Transnational tobacco, alcohol and ultra-processed food corporations use the international trade regime to prevent policy action on non-communicable diseases (NCDs); i.e. to promote policy ‘non-decisions’. Understanding policy non-decisions can be assisted by identifying power operating in relevant decision-making spaces, but trade and health research rarely explicitly engages with theories of power. This realist review aimed to synthesize evidence of different forms and mechanisms of power active in trade and health decision-making spaces to understand better why NCD policy non-decisions persist and the implications for future transformative action. We iteratively developed power-based theories explaining how transnational health-harmful commodity corporations (THCCs) utilize the international trade regime to encourage NCD policy non-decisions. To support theory development, we also developed a conceptual framework for analysing power in public health policymaking. We searched six databases and relevant grey literature and extracted, synthesized and mapped the evidence against the proposed theories. One hundred and four studies were included. Findings were presented for three key forms of power. Evidence indicates THCCs attempt to exercise instrumental power by extensive lobbying often via privileged access to trade and health decision-making spaces. When their legitimacy declines, THCCs have attempted to shift decision-making to more favourable international trade legal venues. THCCs benefit from structural power through the institutionalization of their involvement in health and trade agenda-setting processes. In terms of discursive power, THCCs effectively frame trade and health issues in ways that echo and amplify dominant neoliberal ideas. These processes may further entrench the individualization of NCDs, restrict conceivable policy solutions and perpetuate policymaking norms that privilege economic/trade interests over health. This review identifies different forms and mechanisms of power active in trade and health policy spaces that enable THCCs to prevent progressive action on NCDs. It also points to potential strategies for challenging these power dynamics and relations. KEY MESSAGESThe international trade regime provides transnational health-harmful commodity corporations with opportunities to use and benefit from instrumental, structural and discursive power in ways that likely contribute to non-communicable disease (NCD) prevention policy non-decisions in both national and international policymaking spaces—and particularly so under a dominant neoliberal paradigm.Recognizing power in all its forms across different political spaces and levels is essential for enabling public health actors to identify and evaluate effective strategies for improving trade and health policy coherence.Understanding how corporations constrain public health policy action, or in other words, promote policy ‘non-decision-making’, has been a growing concern for nearly half a century. Over the past few decades, public health researchers have exposed multiple strategies used by transnational health-harmful commodity corporations (THCCs) to prevent regulation of health-harmful commodities  policy non-decisions persist as well as why, so far, relatively limited progress has been made towards increasing attention to NCD risk factors in trade policy. By making visible the different forms, mechanisms and spaces of power at the nexus of trade and health, it becomes possible to identify and evaluate strategies that may generate the necessary changes in power relations between health, trade and corporate actors to drive transformative policy change .This realist review attempts to fill this gap in the literature. Building on established theories of power, we develop a conceptual framework for analysing the interrelationship between different forms, mechanisms and spaces of power in health policymaking. We then map existing evidence against theories grounded in the framework with the aim of better understanding how the power relations between trade, health and corporate actors have emerged and as such, why NCD policy non-decisions persist. By exposing power in this way, it also becomes possible to start identifying strategies to effectively challenge it. While evidence is included from countries across all income groups, we focus, where possible, on low- and middle-income countries (LMICs) since they have become the focus for expansion by many THCCs . Thus, it is useful for expanding trade and health policy analysis beyond a description of problematic trade rules, towards gaining insights into the political economy of trade and health policy.The realist review methodology is based on identifying, interpreting and synthesizing a wide range of evidence to develop and refine explanatory theories about how and why a complex situation results in specific outcomes in certain contexts ; values ; knowledge and evidence ; perception and preference-shaping ; organizational structures ; relationships ; rules (e.g. trade agreements and investment treaties); and norms .Dimensions of power include the different levels—international, national or sub-national where power resides or is contested. Dimensions of power also include different spaces, defined here as formal or informal opportunities where actors can ‘potentially affect policies, discourses, decisions and relationships’ relevant to their interests ; an involuntary failure to act ; or inaction due to a psychological boundary issue .The contexts—political, economic, socio-cultural or situational—can inhibit or activate different mechanisms of power generating different outcomes. For example, LMICs very often have limited capacities—human, financial, organizational, technical and strategic—to exercise instrumental power in relation to negotiating trade rules or agreements in such a way that balances both their economic and health objectives. Lobbying as a form of instrumental power may be constrained where there are clear processes for managing conflicts of interest or restrictions on lobbying in governance spaces. The rule-setting  power of THCCs may be enabled in contexts where there is a strong preference for