Tifanie Valentin Bauer
email: valentin.bauer@limsi.fr

Bouchara Bouchara

Olivier Duris

Charlotte Labossière

Marie-Noëlle Clément

Patrick Bourdot

Tifanie Bouchara

Evaluating the Acceptability and Usability of a Head-Mounted Augmented Reality Approach for Autistic Children With High Support Needs

Keywords: Augmented Reality, User Experience, Multisensory, Autism Spectrum Disorder, Acceptability, Usability, Well-being, Children

published or not. The documents may come

Introduction

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition which concerns 1% of people worldwide [START_REF] Lord | Autism spectrum disorder[END_REF]. It is mainly characterized by social communication and interaction difficulties, restricted interests, and repetitive behaviours [START_REF]American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders[END_REF][START_REF] Lord | Autism spectrum disorder[END_REF]. Autistic people 3 display these traits in various proportions. Some individuals have low support needs (LS) (e.g., to perform academic tasks), and others have high support needs (HS) (e.g., to perform daily tasks), being minimally verbal and with associated Intellectual Disability (ID) [START_REF] Lord | Autism spectrum disorder[END_REF]. Recent research has largely overlooked children with HS [START_REF] Happé | Annual Research Review: Looking back to look forward -changes in the concept of autism and implications for future research[END_REF], being absent from 94% of autism research published in 2016 [START_REF] Russell | Selection bias on intellectual ability in autism research: a cross-sectional review and meta-analysis[END_REF]. This paper proposes to complement practitioners' interventions for children with HS through a technology-based approach.

Interventions for children with HS largely consist in reassuring the child and reinforcing the dyadic child-practitioner relationship, prior to work on other abilities (e.g., social abilities) [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF][START_REF] Sandbank | Project AIM: Autism intervention meta-analysis for studies of young children[END_REF]. To that end, mediation sensory-based interventions are common, such as Sensory Integration Therapy [START_REF] Schoen | A systematic review of ayres sensory integration intervention for children with autism[END_REF][START_REF] Ayres | Sensory integration and learning disorders[END_REF] or Snoezelen [START_REF] Lancioni | Snoezelen: an overview of research with people with developmental disabilities and dementia[END_REF][START_REF] Novakovic | Effects of Snoezelen-Multisensory environment on CARS scale in adolescents and adults with autism spectrum disorder[END_REF]. Sensory Integration Therapy seeks to train multisensory interactive processes to gradually enhance developmental abilities, with various playful sensory objects (e.g., sensory balls). Snoezelen aims at reassuring and relaxing children to gradually reinforce the dyadic relationship, with multisensory spaces often including bubble columns. It is particularly promising for children with HS [START_REF] Novakovic | Effects of Snoezelen-Multisensory environment on CARS scale in adolescents and adults with autism spectrum disorder[END_REF]. Yet, practitioners can struggle to conduct such interventions, especially with children with HS [START_REF] Kouo | A Scoping Review of Targeted Interventions and Training to Facilitate Medical Encounters for School-Aged Patients with an Autism Spectrum Disorder[END_REF]. Difficulties can come from a lack of resources, of flexibility of the tools and environments, time constraints, or expensive cost [START_REF] Kouo | A Scoping Review of Targeted Interventions and Training to Facilitate Medical Encounters for School-Aged Patients with an Autism Spectrum Disorder[END_REF].

Practitioners often use digital tools to complement their interventions, as being individualizable, controllable [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF][START_REF] Sandbank | Project AIM: Autism intervention meta-analysis for studies of young children[END_REF], and often appealing [START_REF] Laurie | An International Survey of Parental Attitudes to Technology Use by Their Autistic Children at Home[END_REF]. They mainly target socio-developmental abilities through various mediums (e.g., tablet, computer, robot). Yet, such interventions lack of multisensory capabilities that are needed to work with autistic children with HS [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF].

Some digitally-augmented multisensory spaces have been designed for autistic children, with successful outcomes over their well-being and the dyadic relationship [START_REF] Basadonne | Working on Cognitive Functions in a Fully Digitalized Multisensory Interactive Room: A New Approach for Intervention in Autism Spectrum Disorders[END_REF][START_REF] Garzotto | Magic Room: A Smart Space for Children with Neurodevelopmental Disorder[END_REF][START_REF] Mora-Guiard | Sparking social initiation behaviors in children with Autism through full-body Interaction[END_REF][START_REF] Pares | Achieving Dialogue with Children with Severe Autism in an Adaptive Multisensory Interaction: The "MEDIATE" Project[END_REF][START_REF] Ringland | Sen-soryPaint: a multimodal sensory intervention for children with neurodevelopmental disorders[END_REF]. Various settings were used: large spaces where physical items are augmented to trigger stimuli based on users' actions [START_REF] Basadonne | Working on Cognitive Functions in a Fully Digitalized Multisensory Interactive Room: A New Approach for Intervention in Autism Spectrum Disorders[END_REF][START_REF] Garzotto | Magic Room: A Smart Space for Children with Neurodevelopmental Disorder[END_REF][START_REF] Pares | Achieving Dialogue with Children with Severe Autism in an Adaptive Multisensory Interaction: The "MEDIATE" Project[END_REF], a projected floor [START_REF] Mora-Guiard | Sparking social initiation behaviors in children with Autism through full-body Interaction[END_REF], or a projected screen and a Kinect camera [START_REF] Ringland | Sen-soryPaint: a multimodal sensory intervention for children with neurodevelopmental disorders[END_REF]. Yet, equipment is often bespoke, bulky, or too expensive for clinical structures.

Virtual (VR) and Augmented (AR) Reality could overcome the limits of autism interventions with or without digital tools, by offering secure multisensory capabilities [START_REF] Dechsling | Virtual reality and naturalistic developmental behavioral interventions for children with autism spectrum disorder[END_REF][START_REF] Karami | Effectiveness of Virtual/Augmented Reality-Based Therapeutic Interventions on Individuals With Autism Spectrum Disorder: A Comprehensive Meta-Analysis[END_REF][START_REF] Mesa-Gresa | Effectiveness of Virtual Reality for Children and Adolescents with Autism Spectrum Disorder: An Evidence-Based Systematic Review[END_REF] through affordable Head-Mounted Displays (HMDs). Moreover, they allow to include all autistic children including those with HS [START_REF] Happé | Annual Research Review: Looking back to look forward -changes in the concept of autism and implications for future research[END_REF]. Yet, children with HS are under-represented, as most studies focus on training socio-emotional abilities [START_REF] Mesa-Gresa | Effectiveness of Virtual Reality for Children and Adolescents with Autism Spectrum Disorder: An Evidence-Based Systematic Review[END_REF][START_REF] Herrero | An immersive virtual reality educational intervention on people with autism spectrum disorders (ASD) for the development of communication skills and problem solving[END_REF][START_REF] Mosher | Immersive Technology to Teach Social Skills to Students with Autism Spectrum Disorder: a Literature Review[END_REF] which can be too advanced for them [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF][START_REF] Bozgeyikli | A Survey on Virtual Reality for Individuals with Autism Spectrum Disorder: Design Considerations[END_REF].

A previous study revealed that autism stakeholders would prefer to use AR for children with HS, in order to augment their familiar surroundings while still seeing their real practitioner, whereas VR could isolate them [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. In particular, AR use cases should focus on reassurance and reinforcing the dyadic child-practitioner relationship [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. Such an AR environment called Magic Bubbles was designed and validated in collaboration with practitioners working on a daily basis with children with HS [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF].

Unknowns remain about HMD's acceptability for autistic children with HS, as their positive acceptability was mainly suggested for children with LS [START_REF] Berenguer | Exploring the Impact of Augmented Reality in Children and Adolescents with Autism Spectrum Disorder: A Systematic Review[END_REF][START_REF] Bradley | Autism and virtual reality head-mounted displays: a state of the art systematic review[END_REF][START_REF] Malihi | Short report: Evaluating the safety and usability of head-mounted virtual reality compared to monitor-displayed video for children with autism spectrum disorder[END_REF][START_REF] Schmidt | A Process-Model for Minimizing Adverse Effects when Using Head Mounted Display-Based Virtual Reality for Individuals with Autism[END_REF]. To our knowledge, only three studies suggested a positive acceptability for individuals with HS: for twelve adults in VR [START_REF] Newbutt | Brief Report: A Pilot Study of the Use of a Virtual Reality Headset in Autism Populations[END_REF], for five children with neurodevelopmental disorders and ID in VR [START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF], and for twenty children with neurodevelopmental disorders and ID in AR [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF]. To complement them, this paper addresses three research questions:

1. Could autistic children with HS accept and use Magic Bubbles AR environment? 2. Could autistic children with HS get engaged with Magic Bubbles AR environment while still communicating with others? 3. Could autistic children with HS get secure when using Magic Bubbles AR environment?

To examine these three research questions, our team of researchers, coming both from the human-computer interaction field and the clinical field (two psychologists and one psychiatrist) conducted a study with 10 autistic children with HS, or similar traits, i.e., neurodevelopmental disorders and associated ID. Thus, this study extends a previous study that was only conducted with 11 practitioners as part of the design process of Magic Bubbles AR environment to ensure its acceptability among the clinical team [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF]. After presenting the methodology that was used, the findings will be outlined, and then discussed.

