Segmentor: a tool for manual refinement of 3D microscopy annotations.

BACKGROUND: Recent advances in tissue clearing techniques, combined with high-speed image acquisition through light sheet microscopy, enable rapid three-dimensional (3D) imaging of biological specimens, such as whole mouse brains, in a matter of hours. Quantitative analysis of such 3D images can help us understand how changes in brain structure lead to differences in behavior or cognition, but distinguishing densely packed features of interest, such as nuclei, from background can be challenging. Recent deep learning-based nuclear segmentation algorithms show great promise for automated segmentation, but require large numbers of accurate manually labeled nuclei as training data. RESULTS: We present Segmentor, an open-source tool for reliable, efficient, and user-friendly manual annotation and refinement of objects (e.g., nuclei) within 3D light sheet microscopy images. Segmentor employs a hybrid 2D-3D approach for visualizing and segmenting objects and contains features for automatic region splitting, designed specifically for streamlining the process of 3D segmentation of nuclei. We show that editing simultaneously in 2D and 3D using Segmentor significantly decreases time spent on manual annotations without affecting accuracy as compared to editing the same set of images with only 2D capabilities. CONCLUSIONS: Segmentor is a tool for increased efficiency of manual annotation and refinement of 3D objects that can be used to train deep learning segmentation algorithms, and is available at https://www.nucleininja.org/ and https://github.com/RENCI/Segmentor .

Influence of Brachial Plexus Birth Injury Location on Glenohumeral Joint Morphology.

PURPOSE: Patient presentation after brachial plexus birth injury (BPBI) is influenced by nerve injury location; more contracture and bone deformity occur at the shoulder in postganglionic injuries. Although bone deformity after postganglionic injury is well-characterized, the extent of glenohumeral deformity after preganglionic BPBI is unclear. METHODS: Twenty Sprague-Dawley rat pups received preganglionic or postganglionic neurectomy on a single forelimb at postnatal days 3 to 4. Glenohumeral joints on affected and unaffected sides were analyzed using micro-computed tomography scans after death at 8 weeks after birth. Glenoid version, glenoid inclination, glenoid and humeral head radius of curvature, and humeral head thickness and width were measured bilaterally. RESULTS: The glenoid was significantly more declined in affected compared with unaffected shoulders after postganglionic (-17.7° ± 16.9°) but not preganglionic injury. Compared with the preganglionic group, the affected shoulder in the postganglionic group exhibited significantly greater declination and increased glenoid radius of curvature. In contrast, the humeral head was only affected after preganglionic but not postganglionic injury, with a significantly smaller humeral head radius of curvature (-0.2 ± 0.2 mm), thickness (-0.2 ± 0.3 mm), and width (-0.3 ± 0.4 mm) on the affected side compared with the unaffected side; changes in these metrics were significantly associated with each other. CONCLUSIONS: These findings suggest that glenoid deformities occur after postganglionic BPBI but not after preganglionic BPBI, whereas the humeral head is smaller after preganglionic injury, possibly suggesting an overall decreased biological growth rate in this group. CLINICAL RELEVANCE: This study expands understanding of the altered glenoid and humeral head morphologies after preganglionic BPBI and its comparisons with morphologies after postganglionic BPBI.

Preganglionic and Postganglionic Brachial Plexus Birth Injury Effects on Shoulder Muscle Growth.

PURPOSE: Brachial plexus birth injury can differ in presentation, depending on whether the nerve ruptures distal to, or avulses proximal to, the dorsal root ganglion. More substantial contracture and bone deformity at the shoulder is typical in postganglionic injuries. However, changes to the underlying muscle structure that drive these differences in presentation are unclear. METHODS: Seventeen Sprague-Dawley rats received preganglionic or postganglionic neurectomy on a single limb on postnatal days 3 and 4. Muscles crossing the shoulder were retrieved once the rats were sacrificed at 8 weeks after birth. External rotation range of motion, muscle mass, muscle length, muscle sarcomere length, and calculated optimal muscle length were measured bilaterally. RESULTS: Average shoulder range of motion in the postganglionic group was 61.8% and 56.2% more restricted at 4 and 8 weeks, respectively, compared with that in the preganglionic group, but affected muscles after preganglionic injury were altered more severely (compared with the unaffected limb) than after postganglionic injury. Optimal muscle length in preganglionic injury was shorter in the affected limb (compared with the unaffected limb: -18.2% ± 9.2%) and to a greater extent than in postganglionic injury (-5.1% ± 6.2%). Muscle mass in preganglionic injury was lower in the affected limb (relative to the unaffected limb: -57.2% ± 24.1%) and to a greater extent than in postganglionic injury (-28.1% ± 17.7%). CONCLUSIONS: The findings suggest that the presence of contracture does not derive from restricted longitudinal muscle growth alone, but also depends on the extent of muscle mass loss occurring simultaneously after the injury. CLINICAL RELEVANCE: This study expands our understanding of differences in muscle architecture and the role of muscle structure in contracture formation for preganglionic and postganglionic brachial plexus birth injury.