market-led approaches to governance. Discourses that promote the primacy of markets and involvement of private sector in governance may be resisted in country contexts with strong human rights norms.Finally, certain A systematic search of the literature was undertaken with the aim of identifying the most relevant evidence to support or dispute the initial set of explanatory theories. The final search strategy included combinations of search and indexed terms for the concepts of international trade and investment liberalization, regulatory chill, policy process, relevant transnational corporations and three trade-sensitive public health policy areas: nutrition, tobacco control and alcohol regulation (Supplementary Text SI). These concepts were developed and refined iteratively with repeated testing in MEDLINE, review of search results, development/refinement of explanatory theories and, in turn, further concept development. The search terms were then developed through repeated testing in six databases: MEDLINE, Global Health, Econlit, SCOPUS, Web of Science and PubMed in order to balance reasonable sensitivity and specificity (given project time constraints) and the realist approach of searching broadly.All six database searches were conducted in January 2020 and limited to English language publications between 1 January 2008 and 15 January 2020. It was considered reasonable to limit the search from 2008 onwards given that engagement with and understanding of trade issues by health academics was relatively limited prior to this , WTO, United Nations Conference on Trade and Development and International Institute of Sustainable Development. All articles were downloaded to an Electronic searches yielded 1585 results. An additional 51 items were identified through bibliography searches, citation tracking and searches of Google/Google Scholar and institutional websites. After duplicates were removed, 991 unique items remained. Given the realist approach and the limited literature, an intentionally inclusive approach was taken throughout the selection process.In a preliminary screening, articles were selected based on the test for inclusion derived from realist principles , as judgFull texts were retrieved for 170 of the 174 articles included after initial screening with four articles not retrievable. The 170 full texts were again assessed for relevance based on the test for inclusion. Full-text review resulted in exclusion of a further 66 articles bringing the final number of relevant articles to 104 . Ten perA screening tool (Supplementary Text SII and SIIb) was used to document the rationale for final inclusion/exclusion in the realist synthesis. This included a set of queries regarding study relevance and reliability based on the test for inclusion adapted from a similar set of constructs  was recorded on the screening tool. NVivo was used not only to improve robustness of data analysis but also to improve transparency by providing an audit of the data analysis process. The data extracted under each node were imported into a Word document for analysis. They were analysed and synthesized using a realist approach that was both deductive and inductive. The findings are presented in a narrative synthesis.non-decisions and the role of power in these outcomes, we identified very few quantitative analyses for inclusion. Furthermore, our enquiry is inherently multi-disciplinary in nature with legal, political and other social science research providing valuable insights. For these reasons, we opted to include analyses based on expert opinion and deductive reasoning, not only empirical research. In most studies, formal power analysis was lacking or limited and understanding contextual elements was generally not included as a primary research objective and typically only discussed superficially.The 104 studies included in the review were from a variety of fields including public health, international law and political science. Accordingly, studies varied in design and methods including prospective analyses of trade and investment agreement texts, analyses of WTO committee meeting minutes and WTO disputes; surveys and key informant interviews; and critical analyses of industry and policy documents. Given that our review question requires investigation of policy decisions but particularly The analysis presents the evidence for each explanatory theory/mechanism under theory areas based on the three power types outlined in the conceptual framework .Economic liberalization has facilitated increases in efficiency, profitability and global reach of THCCs . As suchA number of studies provided evidence of THCC lobbying across multiple trade and health political fora. For example, during China’s WTO accession negotiations British American Tobacco (BAT) intensively lobbied the UK, EU and US officials to petition for among other things, lower tariffs on tobacco products and no restrictions on tobacco advertising . More reDecision-makers can be motivated to grant certain business actors privileged access to decision-making spaces given the complexity of trade rules and concerns for economic growth. During both the TPPA and Transatlantic Trade and Investment Partnership agreement (TTIP) negotiations e.g. tobacco companies met privately with US and European Commission (EC) trade officials to discuss the proposed agreements . A CanadHowever, as their legitimacy declines, THCC’s access to certain decision-making spaces can diminish  and ‘WTOWhile THCCs cannot themselves bring claims against governments at WTO for violating international trade obligations, there is evidence that corporations use international trade-related legal threats in an attempt to force involuntary public health policy non-decisions and prevent policy transfer regionally or globally, especially for tobacco control . More reWhen necessary, the alcohol industry is also adopting similar strategies. For example, the alcohol industry threatened a WTO dispute against Thailand if it adopted a proposed ban on alcohol advertising  and arguTrade-related legal threats may be effective tools for THCCs to drive involuntary non-decisions by governments due to the complexity of establishing an adequate defence