Method

Participants

Two psychologists from our research team recruited 10 children with neurodevelopmental disorders and associated ID among the patients of the day hospital André Boulloche, in agreement with the clinical team. They include six boys and four girls, from 11 to 14 (MA:12.5, SD:0.98). Eight were minimally verbal and two were non-verbal. All children displayed significant intellectual disability, their Intellectual Quotient (IQ) ranging from 50 to 70. Four children had already experienced VR during cultural outings. None had experienced AR. The low number of children is imposed by the following inclusion criteria: children had to display a neurodevelopmental condition according to the ICD-10 [START_REF]ICD-10: international statistical classification of diseases and related health problems : tenth revision[END_REF], an associated ID, not display risks of epilepsy, and be at least 11 years old for using HMDs in line with recent AR/VR studies [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF][START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF][START_REF] Malihi | Short report: Evaluating the safety and usability of head-mounted virtual reality compared to monitor-displayed video for children with autism spectrum disorder[END_REF][START_REF] Newbutt | Using Virtual Reality Head-Mounted Displays in Schools with Autistic Children: Views, Experiences, and Future Directions[END_REF]. As this low number is common in AR/VR studies for autism, mainly because this population is hardto-reach [START_REF] Karami | Effectiveness of Virtual/Augmented Reality-Based Therapeutic Interventions on Individuals With Autism Spectrum Disorder: A Comprehensive Meta-Analysis[END_REF][START_REF] Mesa-Gresa | Effectiveness of Virtual Reality for Children and Adolescents with Autism Spectrum Disorder: An Evidence-Based Systematic Review[END_REF], it was deemed suitable to investigate our research questions. Moreover, according to the psychologists, the 3:2 male to female ratio among the recruited children would not influence the findings. Children's profiles are detailed in Table 1.

HMD-based AR environment tested with children

Magic Bubbles is a multisensory HMD-based AR environment which aims at complementing common practitioners' interventions for autistic children with HS, such as Snoezelen or Sensory Integration Therapy. The design process was first informed by 34 interviews with autism stakeholders [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF], as suggested by Parsons et al. [START_REF] Parsons | Whose agenda? Who knows best? Whose voice?' Co-creating a technology research roadmap with autism stakeholders[END_REF]'s research agenda. The design was then adapted for a day hospital context in collaboration with two psychologists, and validated through testings with 11 practitioners [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF]. This subsection only summarizes the main features of Magic Bubbles, as a previous paper presents its full description [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF].

Children can explore an augmented interaction space, while perceiving their practitioner, and interacting with audio, visuals, and controllers' vibrations. Stimuli are presented through common appealing objects (e.g., bubble columns) drawing upon common interventions. Stimuli are generic (e.g., bubbles) or individualized (e.g., music). Practitioners can prompt exploration and shared play by standing physically close to the child while perceiving what they see and hear through a screen monitor. The design is presented on Figure 1. Two conditions exist: spectator and actor. When spectator, the child can move in space without interacting with objects, but the practitioner can trigger all stimuli. When actor, the child can move and interact with everything.

Augmentation is presented through an AR see-through setup, by using a Zed-Mini camera plugged onto a HTC Vive Pro HMD, that "captures a live feed of the real environment, and then makes it visible inside the HMD, supplemented by virtual objects" [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF]. Two HTC Vive controllers are used, as well as four Vive lighthouse outside-in tracking systems.

Development of the semi-structured questionnaire

To assess the experience of autistic children who cannot complete self-report questionnaires according to the psychologists, we had to build a new questionnaire that could be filled by their respective practitioners, as in Aruanno et al. [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF]'s study. Hence, a two-part semi-directed questionnaire was devised to be filled by the practitioner in 5 to 10 minutes at the end of every child's AR session. Most questions use 1-5 likert scales, apart from questions addressing agency and engagement that use 1-7 likert scales, as drawing upon questionnaires with such scales [START_REF] Schubert | The sense of presence in virtual environments:: A three-component scale measuring spatial presence, involvement, and realness[END_REF][START_REF] Witmer | Measuring Presence in Virtual Environments: A Presence Questionnaire[END_REF]. Practitioners can write additional details if necessary.

In the first part, six questions address the child's state, drawing upon stakeholders' interviews [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. Question 1 (Q1) and Q2 ask if the child was anxious and tired prior to start, and Q3 and Q4 ask the same questions about the end. Q5 asks if the child got resourced and Q6 if they could start another activity at the end. As the questionnaire has to be filled in 10 minutes maximum so that practitioners can complete it, these questions intend to be quickly answered, and to only provide an overall picture of the child's state. Yet, they seek to collect key elements by relying on psychologists' expertise in autism and their knowledge of these children to integrate multiple aspects in order to provide a representative answer for each question.

In the second part, nineteen questions address the acceptability, usability, control, presence, engagement, and social interaction, inspired from questionnaires [START_REF] Brooke | SUS-A quick and dirty usability scale[END_REF][START_REF] Schubert | The sense of presence in virtual environments:: A three-component scale measuring spatial presence, involvement, and realness[END_REF][START_REF] Witmer | Measuring Presence in Virtual Environments: A Presence Questionnaire[END_REF][START_REF] Kennedy | Simulator Sickness Questionnaire: An Enhanced Method for Quantifying Simulator Sickness[END_REF], studies investigating the use of digital tools for autism [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF][START_REF] Garzotto | Magic Room: A Smart Space for Children with Neurodevelopmental Disorder[END_REF][START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF], and interviews with stakeholders [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. First, three questions focus on acceptability [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF][START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF], about: the easiness to wear the HMD (Q7), the annoyance due to wearing/removing it (Q8), and cybersickness' symptoms (e.g., nausea, oculomotor symptoms, disorientation)(Q9). Then six questions address usability [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF][START_REF] Brooke | SUS-A quick and dirty usability scale[END_REF][START_REF] Witmer | Measuring Presence in Virtual Environments: A Presence Questionnaire[END_REF], about: the platform's complexity (Q10), the amount of information (Q11), the need for support (Q12), the confidence (Q13) and easiness (Q14) when using the system, and the ability to interact and to move at the end (Q15). Then, agency is questioned [START_REF] Witmer | Measuring Presence in Virtual Environments: A Presence Questionnaire[END_REF] regarding the ability to identify stimuli (Q16), anticipate reactions (Q17), or actively control the environment (Q18). After that, presence is questioned [START_REF] Schubert | The sense of presence in virtual environments:: A three-component scale measuring spatial presence, involvement, and realness[END_REF][START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF] about: the understanding of real versus virtual (Q19), the real-world awareness (Q20), the feeling of being captivated (Q21), and the consistency of the experience with respect to their usual realworld experience (Q22). Then engagement is addressed [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF], about fun (Q23), and involvement (Q24). After that, the level of communication is questioned [START_REF] Garzotto | Magic Room: A Smart Space for Children with Neurodevelopmental Disorder[END_REF] (Q25), and practitioners can add something if needed (Q26).

Procedure

All procedures were approved by Poléthis Ethics Research Committee of Paris-Saclay University under reference 226.

Before the experiment A psychiatrist explained the protocol to the child's legal tutors and collected their informed consent. Indeed, children were unable to do it by themselves according to the clinical team. As this study did not assess the differences induced by the actor and spectator conditions (see Subsection 2.2), practitioners affected children to each condition depending on their common interventions, and if they were mainly passive or active. Thus, ID4 and ID9 were affected to the spectator condition, and the others to the actor condition.

During the experiment

The child tested the AR environment in a large room of the day hospital which is normally used for sensory-based activities. Four investigators were always present: two psychologists, one investigator being here for technical support, and one psychology intern. The child's educator could also come for reassurance or if interested, following common clinical practices. Sessions lasted between 5 minutes (minimum) and 20 minutes (maximum), depending on the child's acceptability, if they wanted to stop, or on practitioners' perception of their experience. Due to day hospital's constraints, unexpected events could impact the session (e.g., child being late as coming from the infirmary). Psychologists were used to the AR platform when starting the testings, as they participated to the AR design process beforehand [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF]. Several sessions with different children were successively conducted, spaced by a 15-minute break to clean the equipment and air the room according to COVID security rules. Equipment was mounted (20 minutes) and dismounted (15 minutes) before and after all sessions.

At the session's beginning, psychologists introduced the child to the AR system and invited them to wear the HMD. Then, children experienced a free-play time, during which practitioners could interact with them (i.e., verbally or nonverbally) while monitoring what they perceived through the screen. Children were never forced to wear the HMD, and could remove/wear it at will. Session could end up in two ways: if children expressed that they wanted to stop (e.g., verbally, or by removing the HMD), or if time ran out. In this second case, practitioners warned that the session would end in one minute (verbally, and by triggering a gong sound in AR) and proposed to do one last action. After removing the HMD (by themselves or with practitioners' support), psychologists invited children to make a real drawing with a sheet of paper and pencils. Children only drew if they wanted to.

After the experiment After the child left, psychologists completed a semidirection questionnaire (presented in Subsection 2.3). Then, we took notes to summarize the session's unfolding, by paying attention to critical incidents (e.g., unusual events). After the sessions of the six first children, to elicit more insights, semi-directed focus group interviews were added and conducted by the first author with the two psychologists and the intern to debrief about the sessions. They happened right after the sessions were conducted. The interviews with the practitioners relied on three main questions, asking if the child had fun, if they seemed to be reassured, and if they were ready to start other tasks when leaving.

Data collection

To assess children's experiences, multiple data sources were used to mitigate the bias due to each source. Data collected include: the semi-structured questionnaire, semi-directed interviews, first author's notes, and three video recordings (two cameras from different angles and the child's view). Moreover, efforts were made to get the child's perspective: by collecting their drawings, as suggested by Spiel et al. [START_REF] Spiel | Experiences of autistic children with technologies[END_REF]'s study about autistic children's experiences with technology, and by filling the questionnaire while asking questions to the child, as in Aruanno et al. [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF]'s study. Yet, only ID16 accepted to draw and only ID7 could answer questions. Thus, data collected mainly accounted for practitioners' perspective. In addition to that, we measured the time during which children wore the HMD. Collecting physiological data was also considered (e.g., heart rate) but abandoned, as the biosensors could hinder children's experience according to the psychologists. All data were anonymized by affecting identifiers to the children (see Table 1).