Abbreviating the Early Start Denver Model for community-based family-centered care.

Introduction: Parent-mediated approaches for young children with or with a higher likelihood of autism have gained traction, with mounting evidence of efficacy, but a research-to-gap practice exists, and community effectiveness remains to be firmly established. Methods: Using a community-participatory framework, a total of 10 parent-child dyads received a five-day workshop and six follow-up sessions of ESDM parent coaching. Intervention was implemented across two phases with in-person and telehealth delivery. Results: From pre to post intervention across both phases, parents improved in their fidelity of intervention implementation and children maid gains on proximal measures of social communication. Discussion: Community delivery of an evidence-based parent-mediated interventions for toddlers on the autism spectrum is feasible and promising. Giving resource efficiencies associated with parent-mediated approaches, particularly when delivered through government-funded programs, findings bolster current efforts to promote earlier and more widespread community access to necessary interventions. Facilitators and barriers to supporting parent learning and behavior change via interactive strategies are discussed.

"Setting Them Up for Success": Including Youth on the Autism Spectrum in 4-H.

Youth on the autism spectrum often face challenges accessing services in rural communities compared to those who live in higher resource areas. There is a particular need for services that support skills that will help youth transition to adulthood and future employment. 4-H is a national youth development program that is well-positioned to address the needs of youth on the autism spectrum; however, minimal empirical evidence exists about the implementation and effectiveness of inclusive practices in 4-H programs. The goal of this study was to better understand barriers to enrollment and to identify gaps in support for youth on the autism spectrum participating in 4-H. Twenty Extension Educators in Indiana participated in two focus groups. Thematic analysis of focus group transcripts identified barriers to enrollment including awareness of 4-H as an inclusive program and difficulties navigating 4-H culture. Our analysis identified themes related to new training content and delivery including a resource portfolio, communication, individualized accommodations, and working within the existing leadership training structure. Findings support the benefits of 4-H as a program that can promote life skills and personal development for youth on the autism spectrum but also highlight a significant need for additional training opportunities and resources to increase uptake and improve the implementation of inclusive practices.

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei. Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases. We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

Sleep Dysregulation and Daytime Electrodermal Patterns in Children With Autism: A Descriptive Study.

Sleep deficiency influences emotion and behavior regulation but the mechanisms of influence are poorly understood. Emotion, behavioral, and sleep theories highlight differences in autonomic function as a potential pathway of influence and research in typical populations draw links between sleep deficiency and autonomic dysregulation (e.g., elevated reactivity within the sympathetic nervous system). In populations at elevated risk for sleep deficiency/problems (i.e., individuals with autism), greater variability in sleep and autonomic/arousal profiles may be particularly informative. Using electrodermal activity (EDA) as an indicator of sympathetic nervous system activation, this descriptive pilot study aimed to document daytime EDA patterns in children with autism and to explore their relations with sleep dysregulation/deficiency. EDA and sleep were measured using ankle and wrist worn sensors in 13 children (Meanage 6.11 years). EDA indices included nonspecific skin conductance responses (NSSCR) and tonic skin conductance levels (SCL). Descriptively, children in the dysregulated sleep group had fewer NSSCRs and lower SCL in the afternoon. This blunted physiological arousal profile/pattern is consistent with previous research, but this is the first study to explore how sleep may be linked. Notably, this pattern may not reflect sleep but an overall dysregulation profile which in this sample included: dysregulated sleep, a blunted afternoon arousal profile, and elevated ASD symptom severity. Replication with larger, more diverse samples is needed to disentangle the complex relations among sleep, arousal, and ASD behavioral features. However, this study represents an important first step in documenting extended daytime arousal patterns.

Autism Heterogeneity in a Densely Sampled U.S. Population: Results From the First 1,000 Participants in the RI-CART Study.