in a WTO dispute and the vagueness of WTO rules . First, Passing the necessity test is particularly challenging and complex due to significant uncertainty regarding evidential requirements to prove the necessity of a health measure. For example, the SPS Agreement states a measure must be ‘based on’ scientific principles, evidence and risk assessment which leaves some scope for interpretation. Furthermore, it may not be possible for a country to produce indisputable scientific evidence of effectiveness , particuVagueness in trade agreement text has resulted in variable interpretations and rulings by dispute panels creating uncertainty when governments evaluate the risk of future potential WTO disputes in light of a trade-related legal threat . For exaIf the significant hurdle of proving necessity is passed, a government must establish that their proposed measure is not unjustifiably discriminatory between countries . SatisfyWhile some anecdotal evidence exists, empirical evidence that THCCs can effectively promote non-decisions by health departments by generating real or perceived risk of a WTO dispute is, so far, limited. A 2014 Canadian case study found that particularly senior health and safety regulators were concerned with avoiding WTO disputes, although it was not generally reported as a key concern . The stuWith the majority of modern economies structured along neoliberal lines to facilitate free market competition , politicWithin international public health regulatory and norm-setting bodies, alcohol and food corporations are increasingly privileged with high levels of participation . For exaThere is also substantial evidence that neoliberal political values are deeply embedded in trade institutional arrangements at both national and international levels . As suchad hoc observer on a number of relevant WTO committees, the WHO can contribute to discussions but are not officially permitted to be involved in decision-making  after opposition by the USA and the EU , 2017b.At the domestic level, policy actors in Australia and Malaysia identified that export interests were often privileged over health objectives . A studyThe dogma that exporting risk commodity industries are essential for economic growth and job creation can also compel governments to pursue the interests of THCCs in trade agreement negotiations and at WTO  and nearAs their legitimacy declines in HICs, tobacco companies have turned to more economically vulnerable LMICs to act on their behalf. LMICs have been encouraged to use WTO forums to make an economic development argument against tobacco control by raising Article 12.3 of the Technical Barriers to Trade (TBT) Agreement that requires the special needs of developing countries to be taken into account. This was used to oppose Canada’s ban on tobacco additives to help mitigate youth smoking, the European Union Tobacco Products Directive (EUTPD), Brazil’s additives ban and Australia’s plain packaging regulation . The tobThe same trade and economic rationale have compelled governments to pursue the interests of the processed food and alcohol industries within WTO forums. Concerns have been raised at WTO’s TBT Committee in the interest of the processed food industry including over Peru, Chile and Thailand’s proposed food labelling regulations  and rules . Challenging industry’s instrumental power over trade policy might include bans on both THCC political funding and lobbying itself as well as closing the revolving door between government and industry. However, such strategies remain largely unexplored in the trade and health literature. Strengthening countervailing public health lobbying will be challenging given existing money and resource imbalances.Post-treaty implementation measures to defend health policy space and minimize the impact of trade-related arguments, legal threats and challenges include strengthening public health coalitions . For exaOther strategies to increase government confidence and ability to design policies that are consistent with trade rules include capacity building within national health departments on trade issues through technical training and cross-departmental collaboration ; 2014. CIn relation to international trade rules themselves, some experts have recently argued that the relevant WTO Agreements do in fact give governments significant space to design and implement, particularly tobacco control measures, but possibly also alcohol and food regulation, provided they are supported by evidence and are non-discriminatory . In TablIt is important to note, however, that satisfying these conditions may not protect novel measures, particularly those not supported by an international convention or set of standards. They do not take into account the politics of policymaking that very often demands compromise resulting in regulations that may discriminate between like products from different countries . They alSecondly, neoliberal-oriented institutional structures, practices and goals mean THCCs are often granted privileged access to trade and health decision-making spaces where their interests limit the scope of the agenda. Mobilizing broad coalitions to claim greater access to trade policy decision-making spaces and increase the visibility and legitimacy of health interests on the agenda will be important to challenge structural power. For example, in Australia, a broad network of tobacco control advocates managed to gain legitimacy within trade policy spaces while the absence of such a network mobilized on unhealthy diets and nutrition was an impediment to generating attention to this issue in Australian trade policy .Limiting industry representation on government trade committees as well as strengthening government institutional capacity for healthy trade policy will also be important to challenge THCC structural power. At the national level, Thailand is often cited as an example of how sustained investment in technical capacity building and inter-departmental co-ordination between trade and health agencies can generate a common understanding of key health and trade policy issues and bring health actors and considerations into trade policy negotiating forums . StrengtAddressing industry