Data analysis

The first author transcribed the data collected. This process allowed to familiarize with it, to ask the psychologists for clarifications, or to check their ratings when not matching their comments. To do so, psychologists looked at the videos and made corrections when appropriate. Then, two main analytic stages were conducted in parallel, by different researchers, and with different methods, to mitigate potential biases. First, questionnaire's answers were analysed quantitatively by the first author, while considering practitioners' comments. Second, the interviews, notes, and videos were analyzed qualitatively. The first author analyzed the notes and interviews, and two other authors who are also clinical psychologists analyzed the videos. To do so, deductive content analysis was mainly used, complemented by inductive content analysis [START_REF] Elo | The qualitative content analysis process[END_REF]. The two main analytic stages are detailed below.

Analyzing the semi-directed questionnaire Descriptive statistics were used to analyze the questionnaire's answers, as more advanced statistics were not meaningful with respect to the low number of participants. Figures 2 and3 respectively display the findings related to the user experience's categories (e.g., acceptability, usability), and to the evolution of the child's state between the beginning and the end of sessions. Findings are presented with respect to practitioners' comments, through different paragraphs accounting for the different categories (see Subsection 3.1). Moreover, an additional paragraph accounts for critical incidents that appeared from practitioners' comments. Questions about the feeling of presence were not analyzed, as not related to the current research objectives. They will be evaluated in a future paper.

Analyzing the notes, interviews and videos Deductive content analysis was mainly used to analyze the data with respect to the questionnaires' categories. The goal was to confirm and draw comparisons with the questionnaires' findings, as well as to gather further insights about the categories being assessed. To complement this approach, inductive content analysis was used, by constructing new categories from the data.

The first author analyzed the interviews and notes by doing open coding, with the data analysis software called MaxQDA4 . To mitigate potential biases, constant comparison techniques were used to compare the initial data with the phrasings and categories that were gradually constructed. The psychologists analyzed the videos, based on their clinical expertise, as in previous studies [START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF]. The qualitative analysis process stopped when reaching data saturation. Findings were finally compared, and presented together in subsection 3.2.

Findings

Findings are presented in two subsections, accounting for the two main analytic stages that were used. First, questionnaires' answers are presented. Then, findings from the video observations, notes and interviews are presented. The times during which children wore the HMD are not presented as they happened to be unusable. Indeed, practitioners often wore the headset during sessions to support children's acceptability, and children often worn and removed the HMD multiple times, making it impossible to collect precise data.

Findings from the semi-directed questionnaire

Answers about the child's experience are first presented (Q7-Q16, Q23-25) (see Figure 2), and followed by answers about the child's state (Q1-Q6) (see Figure 3). Q17 and Q18 were removed as unanswered for most children. Indeed, practitioners deemed them too advanced for this discovery session, and thus impossible to answer. At last, critical incidents are presented. Acceptability Most children easily accepted the HMD (Q7): seven very easily (5/5) and ID9 easily (4/5). Among them, ID8 wore it nearly alone, ID4 played to wear/remove it, and ID16 expressed a slight discomfort that was not precisely identified. Yet, ID6 had some difficulties (3/5), requiring support and preferring the HMD to be a bit unscrewed. ID17 wore it for a very short time (1/5), which is normal as he usually needs time to get used to new elements. Seven children had no discomfort (Q8-5/5), contrary to ID6 and ID8 (4/5). Indeed, ID16 removed the HMD after six minutes, and ID8 was frustrated to spend so much time to adjust it. Seven children did not experience cybersickness (Q9-5/5), but ID5 said that the image was "blurry" at the start. While no answer was given for ID6, he did not have a negative experience but looked surprised after removing the HMD. ID9 had cybersickness (1/5) and said that the image was blurry. Blurry images could be perceived due to the HMD not being screwed enough, and point at the difficulty to correctly adjust the HMD with such children.

Usability Magic Bubbles' complexity (Q10) was adapted to all children (5/5), and not mentioned for ID17 as he only wore it for a very short time. The psychologists advised using more complexity for three children, to not induce boredom over time (ID1), and for the spectator condition: "if actor, ID4 would have stayed longer". The amount of information (Q11) suited all children (5/5), but adding features was suggested for two of them. Most children required little practitioners' support (Q12): none for seven (5/5), some verbal reassurance for ID14 (4/5), and moderate for ID16 (3/5). Most children were confident (Q12): five entirely (5/5) and two nearly entirely (4/5). ID6 easily explored but no rating was given. Yet, ID9 was scared (1/5), but practitioners said that she expected to be scared when entering. Moreover, she may have been distressed due to feeling watched, and to not controlling the environment due to the spectator condition. Eight children very easily used the system (Q13-5/5), and ID16 easily used it (4/5). Moreover, ID1 was "immersed and discovered everything alone", and ID8 "did not move but everything was at arm length". At the end, apart from ID9, most children interacted well (Q14): totally for seven (5/5), or well for ID7 (4/5).

Agency Most children who wore the HMD could identify the stimuli (Q16): five with great ease (7/7), and ID1 (6/7) and ID16 (5/7) with ease. In particular, ID6 actively switch off the music when wanting to. Though, practitioners were unsure about ID4's ability to identify stimuli (3.5/7), as focusing a lot on the music and not exploring much. No answer was provided for ID9 who got worried.

Engagement Apart from the ID9 who got worried, most children had fun (Q23): seven a lot (7/7) and ID4 nearly a lot (6/7). ID1 was not amused but relaxed (4/7). About involvement (Q24), apart from ID9 (3/7), most children were involved, whether entirely for seven (7/7) or nearly entirely (6/7).

Social Interaction Most children communicated with the adults (Q25): five a lot (5/5) (including ID17 who wore the HMD for a short time), and three moderately (4/5). In particular, ID4 interacted more than usual with people around (e.g., practitioners were struck when she asked them to dance together). ID14 also initiated shared play by giving the HMD to the practitioners. Moreover, ID17 communicated through the monitor while the practitioner was wearing the HMD. At last, ID1 (3/5) and ID6 (2.5/5) also communicated but non-verbally, maybe because not hearing well what practitioners said with the HMD. Evolution of the child's state When starting, all children were in a good physical state, apart from ID17 who was tired (Q1-4/5) and ID9 (Q1-2.5/5). Six children were not anxious (Q2): five not at all (5/5) and ID17 slightly (4/5). Yet, four children were a bit worried: ID16 asked what would happen (3.5/5), ID6 if "it was a trap" (3/5), ID14 displayed apprehension (2.5/5), and ID9 asked if practitioners would "frighten her" (2/5). At the end, no child was tired (Q3-5/5), and nine children were not anxious (Q4-5/5). Hence, four children got secure (ID6, ID14, ID16, ID17). Yet, ID9 got scared (1/5) due to acceptability issues: running out of the room and throwing the HMD. Six children got resourced (Q5-5/5), and two were as before (2.5/5). Practitioners highlighted that ID7 got calmer even if anxious the day before. At last, apart from ID9 (1/5), most children were ready to start another activity when leaving (Q6): seven entirely (5/5), and two with no answer (indicating no detrimental effects).

Critical incidents Psychologists mitigated the fact that ID9 ran out by evoking her condition and impulsiveness. Although ID6 was slightly worried when starting, he asked to come back every week. This request is uncommon for him and accounts for his engagement. ID7 could "unload" when playing while being in control (see Figure 5B), which is uncommon for him. ID7, ID14 and ID17, socially interacted more than usual: ID7 kept eye contact with the adults, ID14 initiated shared play by giving them the HMD (see Figure 5E), and ID17 interacted threw the screen. At last, ID14 went on the floor when first wearing the HMD, and ID5 stepped over the column's border to go inside it (see Figure 5D).

Findings from the Interviews, Notes, and Observations

Findings coming from the deductive content analysis are first presented. Then, three categories that were built through the inductive content analysis are presented: Real vs. Virtual, Exploration of the Body and Space, and New Hypothesis.

Acceptability All children could wear the HMD with practitioners' support. Only ID9 got very anxious, and quickly asked to stop. However, she was already anxious when entering the room. Four children perceived a slight discomfort, due to the HMD being too tight, heavy, or to feeling hot. Half of the children emphasized the institutional aspect, asking questions about the other children who participated. Five children described their experience as "weird" but pleasant. Yet, some children displayed some apprehension: three children only interacted "at arm length", not daring to move too much, and two children asked the practitioners to wear the HMD before them for reassurance. Doing so, they observed the practitioners while looking at what they saw through the monitor.

Usability Eight children easily used the equipment. For instance, ID1 was "immersed and discovered everything alone". Yet, ID14 and ID16 required a lot of guidance. As psychologists said: "We proposed her [ID14] to stand up [. . . ].

Then she held Olivier's arm [one practitioner] to move in space, with still some difficulties", or "I made her [ID16] try things". ID16 verbally confirmed that she preferred to explore with Olivier, and even drew herself next to him (see Figure 4). Children were not bothered by some bugs that happened, even if possibly slowing down their discovery process. The microphone bubble was hard to use, as requiring to perform two actions (touch then speak). Eight children respected the limits of the AR space, and two went beyond to observe the limits of the real room. Five children understood that the vibrations came from the controllers. ID4 could explore alone although she is usually very passive. Agency While three children enjoyed the controllers' vibrations, other children were more interested in the audiovisual stimuli. Three children particularly enjoyed one stimulus: the bubble column (ID1), the music (ID4), and the music panel (ID7). They seemed to use it to: get secure, and/or to refocus on their body experiences after exploring. All children looked at their bodies (mainly their hands) and eight children went through virtual elements with their head and hands to see them disappear.