The objective of this study was to establish a large, densely sampled, U.S. population-based cohort of people with autism spectrum disorder (ASD). The Rhode Island Consortium for Autism Research and Treatment (RI-CART) represents a unique public-private-academic collaboration involving all major points of service for families in Rhode Island affected by ASD. Diagnosis was based on direct behavioral observation via the Autism Diagnostic Observation Schedule, Second Edition. For the first 1,000 participants, ages ranged from 21 months to 64 years. Using Geographic Information System and published prevalence rates, the overall cohort is estimated to represent between 20% and 49% of pediatric age persons in Rhode Island with ASD, with demographics representative of U.S. Census. We observed a high rate of co-occurring medical and psychiatric conditions in affected individuals. Among the most prominent findings of immediate clinical importance, we found that females received a first diagnosis of ASD at a later age than males, potentially due to more advanced language abilities in females with ASD. In summary, this is the first analysis of a large, population-based U.S. cohort with ASD. Given the depth of sampling, the RI-CART study reflects an important new resource for studying ASD in a representative U.S. population. Psychiatric and medical comorbidities in ASD constitute a substantial burden and warrant adequate attention as part of overall treatment. Our study also suggests that new strategies for earlier diagnosis of ASD in females may be warranted. Autism Res 2020, 13: 474-488. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The Rhode Island Consortium for Autism Research and Treatment (RI-CART) represents a unique public-private-academic collaboration involving all major points of service for families in Rhode Island affected by autism spectrum disorder (ASD). In this article, we provide results from the first 1,000 participants, estimated to represent >20% of affected families in the state. Importantly, we find a later age at first diagnosis of ASD in females, which potentially calls attention to the need for improved early diagnosis in girls. Also, we report a high rate of co-occurring medical and psychiatric conditions in affected individuals.

Extending the Parent-Delivered Early Start Denver Model to Young Children with Fragile X Syndrome.

This is the first study to evaluate an autism intervention model, the parent-delivered Early Start Denver Model (P-ESDM), for young children with fragile X syndrome (FXS), a known genetic disorder associated with autism spectrum disorder. Four parent-child dyads participated in a low-intensity, parent coaching model of the P-ESDM to evaluate initial efficacy and acceptability. Parents improved in P-ESDM fidelity, implemented intervention goals to increase child learning, and found the experience moderately to highly acceptable. Visual examination and Baseline Corrected Tau effect sizes revealed mixed results across child measures. Findings suggest a potential therapeutic opportunity in need of larger, well-controlled studies of P-ESDM and other interventions for families of young children with FXS who face limited empirically-supported intervention options.

Developmental trajectories of autonomic functioning in autism from birth to early childhood.

Deficits in social engagement emerge in autism during the infant and toddler period and may be related to emotion regulation and stress response systems. This study examined patterns of growth in autonomic functioning related to autism diagnosis and addresses the hypothesis that there are differences in autonomic functioning related to autism in infancy. Heart rate (HR) and respiratory sinus arrhythmia (RSA) were measured at 8 time points from 1 to 72 months of age in infants later diagnosed with autism (n = 12) and a non-autistic comparison group (n = 106). Multilevel models were used to describe the developmental course of HR and RSA and to test the effect of autism diagnosis on growth trajectories. Both groups showed an expected age-related decrease in HR and increase in RSA. Groups did not differ in the rate of decrease of HR over time. However, infants with autism demonstrated a smaller linear increase in RSA, indicating slower growth in RSA over time in comparison to controls. These results suggest that differences in physiological regulation may develop with age in autism. The slowed RSA growth in autism was most evident after 18 months of age, at a time when behavioral symptoms become prominent. This is consistent with the view that RSA is a marker of functional status in autism rather than a cause of social deficits in autism.

Brief report: aggression and stereotypic behavior in males with fragile X syndrome--moderating secondary genes in a "single gene" disorder.

