larger n per group may successfully delineate marginal effects such as the trend toward an auditory processing enhancement (i.e., Embedded Tone 0-100:10.5kHz and NST tasks) and specific characteristics of ultrasonic vocalizations (i.e., Syllable Pitch).

| Motor Assessment (Rotarod & Modified Three-Chamber Social Task)
Using a 2 × 3 repeated measures ANOVA, we found a main effect of

| Modified three-chamber social task
Results from the Modified Three-Chamber Social task showed a main effect of Genotype [F(1, 37) = 5.219, p < .05] on percent time interacting with conspecific. AS mice spent significantly more time interacting with the conspecific mouse, indicating atypical social behavior ( Figure 3).

| Ultrasonic Vocalizations
USV measures included syllable duration, syllable volume, syllable pitch, the total number of syllables produced, and total time spent vocalizing were analyzed. A complete breakdown of the statistical analyses can be found in Tables 1, 2, 3, 4, and 5. We found no statistical differences between groups in overall Syllable Duration Control mice. However, we did find that AS mice trended to pro-

| Normal Single Tone
Analysis of NST 8kHz did not reveal a main effect of Genotype cue detection ability at ultrasonic frequencies. (Figure 9a & 9b).

| Embedded Tone 100
EBT 100 was analyzed using NST 8kHz as a covariate to account for

| Single Arbitrary Waveform
A univariate ANOVA revealed that AS mice trended better than Control mice on SAW [F(1, 37) = 3.207, p < .10], suggesting AS mice have a modest enhancement when detecting and responding to ultrasonic vocalizations, specifically a C57 wild-type Chevron (Figure 9d).

| Path modeling, USV Production
To develop a baseline for comparison, we analyzed relationships between behavioral scores obtained from adult wild-type C57 mice  0.013 for multiple tests) (Figure 10a). We also used linear multiple Importantly, the subset of data from wild-type females only (n = 17) was separately re-assessed, since the mice in the current study were all female. Analyses confirmed that Auditory

TA B L E 2 Statistics-syllable volume
Processing scores did not relate to predict USV production in wild-type females, whereas significant positive linear bivariate cor- Again, Motor, Social, and Auditory indices showed no co-linearity and auditory processing scores did not relate to USV production. Motor Learning scores continued to show a trend toward positive correlation with USV production that did not survive α-correction (+0.45, p < .04).
However, a correlation between Motor and the specific production specifically of complex USV calls (chevron, 2-frequency step, Complex and Noisy only; Table 5

| DISCUSSION
The current study was designed to further explore communicative anomalies associated with AS using an established mouse model. We replicated evidence that disruptions to maternally expressed

TA B L E 3 Syllable pitch
Ube3a are associated with impaired motor coordination and learning, and atypically enhanced social behaviors. Novel results show enhanced receptive auditory processing ability in baseline prepulse inhibition and social-communicative stimuli. We also report significantly decreased ultrasonic vocalization production, consistent with an AS phenotype. Finally, statistical analyses reveal that motor deficits-but not social or acoustic processing anomaliesare strongly correlated to atypical vocal output in the AS model, suggesting that neurologic anomalies in the motor system are the primary contributor to communicative impairments in the mouse model of AS.

F I G U R E 4
Ultrasonic Vocalizations-Syllable Duration. a) There was no main effect of Genotype on syllable duration when collapsed by category. b) Ube3a m−/p+ mice produced shorter Flat syllables when compared to Control mice. All other syllable categories did not show a main effect of Genotype. # p < .10 F I G U R E 5 Ultrasonic Vocalizations-Syllable Volume. a) There was no main effect of Genotype on syllable volume when collapsed by category. b) Ube3a m-/p+ mice produced louder Short, Noisy, and Complex syllables when compared to Control mice. All other syllable categories did not show a main effect of Genotype. # p < .10; **p < .01; *** p < .001

| AS mice display AS-like phenotypes
Our results validate the use of an Ube3a AS mouse to study the behavioral consequences of maternal-UBE3A disruption, as we report phenotypes consistent with individuals with AS. Specifically, AS mice displayed motor impairments on the Rotarod task and within the Modified Three-Chamber Social Task-these results indicate that AS model mice show motor learning, with a learningcurve comparable to Control mice, but also demonstrate a baseline motor coordination deficit that persists across learning. These results are important in that they suggest AS model mice can attain motor performance levels of controls with additional training. AS