Engagement Eight children were involved and enjoyed the experience. For instance, ID7 sang, laughed, or danced while touching the virtual panels, although being in a bad state the day before according to the practitioners. These children focused on their inner experiences (little social interaction), which is a significant clinical finding according to practitioners. Seven children verbally expressed their joy, asking to come back the week after. Surprisingly, ID1 said that he had fun, but that he did not want to come back the week after. ID9 expressed anxiety.

Social Interaction All children interacted with the adults, but eight had inner experiences. Indeed, these eight children mainly asked questions when requiring support or answered practitioners' questions. Yet, some of them included practitioners in their experience: ID1 pointed at them with the controllers, or ID8 described everything aloud. At last, two children were more interested in the other rather than in technology (ID4, ID17). In particular, ID17 interacted with practitioners through the screen, and ID14 interacted "without disappearing in the relationship with the adult", which is uncommon for her.

Evolution of the child's state Practitioners did not comment on this evolution, except for ID9 who got worried. Yet, they highlighted that the setting was holding for four children. This clinical concept is used to describe a supportive environment for generating and supporting interactions between the child and the practitioner [START_REF] Winnicott | Playing and reality[END_REF]. Indeed, these four children got more secure than usual: ID1 got very calm, ID4 got calm even if several adults surrounded her, ID7 kept in control while being excited, and ID14 could socially interact.

Real versus Virtual Five children questioned the differences between real and virtual. They asked if the physical elements in the room were real, and if the virtual elements could have an impact on the adults being in the room. Three children also experienced a possible feeling of presence, i.e., feeling of being here. Indeed, ID8 asked if the water from the column could go over the floor, ID6 hit the ponds with his feet (see Figure 5A), and ID5 stepped over the border of the column (see Figure 5D).

Exploration of the body and space

Seven children performed gentler gestures than usual, and three children mainly focused on one stimulus. Children focused on their body image: all looked at their hands, ID4 asked to look at herself in the mirror, ID5 asked to be photographed, and ID16 drew herself with the practitioner on her side. Five children made uncommon movements: softer, more hesitant, or, conversely, dancing more for ID16. Four children adopted a different gait. Indeed, three of them lied on the floor: ID4 possibly due to anxiety, while the two others may have done it to calmly explore their body and the AR space. Moreover, ID5 behaved like a robot when entering and leaving.

New Hypothesis Practitioners raised four new hypothesis about using AR for children with neurodevelopmental disorders and associated ID. First, HMD could help to make longer eye contacts. This was raised after ID6 made long eye contacts. Second, AR could help to better understand the others' mental states, by perceiving through the monitor the practitioner's AR view. Indeed, ID17 could understand that the screen represented the practitioner' view. Third, AR could enhance body awareness. Indeed, ID14 and ID16 socially interacted without "disappearing", although uncommon for them. At that, adjusting the proportion between virtual and real elements could prompt reassurance, especially regarding the body presence of the others. This was raised after ID7 kept in control while being excited, although uncommon for him.

Discussion

This paper explored the use of the HMD-based AR environment Magic Bubbles in a day hospital setting with ten children with autism and HS, or neurodevelopmental disorders and associated ID. As little was known so far about how these children would react when exposed to HMD-based VR/AR [START_REF] Berenguer | Exploring the Impact of Augmented Reality in Children and Adolescents with Autism Spectrum Disorder: A Systematic Review[END_REF][START_REF] Bradley | Autism and virtual reality head-mounted displays: a state of the art systematic review[END_REF][START_REF] Malihi | Short report: Evaluating the safety and usability of head-mounted virtual reality compared to monitor-displayed video for children with autism spectrum disorder[END_REF][START_REF] Schmidt | A Process-Model for Minimizing Adverse Effects when Using Head Mounted Display-Based Virtual Reality for Individuals with Autism[END_REF], the paper has four main contributions. Three correspond to the three research questions, about acceptability and usability (RQ1), engagement (RQ2), and reassurance (RQ3), and one appeared from the data analysis process. First, most children displayed positive acceptability and usability (RQ1). The inductive analysis process also showed that children explored two aspects in addition to the virtual interactions: the difference between real and virtual elements and the shift in their self perception. Second, children were engaged with an inner experience, but still communicated (RQ2). Third, apart from ID9 who experienced cybersickness, four children got more secure, and the others were in the same state after than before the experience (RQ3). At last, four new hypothesis emerged in relation to practitioners' concerns which deserve future investigation. The validation of the three research questions prompt to conduct future testings with Magic Bubbles during a longer period with autistic children with HS. After presenting the findings related to the three research questions, limitations and future perspectives are drawn, which include the new hypothesis that emerged.

Accepting and Using Magic Bubbles

All children accepted to wear the HMD, as in previous VR [START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF][START_REF] Newbutt | Brief Report: A Pilot Study of the Use of a Virtual Reality Headset in Autism Populations[END_REF] and AR [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF] studies. To that end, they required practitioners' support. For instance, two children asked practitioners to wear the HMD before them for reassurance, as in Garzotto et al. [START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF]'s study where two out of five children made the same request. In our study, three children experienced discomfort, including cybersickness for one of them. This finding echoes Newbutt et al. [START_REF] Newbutt | Brief Report: A Pilot Study of the Use of a Virtual Reality Headset in Autism Populations[END_REF]'s findings, where four out of twenty-nine participants felt unwell and had to stop. In our study, despite their ID and limited verbal abilities, most children could express when feeling unwell. Though, they could hardly express if the HMD was correctly adjusted. This finding calls for devising future protocols to minimize potential adverse affects with HMDs for autistic children with HS, thus complementing Schmidt et al. [START_REF] Schmidt | A Process-Model for Minimizing Adverse Effects when Using Head Mounted Display-Based Virtual Reality for Individuals with Autism[END_REF]'s process-model that was mainly designed for autism with LS. At last, the methods for measuring acceptability through caregivers vary between previous studies [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF][START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF], and between our study and previous studies, thus making definitions of acceptability to vary. To make sure that we actually measured acceptability features and not other features (e.g., engagement), future research should focus on designing new standardized acceptability and usability measures for children with neurodevelopmental disorders and ID that would allow to collect their views.

Most children easily used Magic Bubbles, apart from ID9 due to acceptability issues and ID17 who wore the HMD for a very short time. Magic Bubbles was well adapted with respect to children's sensorimotor and understanding abilities. For instance, despite understanding difficulties, ID7 could explore and have fun. Then, practitioners supported children during their discovery, as in [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF][START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF]. Guidance was individualized, and ranged from low levels (e.g., for ID1) to moderate levels (e.g., ID16 needed physical guidance). Our positive acceptability and usability findings complement the findings from previous HMD-based studies conducted with autistic individuals with HS [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF][START_REF] Garzotto | Wearable Immersive Virtual Reality for Children with Disability: a Case Study[END_REF][START_REF] Newbutt | Brief Report: A Pilot Study of the Use of a Virtual Reality Headset in Autism Populations[END_REF].

Children explored three main aspects: virtual interactions, real versus virtual elements, and self perception. First, they explored the virtual interactions alone or with practitioners' support. To do so, they often displayed uncommon behaviours: seven used gentler gestures than usual, and three mainly focused on one stimulus. This insight calls for more research to better understand these behaviours. In particular, focusing on one stimulus may be linked with selfregulation strategies during the discovery process (to not get overwhelmed by too many information). Hence, parallels could be drawn with VR/AR derivatives of repetitive behaviours that children often use to get resourced, that previous research already suggested to investigate [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. Gentler behaviours may also be linked with a shift in their self perception. Second, children used various strategies to understand the difference between real and virtual elements. Future research should examine their understanding of real versus virtual, with respect to these various strategies and children's profiles. At last, children were highly interested in their self perception (e.g., looking at their hands). This unexpected insight accounts for a shift in self perception that deserves more investigation.

Communicating with practitioners

All children socially interacted with the adults. Yet, most of them displayed inner experiences. For instance, ID1 and ID6 mainly communicated non-verbally, possibly to remain in the virtual environment (ID1, ID6), or "to benefit from the effect of immersion", as practitioners said (ID1). Moreover, three children displayed unusual social interaction behaviors. First, ID4 and ID17 were more interested in the other than in technology (e.g., ID4 communicated more than usual). Then, ID14 socially interacted without "disappearing" and drew herself next to a practitioner (see Figure 4). These experiences highlight the social interaction potential of Magic Bubbles, when used with practitioners in a clinical setting. This finding contradicts potential AR risks of isolation [START_REF] Berenguer | Exploring the Impact of Augmented Reality in Children and Adolescents with Autism Spectrum Disorder: A Systematic Review[END_REF], that practitioners can be afraid of when working with autistic children with HS [START_REF] Bauer | Designing an Extended Reality Application to Expand Clinic-Based Sensory Strategies for Autistic Children Requiring Substantial Support: Participation of Practitioners[END_REF].

Getting resourced

All children who wore the HMD were engaged, apart from ID9 due to acceptability issues. Most children had fun, and ID1 got relaxed. These findings confirm the positive results that were observed in previous HMD-based VR [START_REF] Newbutt | Brief Report: A Pilot Study of the Use of a Virtual Reality Headset in Autism Populations[END_REF] and AR [START_REF] Aruanno | HoloLearn: Wearable Mixed Reality for People with Neurodevelopmental Disorders (NDD)[END_REF] studies. In our study, seven children also asked to come back the week after. Two children displayed unexpected behaviours accounting for their engagement: ID14 drew the shared experience and ID16 danced a lot. Moreover, Magic Bubbles enabled ID4 to be resourced enough to communicate. Furthermore, children who were engaged were still in control of their actions. In particular, ID7 could "unload" without being over-aroused, which is unusual for him. Hence, practitioners said that Magic Bubbles was holding [START_REF] Winnicott | Playing and reality[END_REF], i.e., a supportive environment for generating and supporting interactions between the child and the practitioner. Indeed, four children got more secure. Thus, such HMD-based AR approaches are promising to complement clinical interventions for children with HS. At last, since some children felt immersed, future studies should examine if reinforcing the feeling of immersion could also reinforce this holding potential.