structural power in relation to domestic health policy and international health standards, norms and laws will require structures and rules governing interactions between THCCs and both governments and international public health standard-setting bodies . For exaThird, our findings suggest THCCs attempt to exercise agency over discursive power through reinforcing various framings of health issues in ways that resonate with neoliberal logic and values. While it is impossible to draw causal inference, there was evidence that decision-makers’ individualized interpretation of health issues, the boundaries around acceptable solutions and resulting dominant policy norms of ‘trade over health’ aligned with industry framings.Counteracting these processes include amplifying and propagating alternative framings of trade and health issues. For example, in Australia, tobacco control advocates focused on framing standardized packaging around the direct harms of tobacco and Australia’s commitment to the FCTC and exposing the manipulative nature of the industry’s previous legal attacks  compare in different contexts e.g. by varying levels of economic development or socio-economic inequality, or under different (and different combinations of) predominant political and economic paradigms? We hope the framework developed in this work is a helpful starting point for shaping a research agenda that covers these and other key questions, as well as providing a useful tool for future analyses of power in health policymaking.Health Policy and Planning online.Penelope Milsom received doctoral funding from the Wellcome Trust [grant number 203286/Z/16/Z ] to support this research.czaa148_SuppClick here for additional data file.


Multielectrode array technology allows researchers to record the spontaneous firing activity of cultured neurons over a period of multiple weeks or months. These data can be valuable for understanding how the functional relationships between neurons evolve as they begin to form connections and wire into a functional network. This technology has been adopted by researchers using stem cells to produce human neurons in culture to study neurodevelopmental disorders. However, the dizzying complexity and scale of the data generated have posed some challenges with the analysis and interpretation of experimental results. Here, we first provide historical context as to why multielectrode array platforms were originally developed, and use this perspective to explore some of the challenges currently facing the field. We then highlight new analysis methods, provide some guidance for improving the analysis of multielectrode array data, and discuss standardizing how these findings are communicated in scientific publications.In vitro multielectrode array (MEA) systems are increasingly used as higher-throughput platforms for functional phenotyping studies of neurons in induced pluripotent stem cell (iPSC) disease models. While MEA systems generate large amounts of spatiotemporal activity data from networks of iPSC-derived neurons, the downstream analysis and interpretation of such high-dimensional data often pose a significant challenge to researchers. In this review, we examine how MEA technology is currently deployed in iPSC modeling studies of neurodevelopmental disorders. We first highlight the strengths of in vitro MEA technology by reviewing the history of its development and the original scientific questions MEAs were intended to answer. Methods of generating patient iPSC-derived neurons and astrocytes for MEA co-cultures are summarized. We then discuss challenges associated with MEA data analysis in a disease modeling context, and present novel computational methods used to better interpret network phenotyping data. We end by suggesting best practices for presenting MEA data in research publications, and propose that the creation of a public MEA data repository to enable collaborative data sharing would be of great benefit to the iPSC disease modeling community. Induced pluripotent stem cell (iPSC) technology has afforded many benefits for the modeling and study of human neurodevelopmental disorders. In this system, somatic cell types such as skin fibroblasts, peripheral blood cells, and urinary epithelial cells can be collected from patients who already have a clinical diagnosis of the disorder under investigation and then reprogrammed into iPSCs through the delivery of pluripotency-associated transcription factors. Using well-established induction protocols, reprogrammed iPSCs can then be differentiated into functional neuronal and glial cell types of interest carrying the same genetic background as the patient they were originally derived from, preserving the complex genetic architecture associated with many neurodevelopmental disorders. They can also be used as a platform to evaluate the effect of genetic rescue intervention. In this way, researchers are afforded a renewable source of patient-derived neurons and glial cells for study and experimentation, without requiring any invasive biopsy of human CNS tissue. These in vitro cultures of iPSC-derived neuronal cells can then be utilized in molecular, morphological, and functional phenotyping experiments, as well as for investigating the effect of rescue genetic manipulations and preclinical drugs.The past decade has seen increasing use of multielectrode arrays (sometimes referred to as microelectrode arrays or MEAs) for functional phenotyping experiments in iPSC disease modeling studies. In MEA systems, special tissue culture plates which contain a grid of small electrodes embedded in the bottom of each well are used to record extracellular action potentials from neuronal cells cultured directly on top of the recording electrodes. Simultaneous recording of spiking activity from multiple spatial locations within a culture allows for the interrogation of population-wide firing activity, and understanding of how network dynamics may be altered in disease states. Newer multi-well MEA systems, which come in 12, 24, 48, and 96-well formats, have been particularly favoured in disease modeling studies as they afford higher throughput than traditional single-cell recording techniques for functional