Challenges and opportunities in mobile e-coaching.

Background: Mobile e-health technologies have proven to provide tailored assessment, intervention, and coaching capabilities for various usage scenarios. Thanks to their spread and adoption, smartphones are one of the most important carriers for such applications. Problem: However, the process of design, realization, evaluation, and implementation of these e-health solutions is wicked and challenging, requiring multiple stakeholders and expertise. Method: Here, we present a tailorable intervention and interaction e-health solution that allows rapid prototyping, development, and evaluation of e-health interventions at scale. This platform allows researchers and clinicians to develop ecological momentary assessment, just-in-time adaptive interventions, ecological momentary intervention, cohort studies, and e-coaching and personalized interventions quickly, with no-code, and in a scalable way. Result: The Twente Intervention and Interaction Instrument (TIIM) has been used by over 320 researchers in the last decade. We present the ecosystem and synthesize the main scientific output from clinical and research studies in different fields. Discussion: The importance of mobile e-coaching for prediction, management, and prevention of adverse health outcomes is increasing. A profound e-health development strategyand strategic, technical, and operational investments are needed to prototype, develop, implement, and evaluate e-health solutions. TIIM ecosystem has proven to support these processes. This paper ends with the main research opportunities in mobile coaching, including intervention mechanisms, fine-grained monitoring, and inclusion of objective biomarker data.

Addendum: The Dutch COVID-19 Notification App: Lessons Learned From a Mixed Methods Evaluation Among End Users and Contact-Tracing Employees.

[This corrects the article DOI: 10.2196/38904.].

Appreciating the Good Things in Life During the Covid-19 Pandemic: A Randomized Controlled Trial and Evaluation of a Gratitude App.

The Covid-19 pandemic has had many negative consequences on the general public mental health. The aim of this study was to test the effectiveness of and satisfaction with an app with gratitude exercises to improve the mental health of people with reduced mental well-being due to the Covid-19 pandemic, as well as potential mechanisms of well-being change and dose-response relationships. A two-armed randomized controlled trial design was used, with two groups receiving the 6-week gratitude intervention app either immediately (intervention group, n = 424) or after 6 weeks (waiting list control group, n = 425). Assessments took place online at baseline (T0), six weeks later (T1) and at 12 weeks (T2), measuring outcomes (i.e., mental well-being, anxiety, depression, stress), and potential explanatory variables (i.e., gratitude, positive reframing, rumination). Linear mixed models analyses showed that when controlled for baseline measures, the intervention group scored better on all outcome measures compared to the control group at T1 (d = .24-.49). These effects were maintained at T2. The control group scored equally well on all outcome measures at T2 after following the intervention. Effects of the intervention on well-being were partially explained by gratitude, positive reframing, and rumination, and finishing a greater number of modules was weakly related to better outcomes. The intervention was generally appealing, with some room for improvement. The results suggest that a mobile gratitude intervention app is a satisfactory and effective way to improve the mental health of the general population during the difficult times of a pandemic.

The Dutch COVID-19 Notification App: Lessons Learned From a Mixed Methods Evaluation Among End Users and Contact-Tracing Employees.