F I G U R E 6
Ultrasonic Vocalizations-Syllable Pitch. a) There was no main effect of Genotype on syllable volume when collapsed by category. b) Ube3a m-/p+ mice produced higher pitched Flat syllables when compared to Control mice. All other syllable categories did not show a main effect of Genotype. # p < .10 F I G U R E 7 Ultrasonic Vocalizations-Syllable Number. a) Ube3a m−/p+ mice produce fewer vocalizations when compared to Control mice. b) Ube3a m−/p+ mice produced less Short and Flat syllables when compared to Control mice. All other syllable categories did not show a main effect of Genotype. *p < .05; **p < .01 mice also displayed atypical social behavior in a Modified Three-Chamber Social Task-AS mice spent more time interacting with the novel, caged mouse. Previous work behaviorally characterizing Ube3a mouse models report impairments on the Rotarod task (Bruinsma et al., 2015;Heck et al., 2008;Huang et al., 2013;Sonzogni et al., 2018), as well as increased social behavior. Using a slightly different protocol, Stoppel and Anderson (2017) reported that female mice with a maternally inherited Ube3a deletion spent prolonged periods of time with a novel social stimulus in the Three-Chamber Social task, exhibiting a hyper-social phenotype.
These motor-and social-related phenotypes parallel human clinical findings-individuals with AS experience movement impairments (i.e., an ataxic gait and tremulous limb movements) and altered social behavior (i.e., frequent laugher/happy demeanor and an easily excitable personality) (Williams et al., 1995;Williams et al., 2006). The combination of the Rotarod an Modified Three-Chamber Social Task results raises an interesting problem-are AS mice traveling less distance and moving slower due to an overall motor impairment or due to a social fixation on the novel mouse?
More fine-tuned rodent behavioral tasks are necessary to fully explore this relationship and the general relationship between maternal UBE3A disruption and motor-and social-related alteration.

| Auditory processing enhancements in AS mice
We evaluated AS mice on additional tasks relevant to human language development and acquisition to better understand how UBE3A impairs language ability. Our study is the first to show that AS mice exhibit enhanced cue detection and prepulse inhibition on auditory processing tasks (i.e., NST 40kHz and SAW). This finding is particularly relevant because early auditory processing scores can accurately predict language outcomes (e.g., vocabulary and other language scores) from human infants .

Low-level perceptual discrimination enhancements have been
reported in other neurodevelopmental disorders, including ASD (Bertone et al., 2005;Mottron et al., 2006;Plaisted et al., 2003) and may contribute to the language-related impairments seen in the disorder. In particular, it has been suggested that enhancements in auditory processing ability can also contribute to poor language outcomes (Eigsti & Fein, 2013). These findings may also hold for AS, but further research is necessary. Research on this topic has been hindered by the frequent lack of oral language in AS, which restricts the use of common language-based outcome measures, although novel studies with mosaic AS populations that show intact communication may provide new insights (Carson et al., 2019;Eigsti and Chamberlain, personal communication).
Further mouse studies can also interrogate the spectrotemporal parameters of any acoustic processing anomalies, and neural histology is needed to quantify possible cellular and structural aspects of auditory-related brain structures in AS model mice.
Indeed, we have previously reported abnormalities in thalamic nuclei and various white matter structures in related ASD model mice (Rendall et al., 2017). This approach could ultimately inform targeted interventions to enhance language development in affected individuals.