phenotyping and drug screening applications. Moreover, as extracellular MEA recording is non-destructive to neuronal integrity, the same neuronal culture can be monitored over multiple weeks or months of in vitro development, potentially providing insights into neurodevelopmental processes which function at longer timescales.The adoption of MEA technology by the iPSC disease modeling field is fairly new, and our intent with this review is not to provide a comprehensive overview of all reported results and disease phenotype from every recent MEA phenotyping study. Instead, we aim to draw attention to some of the challenges that come with deploying MEA technology in an iPSC disease modeling context, and highlight opportunities for improving their use. We begin by providing an overview of the historical background through which MEA technology has developed. We then discuss some of the more recent developments applying MEA technology to iPSC disease modeling studies, as well as the challenges that come with the analysis and interpretation of complex multichannel data.While MEA technology only began to see use in the field of iPSC disease modeling within the last decade, the development of the first MEA systems actually date back to the early 1970s ,3. We feNeurons in tissue culture differ from those in vivo in one important aspect. A neuron in vivo is always part of a neural network, whereas a neuron in tissue culture no longer has synaptic connections with other neurons” . AnWhile a multitude of distinct differentiation protocols exist for generating functional neuronal cell types from human iPSC lines, the majority of these can be broadly categorized as either directed differentiation protocols, or transcription factor programming methods. In commonplace directed neuronal differentiation protocols, iPSCs are first lifted from 2D monolayer cultures and allowed to aggregate into three-dimensional structures known as embryoid bodies (EBs). Various mitogenic and morphogenic compounds are then carefully added to cell culture media over a period of multiple weeks, mimicking extracellular signalling cues involved in normal embryonic development ,45. In pOne significant downside of directed differentiation for producing MEA cultures is the relatively slow speed of neuronal maturation. It takes approximately 4 weeks to establish NPC cultures from iPSCs, and an additional 4–12 weeks of differentiation for neurons to mature into electrophysiologically active cells ,47,48. INgn2 produces a more homogenous population of excitatory layer 2/3 cortical neurons which begin to show signs of electrophysiological activity in as little as one week after inducing transgene expression . . 111]. WDifferentiating between single-neuron and multiunit activity may be particularly important for interpreting changes in functional measurements in the context of drug screens and phenotypic rescue assays, such as increases or decreases in the number of spikes detected in a given channel. For example, drugs may act on single-neuron firing via changes in membrane excitability, or at the level of synapses by increasing transmission. In the former scenario, we may observe increases in global spiking activity due to increases in the spiking activity of single neurons. In the latter, we may observe increases in global spiking activity due to a greater number of neurons becoming responsive in a network C. WithouSpike sorting methods may also be useful to reveal interesting aspects of correlated firing between single neurons within a network. Such single-unit analyses may be utilized to capture differences in activation latencies in the scale of milliseconds and reveal directionality of signal flow within a network, as well as measures of spike propagation speed. While such analyses can also be performed using unsorted multiunit data, single-unit analysis may be useful for further probing the interactions and dynamics that arise between different neuronal subtypes in cultures with mixed cell type composition . Both spike propagation speed and single-unit directional correlation metrics provide more fine-grained insights into intra-network dynamics which could serve as informative phenotypic measures for a variety of neurological disease models.It should be noted that both manual and automated spike sorting methods are susceptible to sorting errors, such as assigning spikes from different neurons to a single unit (type I errors) or missing the assignment of true spikes to a real unit (type II errors) . These sWith the inclusion of MEA phenotyping assays becoming more commonplace in iPSC disease modeling studies, there is a need for discussion within the field about best practices when it comes to the reporting and publication of MEA phenotyping data. In our brief survey of the literature, we found considerable variation in how different studies define biological versus technical replicates, as well as the total number of replicates used . While mAnother important consideration for researchers involves the proper statistical handling of MEA phenotyping data. In particular, “shotgun style” phenotyping approaches—which identify phenotypic differences in network behaviour by leveraging commercial analysis software to extract and compare large numbers of network metrics in parallel—should make use of a multiple testing correction when interpreting results. Researchers should be sure to report whether or not this approach was used in their methodology. Our brief survey of the current literature also uncovered discrepancies in whether parametric or non-parametric statistical tests were used to analyze activity metrics such as MFR. Another recent study by Negri et al. (2020) found similar discrepancies in statistical methodology among studies reporting MEA data, and showed that the distribution of MFRs recorded from primary cultures of rat cortical neurons is decidedly non-normal . While tFinally, MEA data reporting could benefit from standardization in how MEA phenotyping data are presented in publication figures. Considering more commonly used experimental techniques such as Western blotting or immunocytological staining for a moment, it has long been standard practice to publish