Limitations and Future Perspectives

As this field study was conducted in a clinical setting, the environment could not be entirely controlled which limits the generalization of the findings. For instance, children's behaviours may have been influenced by external noise (children shouting in the corridors). Yet, conducting the same experience in a laboratory setting would be impossible, as changing children's environment could disturb them and induce anxiety [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. Hence, new methods must be devised to guarantee the ecological validity of AR autism research, by conducting field studies in clinical settings while precisely controlling specific environmental aspects. To that end, some implicit measures could be used if relevant for the study and the children (e.g., number of interactions). Moreover, physiological data could also be used (e.g., skin conductance) if the clinical team and/or children's relatives deemed that biosensors could be well accepted, even if in our study the psychologists thought that they would not have been accepted by these children. In that case, analyzing physiological data would require to compare it with psychologists' insights, so that to understand the parameters being representative of children's behaviours. Although this approach remains under-used in AR/VR studies [START_REF] Karami | Effectiveness of Virtual/Augmented Reality-Based Therapeutic Interventions on Individuals With Autism Spectrum Disorder: A Comprehensive Meta-Analysis[END_REF], it represents promising research avenues [START_REF] Sharma | Sensing technologies and child-computer interaction: Opportunities, challenges and ethical considerations[END_REF]. The second limitation is due to the fact that the results mainly account for practitioners' perspective. Indeed, children's limited verbal abilities prevented from directly getting their views, despite efforts that were made (e.g., using drawings). Future research should further investigate how to collect the perspective of autistic children with HS, by devising methodologies as previously proposed by Spiel et al. [START_REF] Spiel | Experiences of autistic children with technologies[END_REF].

Four hypothesis emerged that deserve more investigation. First, HMD could allow children with neurodevelopmental disorders and associated ID to make longer eye contacts, as not direct but mediated through AR. Second, AR could prompt the understanding of the others' mental states for autistic children with HS, by perceiving through the monitor the practitioner's AR view. Third, AR could enhance body awareness for children with neurodevelopmental and associated ID. Fourth, adjusting and individualizing the proportion between virtual and real elements, in particular regarding the body presence of others, could prompt reassurance for these children. This fourth hypothesis extends the findings from previous studies, suggesting to first work in VR and then go to AR, so that to gradually fade prompts while encouraging the generalization of the skills learned [START_REF] Bauer | Extended Reality Guidelines for Supporting Autism Interventions Based on Stakeholders' Needs[END_REF]. Moreover, creating use cases as proposed in the last hypothesis could also help to test the other hypothesis.

Fig. 1 .

 1 Fig. 1. Design of Magic Bubbles -Content: 1 Bubble; 2 Bubble column; 3 Panel; 4 Water pond; 5 Music bubble; 6 Image panel; 7 Drawing panel; 8 Recording bubble -Practitioner's UI (E,F): 1 Add/Remove objects; 2 Trigger stimuli; 3 Add/Remove feedback; 4 Show contextual information; 5 Show the UI (these images come from [5]).

Fig. 2 .

 2 Fig. 2. Questionnaire's answers regarding the acceptability, usability, agency, engagement, and social interaction, for the 10 children with neurodevelopmental disorders and intellectual disability who tested Magic Bubbles. Q16, Q23, Q24 were displayed on 1-5 scales rather than 1-7 scales for enhanced readability. Bars represent the median, rectangles represent the interquartile range (IQR) (50% of the sample's values), and circles represent outliers.

Fig. 3 .

 3 Fig. 3. Questionnaires' answers about children's state. Bars represent the median, rectangles represent the interquartile range (IQR) (50% of the sample's values), and circles represent outliers. (A). Evolution of tiredness and anxiousness between the beginning and the end., (B) Being resourced at the end and ready to start another activity.

Fig. 4 .

 4 Fig. 4. Drawing that ID16 made, representing herself next to the practitioner.

Fig. 5 .

 5 Fig. 5. Photographs of critical incidents for four children, (A) ID6 hitting a pond with his foot, (B) ID7 unloading on the music panel while keeping in control, (C) ID8 trying to write his name on the drawing panel, (D) ID5 stepping over the border of the column, (E) ID14 giving the controller to the practitioner after proposing him to test the HMD.

Table 1 .

 1 Profiles of the children who participated to the study. M/F stands for Male/Female. IQ stands for Intellectual Quotient.

	ID M/F Age Condition (ICD-10)	IQ
	1 M	13 Atypical autism (F841)	50< IQ<70
	4 F	11 Other childhood disintegrative disorder (F843)	50< IQ<70
	5 M	13 Other pervasive developmental disorders (F84.8)	50< IQ<70
	6 M	13 Pervasive developmental disorder, unspecified (F849)	50< IQ<70
	7 M	13 Pervasive developmental disorder, unspecified (F849)	50< IQ<70
	8 M	14 Pervasive developmental disorder, unspecified (F849)	50< IQ<70
	9 F	12 Mixed disorder of conduct and emotions, unspecified (F929) 50< IQ<70
	14 F	12 Mixed specific developmental disorders (F83)	50< IQ<70
	16 F	13 Atypical autism (F841)	50< IQ<70
	17 M	11 Autistic disorder (F840)	50< IQ<70

This paper uses autism stakeholders' preferences in terms of terminology, e.g., identity first-language, with terms such as "autistic individual"[START_REF] Bottema-Beutel | Avoiding Ableist Language: Suggestions for Autism Researchers[END_REF]

MaxQDA software: https://www.maxqda.com/

Acknowledgements

The authors would like to thank the children who participated to this research and their families, as well as the clinical team of the day hospital André Boulloche for their support. This work is part of the AudioXR4TSA project, funded by the DIM RFSI Ile de France. This work was also supported by French government funding managed by the National Research Agency under the Investments for the Future program (PIA) under grant ANR-21-ESRE-0030 / CONTINUUM.


Henrietta Szappanos

Sophia Smida-Rezgui

Julianna Cseri

Cecilia Simut

Jean-Marc Sabatier

Michel De Waard

László Kovács

László Csernoch

Michel Ronjat
email: mronjat@cea.fr

Differential effects of maurocalcine on Ca 2+ release events and depolarisation-induced Ca 2+ release in rat skeletal muscle by

Keywords: Ca 2+ sparks, EC coupling, ryanodine receptor

SUMMARY

Maurocalcine (MCa) a 33 amino acid toxin from a scorpion venom has been shown to interact with the isolated skeletal type ryanodine receptor (RyR1) and to strongly modify its calcium channel gating. In this work, we explore the effects of MCa on RyR1 in situ to establish whether the functional interaction of RyR1 with the voltage sensor dihydropyridine receptor (DHPR) would modify the ability of MCa to interact with RyR1. In developing skeletal muscle cells the addition of MCa into the external medium induces a calcium transient resulting from RyR1 activation and strongly inhibits the effect of the RyR1 agonist chloro-mcresol. In contrast, MCa fails to affect the depolarisation-induced Ca 2+ release. In intact adult fibres MCa does not induce any change in the cytosolic Ca 2+ concentration. However, when the surface membrane is permeabilised and calcium release events are readily observable, MCa has a time-dependent dual effect: it first increases event frequency, from 0.060 ± 0.002 to 0.150 ± 0.007 sarc -1 s -1 , and reduces the amplitude of individual events without modifying their spatial distribution. Later on it induces the appearance of long lasting events resembling embers observed in control conditions but having a substantially longer duration. We propose that the functional coupling of DHPR and RyR1 within a Ca 2+ release unit prevents MCa from either reaching its binding site or from being able to modify the gating not only of the RyR1 physical coupled to DHPR but all RyR1 within the Ca 2+ release unit.

INTRODUCTION

In skeletal muscle fibres, the excitation contraction (EC) coupling process requires the functional interaction of two calcium channels, the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) residing in the transverse (t-) tubular membrane and in the membrane of the terminal cisternae of the sarcoplasmic reticulum (SR), respectively. This process is made possible by the specific and correlated arrangement of these two proteins in their respective membranes. Indeed, electron microscopy images of the t-tubule membrane show that DHPR-s organise into clusters of four molecules, called tetrads, located just opposite of RyR1 molecules embedded in the SR membrane [START_REF] Block | Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle[END_REF]). In the SR membrane RyR1 channels also organise in a highly ordered array with every other RyR1 being associated with a DHPR tetrad. Such an organisation supports the leading hypothesis that EC coupling involves the physical i nteraction of DHPR and adjacent RyR1. This hypothesis has been strengthened by studies showing that expression of cardiac isoform of the DHPR α1 subunit in dysgenic mice (which lack the α1 subunit) led to a cardiac type of EC coupling strictly dependent on Ca 2+ entry through DHPR (Tanabe et al. 1990a;[START_REF] Garcia | Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors[END_REF]. Moreover, co-purification and co-immunoprecipitation experiments demonstrated the existence of a complex involving DHPR and RyR1 [START_REF] Marty | Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle[END_REF].

Upon depolarisation of the t-tubular membrane, the DHPR-s undergo a conformational change that activate adjacent RyR1-s (orthograde signal) inducing the release of stored calcium ions from the lumen of the SR [START_REF] Schneider | Voltage dependent charge movement in skeletal muscle: a possible step in excitation-contraction coupling[END_REF][START_REF] Schneider | Membrane charge movement and depolarization-contraction cooupling[END_REF].

