Brain-wide circuitry underlying altered auditory habituation in zebrafish models of autism.

Auditory processing is widely understood to occur differently in autism, though the patterns of brain activity underlying these differences are not well understood. The diversity of autism also means brain-wide networks may change in various ways to produce similar behavioral outputs. We used larval zebrafish to investigate auditory habituation in four genetic lines relevant to autism: fmr1, mecp2, scn1lab and cntnap2. In free-swimming behavioral tests, we found each line had a unique profile of auditory hypersensitivity and/or delayed habituation. Combining the optical transparency of larval zebrafish with genetically encoded calcium indicators and light-sheet microscopy, we then observed brain-wide activity at cellular resolution during auditory habituation. As with behavior, each line showed unique alterations in brain-wide spontaneous activity, auditory processing, and adaptation in response to repetitive acoustic stimuli. We also observed commonalities in activity across our genetic lines that indicate shared circuit changes underlying certain aspects of their behavioral phenotypes. These were predominantly in regions involved in sensory integration and sensorimotor gating rather than primary auditory areas. Overlapping phenotypes include differences in the activity and functional connectivity of the telencephalon, thalamus, dopaminergic regions, and the locus coeruleus, and excitatory/inhibitory imbalance in the cerebellum. Unique phenotypes include loss of activity in the habenula in scn1lab, increased activity in auditory regions in fmr1, and differences in network activity over time in mecp2 and cntnap2. Comparing these distinct but overlapping brain-wide auditory networks furthers our understanding of how diverse genetic factors can produce similar behavioral effects through a range of circuit- and network-scale mechanisms.

Reporting community involvement in autism research: Findings from the journal Autism.

LAY ABSTRACT: There has been a growing push for the Autistic and autism communities to be more actively involved in autism research. From January 2021, the journal Autism made it a rule for authors to report whether they involved community members in their work; and if they did, how they had done so. In this study, we wanted to see how this new rule has changed things. Our team of Autistic and non-autistic researchers read all 283 articles published in Autism in 2019, about 2 years before the rule was in place, and in 2022, about 1 year after. We recorded what each article was about and how the community was involved. We found there was an increase in how often articles talked about community involvement - from about 10% before the rule to over 50% after. Most of these studies, however, only involved community members giving advice, with the researchers making most decisions about the research. This was especially true for applied research (like wellbeing) rather than basic science (like causes of autism). Also, some of these articles were unclear or did not give enough information for us to understand how the community was involved. This tells us that while it is promising that more community involvement is reported, researchers need to describe this involvement more clearly. It is also important for community members to have a bigger say in research by sharing power with the researchers or even leading the research themselves.

EVIDENCE FOR AUDITORY STIMULUS-SPECIFIC ADAPTATION BUT NOT DEVIANCE DETECTION IN LARVAL ZEBRAFISH BRAINS.

Animals receive a constant stream of sensory input, and detecting changes in this sensory landscape is critical to their survival. One signature of change detection in humans is the auditory mismatch negativity (MMN), a neural response to unexpected stimuli that deviate from a predictable sequence. This process requires the auditory system to adapt to specific repeated stimuli while remaining sensitive to novel input (stimulus-specific adaptation). MMN was originally described in humans, and equivalent responses have been found in other mammals and birds, but it is not known to what extent this deviance detection circuitry is evolutionarily conserved. Here we present the first evidence for stimulus-specific adaptation in the brain of a teleost fish, using whole-brain calcium imaging of larval zebrafish at single-neuron resolution with selective plane illumination microscopy. We found frequency-specific responses across the brain with variable response amplitudes for frequencies of the same volume, and created a loudness curve to model this effect. We presented an auditory 'oddball' stimulus in an otherwise predictable train of pure tone stimuli, and did not find a population of neurons with specific responses to deviant tones that were not otherwise explained by stimulus-specific adaptation. Further, we observed no deviance responses to an unexpected omission of a sound in a repetitive sequence of white noise bursts. These findings extend the known scope of auditory adaptation and deviance responses across the evolutionary tree, and lay groundwork for future studies to describe the circuitry underlying auditory adaptation at the level of individual neurons.

'We have so much to offer': Community members' perspectives on autism research.

LAY ABSTRACT: Autism research is changing. Autistic activists and researchers want Autistic people in the community to have more of a say about what is researched and how. But we haven't asked people in the community what they think. This study used the information obtained from 55 community members, including Autistic people, their families, and professionals working with Autistic people, from an existing study on their priorities for autism research. We re-looked at what was said to see if we could understand community members' views and experiences of autism research. People agreed strongly that research can play a powerful role in shaping good Autistic lives. They also felt that big changes were needed for research to do this. Some of these changes were that researchers should stop thinking about autism narrowly and in a negative way, where Autistic people are seen as the problem. Researchers need to think more about how to improve systems, experiences and how other people respond to Autistic people. They also want the autism community to be more involved in what is researched and how it is researched. The findings from our study here highlight the potential for research to be positive when Autistic people and their families are listened to, approached with understanding, and are respected and valued as individuals in the research process.