representative images alongside any plots of quantitative metrics such as signal intensities. These representative images both provide some more intuitive understanding of what changes in abstract metrics such as average pixel intensity actually look like, while also allowing other researchers to quickly assess the quality of the raw data presented, ensuring there are no technical artifacts such as high background noise and non-specific western bands which may have impacted the downstream quantification. We believe that a similar standard practice should be adopted for MEA data reporting and suggest that raster plots of representative network activity should always be included as a supplement to any quantitative metrics used to characterize phenotypic differences in network behaviour. Moreover, representative raster plots should display activity from all available recording channels so potential data quality concerns such as large numbers of inactive electrodes or excessively noisy channels e.g., A can be The inclusion of representative raster plots is particularly important in studies which include phenotypic rescue assays. The effectiveness of certain drug interventions can easily be misrepresented by selective reporting of only those network metrics which see substantial improvement with drug treatment, even if that improvement does little to change the major qualitative differences in the network activity patterns observed between control and disease states. To illustrate this point, we produced example raster plots by randomly generating spike trains with different firing characteristics, representing 60 s of network activity from an imagined control and disease condition, as well as two hypothetical drug interventions . We geneExamining this plot in isolation, it can be seen that the mutant condition (MUT) has a significantly reduced MFR when compared to the wild-type (WT) condition. Additionally, it can be seen that Drug A appears to be an effective therapeutic intervention, clearly rescuing the MFR network phenotype, while Drug B and Drug C appear to have no effect. However, a much different story emerges when we include the representative raster plots A–E. We cWhile the inclusion of representative raster plots in MEA phenotyping studies is clearly a step in the right direction towards ensuring high standards of scientific integrity and transparency in data reporting, we also believe that a greater effort should be made to make all raw MEA recording files freely available for other researchers to utilize. This has been a difficult task for the field, as current multi-well MEA systems are capable of generating multiple gigabytes of data per minute of recording. Large phenotyping studies can therefore generate multiple terabytes worth of raw data, and there is currently a lack of a convenient platform for storing and curating these large recording files. We would like to suggest that the creation of a public MEA data repository should be a goal of the field.Finally, an issue with the analyses of MEA data is that commonly used electrophysiological analyses techniques such as spike sorting, multidimensional analyses of data, and machine learning are not part of the training repertoire of developmental and molecular biologists. A possible solution to this problem is to train the new generations of researchers in a diversity of data analyses and visualization techniques for electrophysiological data. Another possible solution is to promote more collaboration between laboratories with different levels of expertise. Our proposal of generating platforms for making MEA data available to the community could aid in that respect.MEA technology stands to yield new insight into disease phenotypes by revealing population-level differences in neuronal firing and communication. As others have noted, MEA phenotyping assays can be highly robust so long as careful consideration is given to proper assay design, analysis, and data reporting. In this review, we have highlighted some of the current challenges with standardizing MEA experiments within the iPSC disease modeling field and provided some recommendations for working towards this goal.In examining assay design options across recent studies in the field, we first noticed that the most common choices of recording durations were 5 and 10 min . This isWe also noticed some discrepancies in how biological replicates were defined and reported in the literature. While we found that most studies defined replicates in terms of the total number of culture wells analyzed, it is important to note that considerable variation in the development of networks from different culture platings and different neuronal differentiations has been reported in both iPSC and primary neuron literature ,40,109. 2 saturation, and relative humidity to help mitigate these concerns; however, care should be taken to schedule recordings around culture media changes to avoid introducing variability from pH differences between fresh and old culture media. Cultures should also be given 5–10 min to baseline activity levels after being physically moved from an incubator to the recording station. In addition, if a transcription factor programming-based differentiation method is being used to generate iPSC-derived neurons, care should be taken to ensure that similar levels of transgene expression are being induced in control and affected cell lines, as differing levels of NGN2 expression have been shown to promote the adoption of different neuronal cell fates [Final considerations for assay design concern important parameters which must be controlled to avoid introducing unwanted variability and noise into recordings. Spontaneous network activity has been shown to be sensitive to changes in environmental factors including media pH and osmolarity, ambient temperature, as well as physical movement or vibration of the MEA plate or recording apparatus ,109. Manll fates . As bestIn this review, we advocate for a more holistic approach to network phenotyping—one which places greater emphasis on describing differences in the spatiotemporal organization of spontaneous firing into characteristic activity patterns rather than focusing on