Conversely, dyspedic cells (which lack RyR1) have been shown to display a strongly reduced DHPR Ca current that can be recovered by re-expression of RyR1 in these cells, highlighting the control of the DHPR Ca 2+ channel behaviour by RyR1 (retrograde signal) [START_REF] Nakai | Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor[END_REF][START_REF] Chavis | Functional coupling between ryanodine receptors and L-type calcium channels in neurons[END_REF]. The interaction of these two key proteins involves a number of cytoplasmic residues on both moieties. Different regions of the DHPR α1 subunit have been shown to interact in vitro with RyR1. One of the most intensively studied region, in this respect, is the cytoplasmic II-III loop connecting the second and third transmembrane repeats of the α1 subunit (Tanabe et al. 1990b). From the four segments into which the II-III loop has been arbitrarily divided domain C (amino acid residues 724-760) has been implicated in the EC coupling process while a synthetic peptide corresponding to the domain A (peptide A; amino acid residues 671-690) was shown to interact with the isolated RyR1 and to modify its gating [START_REF] Nakai | Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling[END_REF]El-Hayek et al. 1995a;[START_REF] El-Hayek | Identification of the minimum essential region in the II-III loop of the dihydropyridine receptor alpha 1 subunit required for activation of skeletal muscle-type excitation-contraction coupling[END_REF][START_REF] O'reilly | FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor[END_REF]. However, the exact functional role of these different domains in EC coupling remains to be established. In addition to the putative direct interactions between RyR1 and DHPR, several proteins have been shown to be able to regulate in vitro the RyR1 and/or DHPR activity and therefore could play a role in the regulation of EC coupling.

Two scorpion toxins, Imperatoxin A (IpTxA) and Maurocalcine (MCa), have been shown to strongly modify RyR1 Ca channel properties in vitro (El-Hayek et al. 1995b;[START_REF] Fajloun | Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca 2+ release channel/ryanodine receptors[END_REF]. Interestingly these two toxins display some amino acid sequence homologies and structural features with domain A of the DHPR α1 subunit [START_REF] Lee | Molecular basis of the high-affinity activation of type 1 ryanodine receptors by imperatoxin A[END_REF][START_REF] Green | The three-dimensional structural surface of two beta-sheet scorpion toxins mimics that of an alpha-helical dihydropyridine receptor segment[END_REF]. We have previously shown that MCa strongly potentiates [ 3 H]-ryanodine binding on SR vesicles, induces Ca 2+ release from SR vesicles and stabilises the purified RyR1 in an open state characterised by a conductance equivalent to 60% of the full conductance (Chen et al. 2003;[START_REF] Estève | Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of Maurocalcine in skeletal muscle cells[END_REF]. More recently we demonstrated that MCa is able to passively cross biological membrane and penetrate various cell types by a mechanism similar to the one described for Cell-Penetrating Peptides [START_REF] Estève | Transduction of the scorpion toxin maurocalcine into cells -evidence that the toxin crosses the plasma membrane[END_REF]. For all these reasons MCa provides a very interesting tool to study the DHPR-RyR1 interactions in skeletal muscle cells.

In amphibian skeletal muscle cells, massive Ca 2+ release from SR has been proposed to result from the summation of calcium release events, termed sparks, corresponding to the opening of a discrete number of ryanodine receptors [START_REF] Tsugorka | Imaging elementary events of calcium release in skeletal muscle cells[END_REF][START_REF] Klein | Two mechanisms of quantized calcium release in skeletal muscle[END_REF]Gonzalez et al. 2000b). In contrast, intact adult mammalian skeletal muscle fibres do not give rise to spontaneous Ca 2+ release events [START_REF] Shirokova | Local calcium release in mammalian skeletal muscle[END_REF]). However, it has recently been shown that mild treatment of adult mammalian skeletal fibres with saponin provokes the appearance of several types of Ca 2+ release events [START_REF] Kirsch | Spark-and ember-like elementary Ca2+ release events in skinned fibres of adult mammalian skeletal muscle[END_REF]Zhou et al. 2003a;[START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF]); long events with constant amplitude called embers (Gonzalez et al. 2000a) and sparks similar to those observed in frog skeletal muscle cells. Interestingly, sparks can be observed in cultured myotubes and have been shown to occur specifically in regions devoid of t-tubule structures, suggesting that conformational coupling between DHPR and RyR1 prevents the generation of spontaneous opening of RyR1 and thus the appearance of sparks [START_REF] Shirokova | Spatially segregated control of Ca2+ release in developing skeletal muscle on mice[END_REF][START_REF] Zhou | Repression of Ca2+ sparks by voltage sensors or other T tubule structures in mammalian muscle[END_REF].

In this work, we investigated the effects of MCa on Ca 2+ release in myotubes and on spontaneous Ca 2+ release events in adult mammalian muscle fibres permeabilised with saponin. We show that in cultured myotubes, MCa induces Ca 2+ release via the ryanodine receptor but has no effect on the depolarisation evoked Ca 2+ transient. In contrast, MCa does not induce any Ca 2+ release or Ca 2+ release events in intact adult cells. In saponin treated adult cells, MCa had a biphasic effect; it first induced a strong increase in calcium release events frequency and then latter promoted the appearance of long lasting embers. These results strongly support the hypothesis that the interaction between DHPR and RyR1 control the opening of all adjacent RyRs located within the array. Part of this work was presented to the Biophysical Society (Csernoch et al. 2004a).

METHODS

Enzymatic isolation of single fibres

Single skeletal muscle fibres from the extensor digitorum communis muscles of rats were isolated enzymatically as described earlier [START_REF] Szentesi | Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres[END_REF]. Briefly, rats of either sex were anaesthetised and killed by cervical dislocation in accordance with the guidelines of the European Community (86/609/EEC) following a protocol approved by the institutional Animal Care Committee. After removing the muscles, they were treated with collagenase (Sigma, Type I) for 1-1½ hours at 37 o C. Fibres were allowed to rest for at least 20 min after the dissociation.

The selected fibre was transferred into the recording chamber filled with Relaxing solution (in mM: 125 K -glutamate, 10 HEPES, 1 EGTA, 6 MgCl 2 , 5 Na 2 -ATP, 10 Naphosphocreatine, 10 glucose, 0.13 CaCl 2 and 8% dextran). The surface membrane was permeabilised using a Relaxing solution containing 0.002% saponin, 50 µM Fluo-3 and 4% dextran. This solution was then exchanged to a K-glutamate or K 2 SO 4 based Internal solution (in mM: 140 K-glutamate or 95 K 2 SO 4 , 10 HEPES, 1 EGTA, 6 MgCl 2 , 5 Na 2 -ATP, 10 Naphosphocreatine, 10 glucose, 0.26 CaCl 2 , 0.1 Fluo-3 and 8% dextran). For intact fibre measurements, 5 µM Fluo-3 AM was added to the Relaxing solution and the loading of the fibre was continuously monitored. Fluo-3 AM was removed from the external solution when the fluorescence inside the cell reached 70% of that measured with permeabilised fibres.

Measurement of elementary calcium release events

Fibres were imaged with the LSM 510 laser scanning confocal microscope (Zeiss, Oberkochen, Germany) as reported earlier [START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF]. Line scan images of fluorescence (F[x,t]) were taken at 1.54 ms/line and 512 pixels/line (pixel size 0.142 µm) parallel to the fibre axis. Fluo-3 was excited at 488 nm (Argon ion laser; 5% laser intensity), emitted light was collected through a band-pass filter and digitised at 12 bit.

Elementary calcium release events were captured using an automatic computer detection method (e.g. [START_REF] Cheng | Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method[END_REF] as described in our previous report [START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF].

Briefly, the program identified elementary events as regions with fluorescence above a relative threshold, calculated from the noise in the images, and having amplitudes greater than 0.3 ∆F/F 0 units. The eventless portion of the image was used to calculate baseline fluorescence (F 0 [x]). The program also determined the amplitude (A, as ∆F/F 0 ), spatial halfwidth measured at the time of the peak (FWHM), duration and rise time for sparks and sparks with embers, while average amplitude, duration and FWHM for lone embers. FWHM was calculated by fitting a Gaussian function to the spatial distribution obtained by averaging 3 lines at the peak for sparks or by all lines except the first and last 10 ms of the event for lone embers.

Cell Culture

Primary cultures of rat skeletal muscle satellite cells were obtained as described previously [START_REF] Marty | Cloning and characterization of a new isoform of skeletal muscle triadin[END_REF]. Cells were seeded on laminin-coated plates, in a proliferation medium composed of Ham's F-10 with glutamax-I (Invitrogen) supplemented with 20% fetal calf serum ( Invitrogen), 2.5 ng/ml basic fibroblast growth factor ( Invitrogen), and 1% penicillin-streptomycin (Invitrogen) at 37 °C and 5% CO 2 . After 2-3 days, differentiation of satellite cells into myotubes was induced with Dulbecco's modified Eagle's medium with glutamax-I (Invitrogen) supplemented with 10% horse serum (Invitrogen).

Ca 2+ Imaging from Culture of Myotubes

Changes in cytosolic calcium levels w ere monitored using the calcium-dependent fluorescent dye Fluo-4. Myotubes were incubated for 1h at room temperature with fluo-4-AM 10 µM, in Krebs buffer (NaCl 136mM, KCl 5mM, CaCl 2 2 mM, MgCl 2 1mM, Hepes 10mM pH 7.4). Uptake of the dye was facilitated by the addition of 0.02% pluronic F-127 acid (SIGMA). After loading, myotubes were washed for 1h at 37°C to allow the de-esterification of the dye. Calcium imaging was performed in Krebs buffer. To obtain a calcium-free Krebs solution, CaCl 2 was left out of, while 1 mM EGTA and 50 µM lanthanum were added. In the depolarization solution, NaCl was replaced by KCl (140mM final concentration).