Although fragile X syndrome (FXS) is a single gene disorder with a well-described phenotype, it is not known why some individuals develop more significant maladaptive behaviors such as aggression or autistic symptoms. Here, we studied two candidate genes known to affect mood and aggression, the serotonin transporter (5-HTTLPR) and monoamine oxidase A (MAOA-VNTR) polymorphisms, in 50 males with FXS ages 8-24 years. Mothers and fathers of participants reported the frequency and severity of aggressive/destructive, self-injurious, and stereotypic behaviors. Polymorphism genotypes were unrelated to age and IQ. Results showed a significant effect of 5-HTTLPR genotype on aggressive/destructive and stereotypic behavior; males with FXS who were homozygous for the high-transcribing long (L/L) genotype had the most aggressive and destructive behavior, and individuals homozygous for the short (S/S) genotype had the least aggression. Those with the L/L genotype also had the highest levels of stereotypic behavior. There was no effect of MAOA-VNTR on behavior; however those with the high-activity, 4-repeat genotype were more likely to be taking SSRI or SNRI medication. This preliminary study prompts consideration of secondary genes that may modify behavioral phenotype expression in neurodevelopmental disorders, even those with a single gene etiology such as FXS.

Diminished respiratory sinus arrhythmia response in infants later diagnosed with autism spectrum disorder.

Indicators of risk for developing Autism Spectrum Disorder (ASD) are difficult to detect within the first year of life. In this study, infants who were originally followed prospectively to examine general developmental risks due to substance exposure interacted with their mother and an unfamiliar experimenter for 2-min episodes at 4 months of age. Electrocardiogram was collected to measure respiratory sinus arrhythmia (RSA) and the session was video recorded for behavioral coding. Two groups of infants were compared: infants with a diagnosed ASD outcome (N = 8) and matched controls (N = 186). Infants were compared on mean RSA and infant behavioral codes for each 2-min episode. No significant group differences were revealed on RSA or behavior during interactions between the infants and mothers. However, in response to play with a stranger, infants with an ASD outcome had lower RSA (M = 2.49, 95% CI [2.30, 2.98]) than controls (M = 3.12, 95% CI [3.06, 3.18]). During the interaction with a stranger, lower RSA response was associated with more protesting behaviors (P < 0.01), whereas higher RSA response was associated with more social monitoring (P = 0.001). Lower RSA suggests that 4-month-old infants later diagnosed with ASD exhibited poorer autonomic regulation during interaction with an unfamiliar adult than did controls. Physiological regulation during interactions with a new social partner may be disrupted early in infancy in children with ASD, as indicated here by lower RSA, and therefore be a potential target for developing early risk screening tools for ASD. Autism Res 2018, 11: 726-731. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Autism Spectrum Disorder (ASD) emerges within the first years of life; however, it is difficult to identify children who will develop autism before 12 months of age based on behavioral measures. In a study of infants who were followed from birth, infants who were later diagnosed with ASD had poorer physiological regulation during play with a new adult. With additional evidence, poorer physiological regulation may function as an early sign of ASD risk.

Sensitivity to audio-visual synchrony and its relation to language abilities in children with and without ASD.

Autism Spectrum Disorder (ASD) is often accompanied by deficits in speech and language processing. Speech processing relies heavily on the integration of auditory and visual information, and it has been suggested that the ability to detect correspondence between auditory and visual signals helps to lay the foundation for successful language development. The goal of the present study was to examine whether young children with ASD show reduced sensitivity to temporal asynchronies in a speech processing task when compared to typically developing controls, and to examine how this sensitivity might relate to language proficiency. Using automated eye tracking methods, we found that children with ASD failed to demonstrate sensitivity to asynchronies of 0.3s, 0.6s, or 1.0s between a video of a woman speaking and the corresponding audio track. In contrast, typically developing children who were language-matched to the ASD group, were sensitive to both 0.6s and 1.0s asynchronies. We also demonstrated that individual differences in sensitivity to audiovisual asynchronies and individual differences in orientation to relevant facial features were both correlated with scores on a standardized measure of language abilities. Results are discussed in the context of attention to visual language and audio-visual processing as potential precursors to language impairment in ASD. Autism Res 2018, 11: 645-653. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Speech processing relies heavily on the integration of auditory and visual information, and it has been suggested that the ability to detect correspondence between auditory and visual signals helps to lay the foundation for successful language development. The goal of the present study was to explore whether children with ASD process audio-visual synchrony in ways comparable to their typically developing peers, and the relationship between preference for synchrony and language ability. Results showed that there are differences in attention to audiovisual synchrony between typically developing children and children with ASD. Preference for synchrony was related to the language abilities of children across groups.

Brief Report: Factors Influencing Healthcare Satisfaction in Adults with Autism Spectrum Disorder.