aggregate firing rate differences alone. Using didactic examples, we illustrate how simple activity metrics such as MFR can fail to capture significant changes in the arrangement of firing activity within a recording , and howOur final considerations concern how MEA data are reported and presented in publications. While many commercial MEA analysis software suites are capable of automatically extracting dozens of different activity metrics in parallel, we noticed that few studies disclosed whether this approach to network phenotyping was used or not, nor whether any additional metrics were investigated but omitted from the final publication. We believe this has important implications for downstream statistical analysis, specifically whether one needs to correct for multiple comparisons, and authors should explicitly state whether this approach was used when describing their methodology.As a standard, we suggest that representative raster plots, raw signal traces, and spike waveforms should always be included alongside any summary plots of MEA phenotyping metrics. We believe this will allow reviewers and other investigators to easily evaluate the quality of the raw recording data, and is particularly important for evaluating claims about the efficacy of different drug compounds in phenotypic rescue experiments . Finally


The real world is uncertain, and while ever changing, it constantly presents itself in terms of new sets of behavioral options. To attain the flexibility required to tackle these challenges successfully, most mammalian brains are equipped with certain computational abilities that rely on the prefrontal cortex (PFC). By examining learning in terms of internal models associating stimuli, actions, and outcomes, we argue here that adaptive behavior relies on specific interactions between multiple systems including: (1) selective models learning stimulus–action associations through rewards; (2) predictive models learning stimulus- and/or action–outcome associations through statistical inferences anticipating behavioral outcomes; and (3) contextual models learning external cues associated with latent states of the environment. Critically, the PFC combines these internal models by forming task sets to drive behavior and, moreover, constantly evaluates the reliability of actor task sets in predicting external contingencies to switch between task sets or create new ones. We review different models of adaptive behavior to demonstrate how their components map onto this unifying framework and specific PFC regions. Finally, we discuss how our framework may help to better understand the neural computations and the cognitive architecture of PFC regions guiding adaptive behavior. Humans and other animals have evolved in a world that is uncertain, ever changing, and constantly presents choice situations that have been seen before rarely. These characteristics of natural environments—uncertainty, non-stationarity (volatility), and open-endedness—pose critical adaptive challenges, which ultimately determine the animal’s ability to learn about sources of food and danger and to take appropriate actions. To successfully tackle these challenges, animals must adapt their learning and decision-making strategies in multiple ways. Mammals are the most adaptive class of species as evident from their success in populating very different environments on the planet. The ability to adapt to different environments mainly relies on the brain cognitive flexibility, and not surprisingly, mammalian brains have evolved in specific ways related to the demand for adaptability   changes constantly. Each type of change requires specific adjustments in learning and decision making . In thisEnvironmental changes include but are not limited to alterations in external contingencies in terms of the rate or probability at which different stimuli, actions provide reward, resulting in uncertainty , 3; how The aforementioned examples highlight important learning and decision-making challenges that animals face in natural environments featuring uncertain, volatile, and open-ended situations. In general, these features require simultaneous updates of different models that the animal uses to link stimuli, actions, reward outcomes , as well as adjustments in how information from these internal models should be combined to make choices. However, updating internal models that contribute to ongoing behavior should differ from previously learned models that do not. Non-stationary or volatile environments require adjustments in learning and/or in weighting different estimates from various learning systems. Finally, open-ended environments necessitate to regulate the generalization and transfer of previously learned models to novel situations and tasks.To understand how the brain resolves these adaptive requirements arising in natural environments, similar situations are recreated in controlled experimental settings. These settings provide evidence that humans and other mammals exhibit different types of flexibility required to tackle them.The probabilistic reversal learning (PRL) task and its variants have often been used to study the effects of uncertainty and volatility on learning and decision making , pointinOther studies have found higher learning rates in volatile compared to stable environments in both monkeys  and humacurrent latent state of environment  without inferring possible higher-order causes of changes in the environment such as the level of volatility ).Mechanistically, associations composing selective models and linking stimuli and actions according to reward values are likely stored at the synaptic level using activity-dependent synaptic plasticity modulated by reward signals . SynaptiThe orbitofrontal cortex (OFC) is present in all mammals and comprises a medial and lateral sector, with the latter (laOFC) receiving most sensory inputs to the PFC . There iIn contrast to the OFC, the posterior dorsomedial PFC (posterior dmPFC) including the presupplementary motor area and caudal parts of the anterior cingulate cortex is linked to the motor system. Although action values (A-Rew) divorced from stimulus are experimentally difficult to dissociate from stimulus values