| Alterations in AS mice ultrasonic vocalizations
To our knowledge, this study provides the first thorough characterization of adult Ube3a AS mouse ultrasonic vocalizations. We found that AS mice produced fewer calls and vocalized for a shorter amount of time than Control mice. These novel results contrast conflicting literature reports on USV patters in AS models suggesting a reduction in calls (Mandel-Brehm et al., 2015;Stoppel & Anderson, 2017). Ultrasonic vocalizations in juveniles reflect a pup's ability to seek maternal care by generating distress calls after being separated from their mother. This paradigm has frequently been used to show reduced vocalizations in ASD mouse models (Fischer & Hammerschmidt, 2011;Scattoni et al., 2009). Prior studies of the AS mouse model using this task include work from Mandel-Brehm et al., (2015), who showed that AS mouse pups produce more USVs at P13 through P15 when compared to wild-type mice. This result was originally interpreted as a general enhancement in vocal communication, which was puzzling since humans with AS show limited or absent vocal speech communication. However, a more finegrained examination of those data reveals a developmental "delay" in peak distress call production in AS mouse pups, such that distress call production peaks at 15-17 days in the AS pups compared with 5-7 days in the wild-type pups. This suggests a maturational lag rather than an overall increase in vocalizations in AS pups, a pattern that may be analogous to the developmental delay of ~4 days in peak USV distress calls in Down-syndrome model mouse pups (Holtzman et al., 1996).
A second potentially conflicting finding was reported by Stoppel and Anderson (2017) (Okobi et al., 2019). Moreover, orthogonal circuitry in the mouse appears to regulate the socio-emotional components of their USV production (periaqueductal gray; Tschida et al., 2019). And, finally, left-hemisphere specialization for the processing of USVs (relative to general spectrotemporal acoustic information) has been documented in mouse auditory cortex (Levy et al., 2019). These findings bolster the value of mouse models of communication to tap precursors to the multiple systems subserving language and communication in the human brain. As such, it becomes critically important to evaluate which circuits in particular appear to be affected in neurodevelopmental disorders such as AS. These studies are difficult to perform in humans, in part because the complex and profound interactive influences of emergent language in humans make the dissociation of domain-specific systems quite difficult. This is because, for example, anomalies in processing input that lead to speech delays may have subsequent impact on social verbal interactions, as well as altering educational experiences and development of higher language skills, making dissociation of "causal deficits" in communicative processes all but impossible at older ages. Though mouse studies also suffer confounds from developmental experience, the simplicity of communicative systems coupled with a highly consistent and controlled environment, and the power of genetic manipulation, mitigate these confounds to some degree.
To directly address the important question of which circuits are affected in the mouse AS model, we assessed the correlations within subjects between scores on motor learning tasks, acoustic processing tasks, social interaction tasks, and USV production. Rather than finding a general intercorrelation of all measures, reflecting broad within-subjects symptom-severity effects, results indicated that motor indices correlated strongly with anomalous USV output. This effect was magnified when only complex USV call production was considered, perhaps due to greater precision in oral-motor control required for more complex vocalizations. Neither social nor acoustic processing measures appeared to relate to USV output in the AS model mice, despite significant AS model group differences on both of those measures. Notably, results from a Shank3b ASD mouse model (unpublished data) follow a distinct pattern, with a more robust statistical contribution to USV production from Social rather than Motor indices. This suggests that the role of motor processes in communicative impairments may be a specific to Angelman Syndrome.
Interestingly, we observed a reduction in vocalization production (i.e., number of vocalizations produced and time spent vocalizing) and atypical social behavior (i.e., increased time spent with novel mouse) in AS mice-two results that seem contradictory-one might expect increased social behavior and increased vocalization production. However, these results are consistent with symptoms of AS (i.e., heightened sociability despite communicative deficits). If motor impairments are driving reductions in vocalizations, perhaps AS mice are compensating via other various methods of communication (i.e., increased sniffing). Future work requires a detailed analysis of how AS mice interact when measuring ultrasonic vocalizations.

| CON CLUS ION
The current findings replicate prior evidence of an AS-like behavioral phenotype in Ube3a m−/p+ mice and provide novel findings into how Ube3a mutations in mice affect rapid auditory processing ability and ultrasonic vocalization production. Evidence from AS model mice suggests a strong motor contribution to communicative impairments. Coupled with evidence that AS model mice show motor learning at rates comparable to control mice (despite baseline deficits that persist), combined findings offer substantial promise for the success of motor interventions and therapies in improving communicative performance of individuals with AS. Furthermore, findings suggest that language-rescue efforts in AS populations should focus on treatments and timeframes that can rescue motor ability. Ongoing studies will further assess the correlation between motor activity and ultrasonic vocalization production-future studies will include more fine-grained measures during ultrasonic vocalization recordings to evaluate the direct motor-and social interaction between subjects.

ACK N OWLED G M ENTS
We would like to thank Dr. Ben Philpot and Dr. Matthew Judson at the UNC Neuroscience Center for providing the Ube3a FLOX/p+ breeding pairs and the Center for Mouse Genome Modification (CMGM) at UConn Health for the rederivation of AS mice.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.1937.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data that support these findings can be made available upon reasonable request.