Contributing to an autism biobank: Diverse perspectives from autistic participants, family members and researchers.

LAY ABSTRACT: A lot of autism research has focused on finding genes that might cause autism. To conduct these genetic studies, researchers have created 'biobanks' - collections of biological samples (such as blood, saliva, urine, stool and hair) and other health information (such as cognitive assessments and medical histories). Our study focused on the Australian Autism Biobank, which collected biological and health information from almost 1000 Australian autistic children and their families. We wanted to know what people thought about giving their information to the Biobank and why they chose to do so. We spoke to 71 people who gave to the Biobank, including 18 autistic adolescents and young adults, 46 of their parents and seven of their siblings. We also spoke to six researchers who worked on the Biobank project. We found that people were interested in giving their information to the Biobank so they could understand why some people were autistic. Some people felt knowing why could help them make choices about having children in the future. People also wanted to be involved in the Biobank because they believed it could be a resource that could help others in the future. They also trusted that scientists would keep their information safe and were keen to know how that information might be used in the future. Our findings show that people have lots of different views about autism biobanks. We suggest researchers should listen to these different views as they develop their work.

The INSAR Community Collaborator Request: Using community-academic partnerships to enhance outcomes of participatory autism research.

Participatory approaches, in which researchers work together with members of the autism community (e.g., autistic people, family members, caregivers, or other stakeholders) to design, conduct, and disseminate research, have become increasingly prominent within the field of autism research over the past decade. Despite growing academic and community interest in conducting participatory studies, stakeholder collaboration remains infrequent in autism research, at least partially due to systemic barriers. To help reduce barriers to engaging in participatory autism research, the International Society for Autism Research (INSAR) Autistic Researchers Committee has launched the INSAR Community Collaborator Request (ICCR; https://www.autism-insar.org/page/iccr), a platform on the INSAR website that allows autism researchers conducting participatory research to seek out stakeholder collaborators from the autism community (including both autistic people and their family members/caregivers, as relevant to a given research project). Interested stakeholders also have the opportunity to subscribe to ICCR posts, allowing them to be alerted of new opportunities for collaboration and potentially increasing their involvement in autism research. Overall, the ICCR provides a venue to connect autism researchers with potential community collaborators, reducing barriers to participatory autism research and increasing the frequency of successful community-academic partnerships within the field. We are hopeful that in the long term, such changes will lead to greater alignment between research outputs and the goals of the greater autism community, and consequently an increase in the overall quality and relevance of autism research.

Experiences of student and trainee autism researchers during the COVID-19 pandemic.

Circumstances surrounding the COVID-19 pandemic have resulted in significant personal and professional adjustments. Students and trainees, including those in autism research, face unique challenges to accomplishing their training and career goals during this unprecedented time. In this commentary, we, as members of the International Society for Autism Research Student and Trainee Committee, describe our personal experiences, which may or may not align with those of other students and trainees. Our experiences have varied both in terms of the ease (or lack thereof) with which we adapted and the degree to which we were supported in the transition to online research and clinical practice. We faced and continue to adjust to uncertainties about future training and academic positions, for which opportunities have been in decline and have subsequently negatively impacted our mental health. Students and trainees' prospects have been particularly impacted compared to more established researchers and faculty. In addition to the challenges we have faced, however, there have also been unexpected benefits in our training during the pandemic, which we describe here. We have learned new coping strategies which, we believe, have served us well. The overarching goal of this commentary is to describe these experiences and strategies in the hope that they will benefit the autism research community moving forward. Here, we provide a set of recommendations for faculty, especially mentors, to support students and trainees as well as strategies for students and trainees to bolster their self-advocacy, both of which we see as crucial for our future careers. LAY SUMMARY: The COVID-19 pandemic has affected students and trainees, including those in autism research, in different ways. Here, we describe our personal experiences. These experiences include challenges. For example, it has been difficult to move from in-person to online work. It has also been difficult to keep up with work and training goals. Moreover, working from home has made it hard to connect with our supervisors and mentors. As a result, many of us have felt unsure about how to make the best career choices. Working in clinical services and getting to know and support our patients online has also been challenging. Overall, the pandemic has made us feel more isolated and some of us have struggled to cope with that. On the other hand, our experiences have also included benefits. For example, by working online, we have been able to join meetings all over the world. Also, the pandemic has pushed us to learn new skills. Those include technical skills but also skills for well-being. Next, we describe our experiences of returning to work. Finally, we give recommendations for trainees and supervisors on how to support each other and to build a strong community.