Fluorescence changes were measured by confocal laser-scanning microscopy using a LEICA TCS-SP2 operating system in the xyt mode. Fluo4 was excited at a wavelength of 488 nm, and the fluorescence was collected from 500 to 570 nm. Images were collected every 1.6 s for 2-4 min and then analyzed frame by frame with the data analysis software provided by Leica.

Fluorescence curves are expressed as a function of time as ? F/F, where F represents the baseline fluorescence and ? F represents the change in fluorescence.

Chemicals

Fluo-3, Fluo-4, Fluo-3 AM and Fluo-4 AM were purchased from Molecular Probes Inc.

(Eugene, OR, USA), while all other chemicals, unless otherwise specified, were from Sigma (St. Louis, MO, USA).

RESULTS

Experiments on differentiating muscle fibres..

MCa is a 33 amino acid basic peptide that was first isolated from the venom of the scorpion Scorpio maurus palmatus. MCa was thus chemically synthesised and shown to retain both the structural and functional properties of the native MCa [START_REF] Fajloun | Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca 2+ release channel/ryanodine receptors[END_REF].

We have previously shown that the application of MCa to cultured myotubes induces both an increase of the cytosolic Ca 2+ concentration, due to the Ca 2+ release from SR, and a strong inhibition of the Ca 2+ release induced by 4-chloro-m-cresol [START_REF] Estève | Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of Maurocalcine in skeletal muscle cells[END_REF].

In order to investigate the effect of MCa on RyR1-DHPR interaction in a cellular context we studied the effect of MCa on Ca 2+ release induced by activation of the DHPR, i.e. by depolarisation of the plasma membrane. I ntact myotubes were loaded with Fluo-4 and depolarisation of the plasma membrane was induced by addition of 140 mM KCl in the extracellular medium, before or after addition of MCa to the extracellular medium. In order to further investigate the effect of MCa at the level of EC complexes, elementary Ca 2+ release events were studied on adult mammalian skeletal muscle fibres.

Experiments on adult muscle fibres.

MCa increases event frequency on permeabilised fibres

We first tested whether MCa was able to induce calcium release or initiate spontaneous calcium release events in intact adult striated muscle fibres. To this end, cells were loaded with the AM form of the calcium indicator as for the measurements on myotubes. Fibres held in Relaxing solution had stable background fluorescence and did not show spontaneous calcium release events in accordance with previous observations [START_REF] Shirokova | Local calcium release in mammalian skeletal muscle[END_REF]Csernoch et al. 2004a). The addition of 50 nM MCa to the bathing medium neither increased the background fluorescence nor promoted the appearance of calcium release events (Fig. 2A andB). To exclude the possibility of an insufficient penetration of the toxin, small notches were cut on the f ibres. Measurements, recording both x -y and line-scan images, were conducted for approximately 10 min in each condition before and after the addition of the toxin near the notch. Figure 2C andD show that, under these conditions, no spontaneous calcium release is observed neither in the absence nor in the presence of 50 nM MCa. These results demonstrate that a mechanical injury on its own does not lead to the appearance of calcium release events and that On the other hand, it suggests the lack of effect of MCa on adult fibres is not due to an insufficient penetration of the toxin.

As a next step, fibres were treated with saponin in order to establish conditions under which spontaneous elementary calcium release events (ECRE) occur [START_REF] Kirsch | Spark-and ember-like elementary Ca2+ release events in skinned fibres of adult mammalian skeletal muscle[END_REF]Zhou et al. 2003a;[START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF]). In contrast to what is observed in intact (or notched) fibres, the addition of MCa into the bathing medium of saponin permeabilised fibres had a dramatic effect. As demonstrated in Fig. 3A andB, shortly after the addition of 50 nM MCa (2 to 10 min) the frequency at which ECRE occurred increased considerably. This effect of the toxin was independent of the major anion of the solution since MCa increased the frequency of ECRE both in the glutamate and in the sulphate based saline. Fig. 3Aa and Ba also show that events in glutamate, under control conditions had a much lower frequency than in sulphate. Although the addition of MCa in glutamate still resulted in an increase in event frequency, the actual frequency remained lower than in MCa treated fibres in sulphate.

The effect of MCa on event frequency was concentration-dependent as presented in Fig. 3D. Both in the glutamate and in the sulphate-based Internal solution, MCa at a concentration of 20 nM already produced a significant (p<0.05) increase in the ECRE frequency. This was further augmented by the addition of 50 nM MCa. It should be noted that when using sulphate, the solution exchange by itself may induce a change in intracellular [Ca 2+ ] ( Zhou et al. 2003a). Therefore, measurements in sulphate at different MCa concentrations were conducted on different fibres. When a glutamate based solution is used this is not the case and successive additions of MCa gave statistically identical results. As a control of the specificity of MCa effect, the addition of the [Ala 24 ]MCa analogue of MCa was found to have no effects on the occurrence of ECRE at a concentration up to 500 nM (Fig. 3D triangles).

MCa alters the morphology of ECRE

Fig. 4 goes on to plot representative events from fibres bathed in the control sulphate based Internal solution without and with 50 nM MCa. Both sparks and embers [START_REF] Kirsch | Spark-and ember-like elementary Ca2+ release events in skinned fibres of adult mammalian skeletal muscle[END_REF][START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF] were readily observable in the absence as well as in the presence of MCa. Their characteristic parameters were, however, altered by the toxin. As demonstrated in Fig. 4C andE, the distribution of event amplitudes, measured 2 to 10 min after the addition of the toxin, was shifted to the left, i.e., ECRE were smaller in the presence of MCa. The average amplitude of sparks was reduced from 0.782 ± 0.003 (mean ± SE, n = 1327) to 0.460 ± 0.002 (mean ± SE, n = 1821) and the average amplitude of embers from 0.281 ± 0.005 (n = 446) to 0.111 ± 0.002 (n =767). Unlike the event amplitude, the spatial spread (Fig. 4D andF) was unaltered by the toxin (1.73 ± 0.02 µm and 1.76 ± 0.02 µm for sparks and 1.81 ± 0.03 µm and 1.84 ± 0.02 µm for embers in the absence and presence of MCa respectively).

As stated before, the above described effects of MCa were derived from images measured right after the addition of the toxin. Approximately twenty minutes after the addition of MCa (values for individual fibres varying between 8 and 25 min), the sparks and the short embers were replaced by long-lasting events that resembled embers in control, but had much longer duration. At the same time the frequency of events decreased. These events were readily observable in both the sulphate (Fig. 5A) and the glutamate (Fig. 5B) based Internal solution and their duration often exceeded 500 ms. Fig. 5C represents the distribution of the duration of these long lasting events (n = 223), together with the corresponding portion of the distribution for embers under control conditions, and shows that all events recorded twenty minutes after addition of MCa were longer than 200 ms. In the presence of MCa, a total of 16 events for which both the start and the end were missing were recognised, by repeating the scan at the same position, to last longer than 1.58 s (i.e. the duration of recording an image) (Fig. 5C, grey bar). Moreover, 17% of the events (longer than 200 ms) had either their starting or ending point missing and, therefore, could not be measured precisely. These events are not represented in Fig. 5C.

DISCUSSION

In this work, we show that MCa induces an increase in the frequency of Ca 2+ release events and the appearance of long lasting embers in saponin treated adult striated muscle fibres while it does not produce any Ca 2+ mobilisation in intact adult fibres. We also show that in developing skeletal muscle cells, i.e. cultured myotubes, MCa does not interfere with DHPR mediated Ca 2+ release via RyR1 although it induces a transient Ca 2+ release from SR.

Effect of MCa on ECRE parameters

In previous works we have characterised the effect of MCa on RyR1. On purified RyR1 reconstituted into planar lipid bilayers, the toxin induces both an increase of the open probability and the appearance of long lasting open events characterised by a reduced conductance [START_REF] Chen | Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism[END_REF][START_REF] Estève | Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of Maurocalcine in skeletal muscle cells[END_REF]. As a consequence of these effects, MCa induces Ca 2+ release from SR vesicles as well as a dramatic increase of the [ 3 H]-ryanodine binding to RyR1. All these effects were completely abolished by the point mutation of the Arg residue in position 24 of MCa [START_REF] Estève | Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of Maurocalcine in skeletal muscle cells[END_REF] supporting the hypothesis that the basic amino acid-rich region is important for the functional effect of MCa on RyR1. In the present work, we show two major effects of MCa on ECRE in saponin treated adult mammalian striated muscle fibres: i) the increase of the frequency of ECRE together with a decrease of the amplitude of the individual events and ii) the induction of long lasting embers with durations usually exceeding 200 ms, occasionally longer than 1.5 s. These effects of MCa on ECRE correlate with those previously observed on SR vesicles or purified RyR1. Indeed, the increase of ECRE frequency would be the consequence of the increase in the open probability of RyR1 channels while long lasting embers would result from the stabilisation of the RyR1 channel in a sub-conductance state. In addition, the [Ala 24 ]MCa analogue of MCa did not show any effect on ECRE strengthening the correlation of the MCa effect on isolated RyR1 and ECRE. These results are in agreement with previous observations illustrating that other activators inducing long lasting sub-conductance state of RyR1 such as imperatoxin A [START_REF] Tripathy | Imperatoxin A induces subconductance states in Ca2+ release channel (ryanodine receptor) of cardiac and skeletal muscle[END_REF], ryanodine [START_REF] Rousseau | Ryanodine modifies conductance and gating behavior of single Ca2+ release channel[END_REF]) or bastadin 10 (Chen et al. 1999) also provoke long lasting ECRE in frog skeletal muscle fibres [START_REF] Shtifman | Effect of imperatoxin A on local sarcoplasmic reticulum Ca2+ release in frog skeletal muscle[END_REF]Gonzalez et al. 2000b). In contrast, modifications of RyR1 that affect only its open probability and not its mean open time or conductance, such as the modification of interdomain interaction or the binding of homer protein, have been shown to induce an increase of spark frequency without the appearance of long lasting events in frog skeletal muscle cells [START_REF] Shtifman | Interdomain interactions within ryanodine receptor regulate Ca2+ spark frequency in skeletal muscle[END_REF][START_REF] Ward | Homer protein increase activation of Ca2+ sparks in permeabilized skeletal muscle[END_REF].