BACKGROUND: The Dutch CoronaMelder (CM) app is the official Dutch contact-tracing app (CTA). It has been used to contain the spread of the SARS-CoV-2 in the Netherlands. It allows its users and those of connected apps to anonymously exchange warnings about potentially high-risk contacts with individuals infected with the SARS-CoV-2. OBJECTIVE: The goal of this mixed methods study is to understand the use of CTA in the pandemic and its integration into the Municipal Health Services (MHS) efforts of containment through contact tracing. Moreover, the study aims to investigate both the motivations and user experience-related factors concerning adherence to quarantine and isolation measures. METHODS: A topic analysis of 56 emails and a web-based survey of 1937 adults from the Netherlands, combined with a series of 48 in-depth interviews with end users of the app and 14 employees of the Dutch MHS involved in contact tracing, were conducted. Mirroring sessions were held (n=2) with representatives from the development (n=2) and communication teams (n=2) responsible for the creation and implementation of the CM app. RESULTS: Topic analysis and interviews identified procedural and technical issues in the use of the CTA. Procedural issues included the lack of training of MHS employees in the use of CTAs. Technical issues identified for the end users included the inability to send notifications without phone contact with the MHS, unwarranted notifications, and nightly notifications. Together, these issues undermined confidence in and satisfaction with the app's use. The interviews offered a deeper understanding of the various factors at play and their effects on users; for example, the mixed experiences of the app's users, the end user's own fears, and uncertainties concerning the SARS-CoV-2; problematic infrastructure at the time of the app's implementation on the side of the health services; the effects of the society-wide efforts in containment of the SARS-CoV-2 on the CM app's perception, resulting in further doubts concerning the app's effectiveness among MHS workers and citizens; and problems with adherence to behavioral measures propagated by the app because of the lack of confidence in the app and uncertainty concerning the execution of the behavioral measures. All findings were evaluated with the app's creators and have since contributed to improvements. CONCLUSIONS: Although most participants perceived the app positively, procedural and technical issues identified in this study limited satisfaction and confidence in the CM app and affected its adoption and long-term use. Moreover, these same issues negatively affected the CM app's effectiveness in improving compliance with behavioral measures aimed at reducing the spread of the SARS-CoV-2. This study offers lessons learned for future eHealth interventions in pandemics. Lessons that can aid in more effective design, implementation, and communication for more effective and readily adoptable eHealth applications.

Integrating Top-down and Bottom-up Requirements in eHealth Development: The Case of a Mobile Self-compassion Intervention for People With Newly Diagnosed Cancer.

BACKGROUND: Psychosocial eHealth interventions for people with cancer are promising in reducing distress; however, their results in terms of effects and adherence rates are quite mixed. Developing interventions with a solid evidence base while still ensuring adaptation to user wishes and needs is recommended to overcome this. As most models of eHealth development are based primarily on examining user experiences (so-called bottom-up requirements), it is not clear how theory and evidence (so-called top-down requirements) may best be integrated into the development process. OBJECTIVE: This study aims to investigate the integration of top-down and bottom-up requirements in the co-design of eHealth applications by building on the development of a mobile self-compassion intervention for people with newly diagnosed cancer. METHODS: Four co-design tasks were formulated at the start of the project and adjusted and evaluated throughout: explore bottom-up experiences, reassess top-down content, incorporate bottom-up and top-down input into concrete features and design, and synergize bottom-up and top-down input into the intervention context. These tasks were executed iteratively during a series of co-design sessions over the course of 2 years, in which 15 people with cancer and 7 nurses (recruited from 2 hospitals) participated. On the basis of the sessions, a list of requirements, a final intervention design, and an evaluation of the co-design process and tasks were yielded. RESULTS: The final list of requirements included intervention content (eg, major topics of compassionate mind training such as psychoeducation about 3 emotion systems and main issues that people with cancer encounter after diagnosis such as regulating information consumption), navigation, visual design, implementation strategies, and persuasive elements. The final intervention, Compas-Y, is a mobile self-compassion training comprising 6 training modules and several supportive functionalities such as a mood tracker and persuasive elements such as push notifications. The 4 co-design tasks helped overcome challenges in the development process such as dealing with conflicting top-down and bottom-up requirements and enabled the integration of all main requirements into the design. CONCLUSIONS: This study addressed the necessary integration of top-down and bottom-up requirements into eHealth development by examining a preliminary model of 4 co-design tasks. Broader considerations regarding the design of a mobile intervention based on traditional intervention formats and merging the scientific disciplines of psychology and design research are discussed.

The Dutch COVID-19 Contact Tracing App (the CoronaMelder): Usability Study.