Broad frequency sensitivity and complex neural coding in the larval zebrafish auditory system.

Most animals have complex auditory systems that identify salient features of the acoustic landscape to direct appropriate responses. In fish, these features include the volume, frequency, complexity, and temporal structure of acoustic stimuli transmitted through water. Larval fish have simple brains compared to adults but swim freely and depend on sophisticated sensory processing for survival.1-5 Zebrafish larvae, an important model for studying brain-wide neural networks, have thus far been found to possess a rudimentary auditory system, sensitive to a narrow range of frequencies and without evident sensitivity to acoustic features that are salient and ethologically important to adult fish.6,7 Here, we have combined a novel method for delivering water-borne sounds, a diverse assembly of acoustic stimuli, and whole-brain calcium imaging to describe the responses of individual auditory-responsive neurons across the brains of zebrafish larvae. Our results reveal responses to frequencies ranging from 100 Hz to 4 kHz, with evidence of frequency discrimination from 100 Hz to 2.5 kHz. Frequency-selective neurons are located in numerous regions of the brain, and neurons responsive to the same frequency are spatially grouped in some regions. Using functional clustering, we identified categories of neurons that are selective for a single pure-tone frequency, white noise, the sharp onset of acoustic stimuli, and stimuli involving a gradual crescendo. These results suggest a more nuanced auditory system than has previously been described in larval fish and provide insights into how a young animal's auditory system can both function acutely and serve as the scaffold for a more complex adult system.

Sound generation in zebrafish with Bio-Opto-Acoustics.

Hearing is a crucial sense in underwater environments for communication, hunting, attracting mates, and detecting predators. However, the tools currently used to study hearing are limited, as they cannot controllably stimulate specific parts of the auditory system. To date, the contributions of hearing organs have been identified through lesion experiments that inactivate an organ, making it difficult to gauge the specific stimuli to which each organ is sensitive, or the ways in which inputs from multiple organs are combined during perception. Here, we introduce Bio-Opto-Acoustic (BOA) stimulation, using optical forces to generate localized vibrations in vivo, and demonstrate stimulation of the auditory system of zebrafish larvae with precise control. We use a rapidly oscillated optical trap to generate vibrations in individual otolith organs that are perceived as sound, while adjacent otoliths are either left unstimulated or similarly stimulated with a second optical laser trap. The resulting brain-wide neural activity is characterized using fluorescent calcium indicators, thus linking each otolith organ to its individual neuronal network in a way that would be impossible using traditional sound delivery methods. The results reveal integration and cooperation of the utricular and saccular otoliths, which were previously described as having separate biological functions, during hearing.

Altered brain-wide auditory networks in a zebrafish model of fragile X syndrome.

BACKGROUND: Loss or disrupted expression of the FMR1 gene causes fragile X syndrome (FXS), the most common monogenetic form of autism in humans. Although disruptions in sensory processing are core traits of FXS and autism, the neural underpinnings of these phenotypes are poorly understood. Using calcium imaging to record from the entire brain at cellular resolution, we investigated neuronal responses to visual and auditory stimuli in larval zebrafish, using fmr1 mutants to model FXS. The purpose of this study was to model the alterations of sensory networks, brain-wide and at cellular resolution, that underlie the sensory aspects of FXS and autism. RESULTS: Combining functional analyses with the neurons' anatomical positions, we found that fmr1-/- animals have normal responses to visual motion. However, there were several alterations in the auditory processing of fmr1-/- animals. Auditory responses were more plentiful in hindbrain structures and in the thalamus. The thalamus, torus semicircularis, and tegmentum had clusters of neurons that responded more strongly to auditory stimuli in fmr1-/- animals. Functional connectivity networks showed more inter-regional connectivity at lower sound intensities (a - 3 to - 6 dB shift) in fmr1-/- larvae compared to wild type. Finally, the decoding capacities of specific components of the ascending auditory pathway were altered: the octavolateralis nucleus within the hindbrain had significantly stronger decoding of auditory amplitude while the telencephalon had weaker decoding in fmr1-/- mutants. CONCLUSIONS: We demonstrated that fmr1-/- larvae are hypersensitive to sound, with a 3-6 dB shift in sensitivity, and identified four sub-cortical brain regions with more plentiful responses and/or greater response strengths to auditory stimuli. We also constructed an experimentally supported model of how auditory information may be processed brain-wide in fmr1-/- larvae. Our model suggests that the early ascending auditory pathway transmits more auditory information, with less filtering and modulation, in this model of FXS.