The current study investigated healthcare satisfaction and factors related to satisfaction in 92 adults with Autism Spectrum Disorder (ASD). Participants or their caregiver completed a survey about their experiences with primary care and specialty physicians. Respondents reported a high level of satisfaction with their healthcare. The only factor significantly associated with satisfaction was age, with participants under age 26 reporting significantly higher levels of satisfaction than participants above age 26. Participants under age 26 also were significantly more likely to live at home, have private health insurance, and have others making their healthcare decisions than participants above age 26. Results indicate that healthcare satisfaction can be high for adults with ASD that have good family and community support.

Sensory symptoms in children with autism spectrum disorder, other developmental disorders and typical development: A longitudinal study.

Sensory symptoms are prevalent in autism spectrum disorder but little is known about the early developmental patterns of these symptoms. This study examined the development of sensory symptoms and the relationship between sensory symptoms and adaptive functioning during early childhood. Three groups of children were followed across three time points from 2 to 8 years of age: autism spectrum disorder, developmental delay, and typical development. At each time point, parents filled out questionnaires regarding their child's sensory symptoms and adaptive functioning. At the initial time point, parents of children with autism spectrum disorder reported more sensory symptoms in their children than parents in the typical development group. Parents in the autism spectrum disorder group reported more sensory symptoms than parents in the developmental delay group within smell, taste, and auditory domains. While the typical development group decreased in reported sensory symptoms across the study period, the clinical groups demonstrated no significant change across assessment points. Sensory symptoms for all groups were not independently predictive of adaptive functioning when verbal mental age was also included in the model. The young age range at the initial assessment and pattern of results suggest that sensory symptoms are present early in the etiology of autism spectrum disorder and other developmental disorders and remain stable over time.

Electrodermal and behavioral responses of children with autism spectrum disorders to sensory and repetitive stimuli.

Parents frequently report that their children with autism spectrum disorders (ASD) respond atypically to sensory stimuli. Repetitive behaviors are also part of the ASD behavioral profile. Abnormal physiological arousal may underlie both of these symptoms. Electrodermal activity (EDA) is an index of sympathetic nervous system arousal. The goals of this study were twofold: (1) to pilot methods for collecting EDA data in young children and (2) to examine hypothesized relationships among EDA, and sensory symptoms and repetitive behaviors in children with ASD as compared with children with typical development. EDA was recorded on 54 young children with ASD and on 33 children with typical development (TD) during a protocol that included baseline, exposure to sensory and repetitive stimuli, and play. Parents completed standardized questionnaires regarding their child's sensory symptoms and repetitive behaviors. Frequency and type of repetitive behavior during play was coded offline. Comparisons between EDA data for ASD and TD groups indicated no significant between-group differences in any measures. Parents of children with ASD reported more abnormal responses to sensory stimuli and more repetitive behaviors, but scores on these measures were not significantly correlated with EDA or with frequency of observed repetitive behaviors. Parent report of frequency and severity of sensory symptoms was significantly correlated with reports of repetitive behaviors in both groups. Although parents of children with ASD report high levels of sensory symptoms and repetitive behaviors, these differences are not related to measured EDA arousal or reactivity.

Preliminary findings of a telehealth approach to parent training in autism.

Telehealth or online communication technologies may lessen the gap between intervention requirements for children with autism spectrum disorders (ASDs) and the available resources to provide these services. This study used a video conferencing and self-guided website to provide parent training in the homes of children with ASD. The first eight families to complete the 12-week online intervention and three-month follow up period served as pilot data. Parents' intervention skills and engagement with the website, as well as children's verbal language and joint attention skills were assessed. Preliminary research suggests telehealth may support parental learning and improve child behaviors for some families. This initial assessment of new technologies for making parent training resources available to families with ASD merits further, in-depth study.

Intact and impaired mechanisms of action understanding in autism.

Typically developing children understand and predict others' behavior by extracting and processing relevant information such as the logic of their actions within the situational constraints and the intentions conveyed by their gaze direction and emotional expressions. Children with autism have difficulties understanding and predicting others' actions. With the use of eye tracking and behavioral measures, we investigated action understanding mechanisms used by 18 children with autism and a well-matched group of 18 typically developing children. Results showed that children with autism (a) consider situational constraints in order to understand the logic of an agent's action and (b) show typical usage of the agent's emotional expressions to infer his or her intentions. We found (c) subtle atypicalities in the way children with autism respond to an agent's direct gaze and (d) marked impairments in their ability to attend to and interpret referential cues such as a head turn for understanding an agent's intentions.