BACKGROUND: Adoption and evaluation of contact tracing tools based on information and communications technology may expand the reach and efficacy of traditional contact tracing methods in fighting COVID-19. The Dutch Ministry of Health, Welfare and Sports initiated and developed CoronaMelder, a COVID-19 contact tracing app. This app is based on a Google/Apple Exposure Notification approach and aims to combat the spread of the coronavirus among individuals by notifying those who are at increased risk of infection due to proximity to someone who later tests positive for COVID-19. The app should support traditional contact tracing by faster tracing and greater reach compared to regular contact tracing procedures. OBJECTIVE: The main goal of this study is to investigate whether the CoronaMelder is able to support traditional contact tracing employed by public health authorities. To achieve this, usability tests were conducted to answer the following question: is the CoronaMelder user-friendly, understandable, reliable and credible, and inclusive? METHODS: Participants (N=44) of different backgrounds were recruited: youth with varying educational levels, youth with an intellectual disability, migrants, adults (aged 40-64 years), and older adults (aged >65 years) via convenience sampling in the region of Twente in the Netherlands. The app was evaluated with scenario-based, think-aloud usability tests and additional interviews. Findings were recorded via voice recordings, observation notes, and the Dutch User Experience Questionnaire, and some participants wore eye trackers to measure gaze behavior. RESULTS: Our results showed that the app is easy to use, although problems occurred with understandability and accessibility. Older adults and youth with a lower education level did not understand why or under what circumstances they would receive notifications, why they must share their key (ie, their assigned identifier), and what happens after sharing. In particular, youth in the lower-education category did not trust or understand Bluetooth signals, or comprehend timing and follow-up activities after a risk exposure notification. Older adults had difficulties multitasking (speaking with a public health worker and simultaneously sharing the key in the app). Public health authorities appeared to be unprepared to receive support from the app during traditional contact tracing because their telephone conversation protocol lacks guidance, explanation, and empathy. CONCLUSIONS: The study indicated that the CoronaMelder app is easy to use, but participants experienced misunderstandings about its functioning. The perceived lack of clarity led to misconceptions about the app, mostly regarding its usefulness and privacy-preserving mechanisms. Tailored and targeted communication through, for example, public campaigns or social media, is necessary to provide correct information about the app to residents in the Netherlands. Additionally, the app should be presented as part of the national coronavirus measures instead of as a stand-alone app offered to the public. Public health workers should be trained to effectively and empathetically instruct users on how to use the CoronaMelder app.

Degenerated Cones in Cultured Human Retinas Can Successfully Be Optogenetically Reactivated.

Biblical references aside, restoring vision to the blind has proven to be a major technical challenge. In recent years, considerable advances have been made towards this end, especially when retinal degeneration underlies the vision loss such as occurs with retinitis pigmentosa. Under these conditions, optogenetic therapies are a particularly promising line of inquiry where remaining retinal cells are made into "artificial photoreceptors". However, this strategy is not without its challenges and a model system using human retinal explants would aid its continued development and refinement. Here, we cultured post-mortem human retinas and show that explants remain viable for around 7 days. Within this period, the cones lose their outer segments and thus their light sensitivity but remain electrophysiologically intact, displaying all the major ionic conductances one would expect for a vertebrate cone. We optogenetically restored light responses to these quiescent cones using a lentivirus vector constructed to express enhanced halorhodopsin under the control of the human arrestin promotor. In these 'reactivated' retinas, we show a light-induced horizontal cell to cone feedback signal in cones, indicating that transduced cones were able to transmit their light response across the synapse to horizontal cells, which generated a large enough response to send a signal back to the cones. Furthermore, we show ganglion cell light responses, suggesting the cultured explant's condition is still good enough to support transmission of the transduced cone signal over the intermediate retinal layers to the final retinal output level. Together, these results show that cultured human retinas are an appropriate model system to test optogenetic vision restoration approaches and that cones which have lost their outer segment, a condition occurring during the early stages of retinitis pigmentosa, are appropriate targets for optogenetic vision restoration therapies.

Perceived Mental Workload Classification Using Intermediate Fusion Multimodal Deep Learning.