Interestingly the long lasting embers observed in the presence of MCa resemble to those elicited by low level depolarisation in mammalian striated fibres (Csernoch et al. 2004b).

Together with the fact that MCa and domain A of the α1 subunit share a common binding domain on RyR1 [START_REF] Altafaj | Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor CAv1.1 subunit share common binding sites on the skeletal ryanodine receptor[END_REF] this result suggests that the RyR1 conformational changes induced by MCa could mimic physiological events taking place during depolarisation.

How does MCa interfere with EC coupling?

A number of studies have shown that developing mammalian skeletal muscle cells display elementary calcium release events resembling calcium sparks measured in adult frog skeletal muscle (e.g. Conklin et al.1999;[START_REF] Chun | Ca2+ sparks are initiated by Ca2+ entry in embrionic skeletal muscle and decrease in frequency postnataly[END_REF]. In addition, Shirokova and coworkers (1999) described another form of calcium release devoid of discrete Ca 2+ release events, following membrane depolarisation, which coexists with the calcium sparks in these cells.

The latter was assumed to be the only form of voltage-evoked calcium release in adult mammalian fibres [START_REF] Shirokova | Local calcium release in mammalian skeletal muscle[END_REF] until we recently demonstrated the presence, in these cells, of discrete events upon membrane depolarisation (Csernoch et al. 2004b).

However, these voltage-evoked events in adult mammalian striated muscle took the shape of embers rather than sparks. Therefore, the interpretation of Shirokova and coworkers (1999), albeit in a slightly altered form, still stands, i.e. in developing mammalian skeletal muscle cells, spontaneous calcium release events are sparks while voltage-evoked release is either eventless or, probably, resembles that described for adult fibres.

In addition to this functional difference in how events are elicited, a spatial segregation of these two types of Ca 2+ release seems also to be present in myotubes. Recent work from Zhou et al. (2004b) showed that calcium sparks in developing myotubes occur only in areas devoid of t-tubules. In this framework, one could extend the above described Ca 2+ release segregation to suggest that RyR-s not involved in junctional complexes give rise to sparks while those associated with DHPR-s don't.

Here (Fig. 1), and in previous works [START_REF] Chen | Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism[END_REF][START_REF] Estève | Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of Maurocalcine in skeletal muscle cells[END_REF], we demonstrate that MCa is capable of inducing calcium release from the SR of developing myotubes and that this release is specific for RyR. However, MCa, as demonstrated in Fig. 1B and C, failed to influence the depolarisation-evoked release of calcium in these cells. These observations strongly support the segregated control of calcium release in these cells, namely, extra-juntional RyR-s are susceptible to MCa while those in junctional complexes are not. We have previously shown that under similar conditions, treatment of myotubes with MCa induces a strong inhibition of 4-chloro-m-cresol induced Ca 2+ release in myotubes (Estève et al. 2003). Alltogether these results s uggest that extra-junctional RyR-s and junctional complexes are located in different Ca 2+ pools. Different results have shown that RyR3 is expressed in differentiating myotubes [START_REF] Shirokova | Spatially segregated control of Ca2+ release in developing skeletal muscle on mice[END_REF]. Therefore the RyR3 containing Ca 2+ pool could be responsible for the MCa-induced Ca 2+ transient while the RyR1 containing pool would be under the control of DHPR and therefore not affected by MCa. Interestingly, [START_REF] Nabhani | Imperatoxin A enhances Ca 2+ release in developing skeletal muscle containing ryanodine receptor type 3[END_REF] have previously reported that imperatoxin A fails to modify Ca 2+ release in myotubes not expressing RyR3. This implies that in adult skeletal muscle cells, where RyR-s are found exclusively in junctional complexes, MCa would not exert any effect. Indeed, this is exactly what we have observed in intact adult cells. The absence of an effect of MCa on notched fibres together with our recent results showing that MCa passively crosses the plasma membrane of various cell types [START_REF] Estève | Transduction of the scorpion toxin maurocalcine into cells -evidence that the toxin crosses the plasma membrane[END_REF] renders very unlikely that the absence of effect of MCa on intact adult fibres could be due to the lack of penetration of MCa.

One would also assume that a destabilisation of the junctional DHPR-RyR1 would allow MCa to activate RyR-s. Previous works have demonstrated that a mild treatment with saponin results in such destabilisation as witnessed by the appearance of spontaneous Ca 2+ release events (Kisch et al. 2001;Zhou et al. 2003a;[START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF]. In line with the above prediction we show here that, after saponin treatment, MCa becomes capable of modifying RyR Ca 2+ channel gating in adult mammalian skeletal cells, as visualised by the significant increase of sparks frequency and the appearance of long lasting embers.

Morphological data show that only every other RyR1 is directly coupled to a tetrad of DHPR, suggesting that "uncoupled" RyR1-s could behave independently of the "coupled" ones and, therefore, be activated by RyR1 agonists such as MCa. Functional approach presented here as well as previously [START_REF] Szentesi | Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle[END_REF]) clearly failed to demonstrate such an effect of MCa and thymol, respectively. A possible explanation for this apparent contradiction could reside in a strong interaction between "coupled" and "uncoupled" RyR-s, namely, "coupled" RyR-s held closed by the DHPR would prevent adjacent RyR-s from opening in the presence of or from interacting with MCa. The coupled gating of RyR-s demonstrated by [START_REF] Marx | Coupled gating between individual muscle Ca2+ release channels (Ryanodine Receptors)[END_REF] could provide the functional background for such interaction. Since MCa and domain A of the II-III loop of the α1 subunit of the DHPR share a common binding site on RyR1 and MCa can displace, in a competitive way, the binding of domain A on RyR1 [START_REF] Altafaj | Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor CAv1.1 subunit share common binding sites on the skeletal ryanodine receptor[END_REF], it is tempting to postulate that in coupled RyR-s, the MCa binding site is occupied by domain A of the DHPR. The different levels of coupling between DHPR-s and RyR-s are schematised in Fig. 6. For embers the average amplitude of the ember was considered. (D and F) Distribution of full width at half maximum for sparks (D) and embers (F). Only events with amplitude > 0.3 ∆F/F (for sparks) or average amplitude greater than 0.05 ∆F/F (for embers) were used to construct the histograms. White traces present the time course of fluorescence at the spatial centre of the events (three neighbouring pixels averaged). (C) Distribution of the duration of long-lasting events (red bars). Events with duration longer than 1.5 s. (longer than the duration of an image) are displayed separately as a filled bar. All long-lasting events (events with duration longer than 200 ms), in glutamate and in sulphate, were included in the distribution. However, events where either the beginning or the end was missed are not graphed. For comparison the corresponding portion of the distribution for embers measured under control conditions (white bars) is also shown.

  Figure 1B (middle trace) shows that the addition of 100 nM MCa into the extracellular medium induces a rapid and transient cytosolic Ca 2+ concentration increase. No significant change of the basal cytolpasmic Ca 2+ concentration was observed following the transient Ca 2+ release induced by MCa. Ten minutes after the addition of MCa, cells were depolarised by the addition to the external medium of KCl (140 mM final concentration). In contrast with what we previously observed on CMC-induced Ca 2+ release, MCa (100 nM) did not affect Ca 2+ release triggered by KCl-induced depolarisation of the plasma membrane of myotubes. Figure 1A shows a series of confocal fluorescence images of Fluo-4 loaded myotubes before and after addition of KCl into the extracellular medium. The time course of Fluo-4 fluorescence shows a rapid increase after KCl addition followed by a decrease back to the basal level, both in the absence and in the presence of MCa (100 nM) (Fig. 1B left and right trace respectively). Quantitative analysis of Fluo-4 fluorescence change induced by KCl shows no significant effect of MCa (mean ∆F/F = 172 ± 14 (n = 77) versus 160 ± 40 (n = 21) in the absence and presence of MCa, respectively, Fig.1C). Similar results were obtained in the absence or presence of Ca 2+ in the external medium. These results highlight the existence of two populations of RyRs differentially accessible to MCa and suggest that MCa preferentially affects RyRs that are not engaged in the voltage-gated EC complex.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS Figure 1. Maurocalcine does not affect the depolarisation-induced Ca 2+ release in

Figure 2 .

 2 Figure 2. Maurocalcine does not induce ECRE in intact or noched mammalian adult

Figure 3 .

 3 Figure 3. MCa increases ECRE frequency in fibres treated with saponin.

Figure 4 .

 4 Figure 4. Depicted events from control and MCa treated fibres

Figure 5 .

 5 Figure 5. Long-lasting events in the presence of MCa

Figure 6 .

 6 Figure 6. Scematic representation of the different coupling states of DHPR-s and RyR-s

Figure 6 (

 6 Figure 1 (Szappanos et al.)

Acknowledgments

The authors are grateful for the technical assistance of Ms. R. Öri. The work was supported by grants from the Hungarian Scientific Research Fund OTKA (TS040773 and T034894), FKFP 0193/2001, ETT 250/2003, INSERM, CEA, UJF and from the European Commission (RTN2-2001-00337).