A lot of research has been done on the detection of mental workload (MWL) using various bio-signals. Recently, deep learning has allowed for novel methods and results. A plethora of measurement modalities have proven to be valuable in this task, yet studies currently often only use a single modality to classify MWL. The goal of this research was to classify perceived mental workload (PMWL) using a deep neural network (DNN) that flexibly makes use of multiple modalities, in order to allow for feature sharing between modalities. To achieve this goal, an experiment was conducted in which MWL was simulated with the help of verbal logic puzzles. The puzzles came in five levels of difficulty and were presented in a random order. Participants had 1 h to solve as many puzzles as they could. Between puzzles, they gave a difficulty rating between 1 and 7, seven being the highest difficulty. Galvanic skin response, photoplethysmograms, functional near-infrared spectrograms and eye movements were collected simultaneously using LabStreamingLayer (LSL). Marker information from the puzzles was also streamed on LSL. We designed and evaluated a novel intermediate fusion multimodal DNN for the classification of PMWL using the aforementioned four modalities. Two main criteria that guided the design and implementation of our DNN are modularity and generalisability. We were able to classify PMWL within-level accurate (0.985 levels) on a seven-level workload scale using the aforementioned modalities. The model architecture allows for easy addition and removal of modalities without major structural implications because of the modular nature of the design. Furthermore, we showed that our neural network performed better when using multiple modalities, as opposed to a single modality. The dataset and code used in this paper are openly available.

Saltatory Conduction along Myelinated Axons Involves a Periaxonal Nanocircuit.

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.

The use of technology in the context of frailty screening and management interventions: a study of stakeholders' perspectives.

BACKGROUND: Health and social care interventions show promise as a way of managing the progression of frailty in older adults. Information technology could improve the availability of interventions and services for older adults. The views of stakeholders on the acceptability of technological solutions for frailty screening and management have not been explored. METHODS: Focus groups were used to gather data from healthy and frail/pre-frail older adults, health and social care providers, and caregivers in three European countries - Italy, Poland and UK. Data were analysed using framework analysis in terms of facilitators or determinants of older adults' adoption of technology. RESULTS: Our findings clustered around the perceived value; usability, affordability and accessibility; and emotional benefits of frailty screening and management technology to stakeholders. We also noted issues relating to social support, previous experience of technology and confidence of stakeholders. CONCLUSIONS: Professionals and caregivers understand the benefits of technology to facilitate frailty care pathways but these views are tempered by concerns around social isolation. Frail older adults raised legitimate concerns about the accessibility and usability of technology, specifically around the potential for their personal information to be compromised. Solutions must be developed within a framework that addresses social contexts and avoids stigma around frailty and ageing.

Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype.

Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.

CRB2 in immature photoreceptors determines the superior-inferior symmetry of the developing retina to maintain retinal structure and function.

The mammalian apical-basal determinant Crumbs homolog-1 (CRB1) plays a crucial role in retinal structure and function by the maintenance of adherens junctions between photoreceptors and Müller glial cells. Patients with mutations in the CRB1 gene develop retinal dystrophies, including early-onset retinitis pigmentosa and Leber congenital amaurosis. Previously, we showed that Crb1 knockout mice developed a slow-progressing retinal phenotype at foci in the inferior retina, although specific ablation of Crb2 in immature photoreceptors leads to an early-onset phenotype throughout the retina. Here, we conditionally disrupted one or both alleles of Crb2 in immature photoreceptors, on a genetic background lacking Crb1, and studied the retinal dystrophies thereof. Our data showed that disruption of one allele of Crb2 in immature photoreceptors caused a substantial aggravation of the Crb1 phenotype in the entire inferior retina. The photoreceptor layer showed early-onset progressive thinning limited to the inferior retina, although the superior retina maintained intact. Surprisingly, disruption of both alleles of Crb2 in immature photoreceptors further aggravated the phenotype. Throughout the retina, photoreceptor synapses were disrupted and photoreceptor nuclei intermingled with nuclei of the inner nuclear layer. In the superior retina, the ganglion cell layer appeared thicker because of ectopic nuclei of photoreceptors. In conclusion, the data suggest that CRB2 is required to maintain retinal progenitor and photoreceptor cell adhesion and prevent photoreceptor ingression into the immature inner retina. We hypothesize, from these animal models, that decreased levels of CRB2 in immature photoreceptors adjust retinitis pigmentosa because of the loss of CRB1 into Leber congenital amaurosis phenotype.

Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

Myelinating satellite oligodendrocytes are integrated in a glial syncytium constraining neuronal high-frequency activity.

Satellite oligodendrocytes (s-OLs) are closely apposed to the soma of neocortical layer 5 pyramidal neurons but their properties and functional roles remain unresolved. Here we show that s-OLs form compact myelin and action potentials of the host neuron evoke precisely timed Ba(2+)-sensitive K(+) inward rectifying (Kir) currents in the s-OL. Unexpectedly, the glial K(+) inward current does not require oligodendrocytic Kir4.1. Action potential-evoked Kir currents are in part mediated by gap-junction coupling with neighbouring OLs and astrocytes that form a syncytium around the pyramidal cell body. Computational modelling predicts that glial Kir constrains the perisomatic [K(+)]o increase most importantly during high-frequency action potentials. Consistent with these predictions neurons with s-OLs showed a reduced probability for action potential burst firing during [K(+)]o elevations. These data suggest that s-OLs are integrated into a glial syncytium for the millisecond rapid K(+) uptake limiting activity-dependent [K(+)]o increase in the perisomatic neuron domain.

An Ultrastructural and Immunohistochemical Analysis of the Outer Plexiform Layer of the Retina of the European Silver Eel (Anguilla anguilla L).

Here we studied the ultrastructural organization of the outer retina of the European silver eel, a highly valued commercial fish species. The retina of the European eel has an organization very similar to most vertebrates. It contains both rod and cone photoreceptors. Rods are abundantly present and immunoreactive for rhodopsin. Cones are sparsely present and only show immunoreactivity for M-opsin and not for L-, S- or UV-cone opsins. As in all other vertebrate retinas, Müller cells span the width of the retina. OFF-bipolar cells express the ionotropic glutamate receptor GluR4 and ON-bipolar cells, as identified by their PKCα immunoreactivity, express the metabotropic receptor mGluR6. Both the ON- and the OFF-bipolar cell dendrites innervate the cone pedicle and rod spherule. Horizontal cells are surrounded by punctate Cx53.8 immunoreactivity indicating that the horizontal cells are strongly electrically coupled by gap-junctions. Connexin-hemichannels were found at the tips of the horizontal cell dendrites invaginating the photoreceptor synapse. Such hemichannels are implicated in the feedback pathway from horizontal cells to cones. Finally, horizontal cells are surrounded by tyrosine hydroxylase immunoreactivity, illustrating a strong dopaminergic input from interplexiform cells.

Astrocytes are central in the pathomechanisms of vanishing white matter.

Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients' tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders.

Gene therapy into photoreceptors and Müller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models.

Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.

Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle.

OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. METHODS: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. RESULTS: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. INTERPRETATION: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.

Myosin VIIA is a marker for the cone accessory outer segment in zebrafish.

The accessory outer segment, a cytoplasmic structure running alongside the photoreceptor outer segment, has been described in teleost fishes, excluding the model organism zebrafish. So far, the function of the accessory outer segment is unknown. Here, we describe the ultrastructure of the zebrafish cone accessory outer segment by electron microscopy. Starting at the connecting cilium, the accessory outer segment runs parallel alongside the cone outer segment (COS). A thin plasma bridge connects the outer segment with the accessory outer segment, whose surface is enlarged by foldings and invaginations. Beside the morphological descriptions, we demonstrate that the Usher protein myosin VIIa (Myo7a) is a specific marker for the zebrafish cone accessory outer segment. Zebrafish cone photoreceptors possess a large and well-differentiated accessory outer segment, in which the unconventional motor protein Myo7a is highly enriched. The direct cytoplasmic contact with the COS as well as the surface enlargement of the accessory outer segment suggests an important role of this structure in transport and exchange of metabolites between the COS and the surrounding retinal pigment epithelium. In future studies of the outer retina, more attention should be paid to this often neglected structure.

Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²âº channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.