that elicit responses, human studies successfully dissociated the two value notions and found action values guiding selection to be encoded in the posterior dmPFC , 102. ThWe note that many studies investigating learning and decision-making use binary rewards that conflate the notion of reward magnitudes and outcome identities. Except in sequential, multi-step tasks, the expectations about binary rewards confound reward magnitudes with outcome probabilities, making the notion of stimulus or action values (S-Rew or A-Rew) indistinguishable from the notion of predictive models comprising S–O and/or (S–)A–O associations. However, several studies that manipulates reward magnitudes independently of reward probabilities allow for identifying the neural bases of predictive models.The ventromedial PFC (vmPFC) refers to the prefrontal region including the medial OFC and the subgenual part of the anterior cingulate cortex. Studies across mammalian brains from rodents to monkeys and humans provide evidence that the vmPFC learns and encodes predictive models that predict action outcomes in response to stimulus  lies between the vmPFC and posterior dmPFC and particularly includes the dorsal anterior cingulate cortex (dACC). There is ample evidence across mammals that the neural activity in the anterior dmPFC reflect multiple value and reliability signals associated with actions, action–outcome, stimulus–action, and stimulus–action-outcome associations  mainly corresponds to BA9 and BA46 located in front of the clPFC. The midlPFC is mainly connected to the clPFC, the laOFC, vmPFC, the anterior dmPFC and the frontopolar cortex (see below) , 130–134Overall, these findings support the idea that the midlPFC encodes the notion of contextual models we defined above as learning contextual cues predicting task set reliability. Contextual models index task sets through external cues that act as proactive predictors of their reliability. The occurrence of such cues enables the brain to proactively update actor task set reliability monitored in the vmPFC contributing to maintain or inhibiting the current actor task set. This further allows proactively building a new context-dependent task set from long-term memory during the process of task set creation. In humans, contextual models may further allow for updating the reliability of counterfactual task sets monitored in the frontopolar cortex (see below).The PFC has further evolved in humans in its most rostral portion with the emergence of a lateral frontopolar region (fpPFC) , 147, whIn this review/perspective, we propose that in order to identify computational components of adaptive behavior and their underlying neural mechanisms, we need to examine what learning in a given task entails in terms of associating stimuli, actions, and outcomes. This allows us to distinguish between learning processes that rely on simple model-free associations  versus those requiring an internal model of how stimuli, actions, and outcomes are linked in the environment A–O associations). This distinction is critical because these different types of processes provide very different levels of adaptability for tackling learning challenges in natural environments. More importantly, these processes rely on different types of synaptic plasticity and learning mechanisms, only some of which are available in certain brain areas or regions. Distinguishing between different types of stimulus, action, and outcome associations further enables disambiguating the contributions of specific brain areas, especially within the PFC to adaptive behavior. Critically, our framework suggests specific types of interactions between multiple systems that can be tested in future experiments.By considering and examining alternative ways that stimuli, actions, and outcomes are linked through leaning processes, one can also pinpoint how inflexibility in learning and choice emerge and what their underlying neural mechanisms are. This has important implications for understanding various behavioral impairments due to neurological disorders and could resolve many disputes about the role of different cortical and sub-cortical areas in maladaptive behavior. For example, impairment in estimating action values ––which rely on no predictive models and thus is less flexible––has a very different impact on adaptive behavior than impairment in predictive models predicting outcomes based on the same actions .In addition, we highlighted shortcomings of most commonly used experimental paradigms to study different aspects of adaptive behavior. More specifically, one-dimensional experimental paradigms––that is, those involving only one reward attribute such as reward probability or reward magnitude––cannot distinguish between different types of learning processes. To carefully examine flexibility in learning and choice behavior, one needs to consider tasks with multiple types of reward information. Only when multiple types of reward information are present, can one tease apart different mechanisms of adaptability and their neural substrates. Nonetheless, distinguishing between these mechanisms also requires utilizing computational models that incorporate multiple components/systems for capturing different types of learning or associations between stimuli, actions, and outcomes. For example, ignoring how reward probability and magnitude are combined can result in erroneous conclusions about the impacts of volatility on learning and choice behavior.Inference is an important component of predictive and contextual models as well as their interactions with other internal models. However, we would like to emphasize that basic inference processes for estimating outcomes or for combining inferential and non-inferential processes can be approximated using very simple mechanisms , 74, 75.As mentioned above, to tackle the challenges of adaptive behavior in uncertain, ever changing, and open-ended environments, the brain has to rely on dynamic interaction between multiple systems that aim to link stimuli, actions, and outcomes in different fashions. This means the amount by which each system contributes to behavior constantly changes over time. As described earlier in details, there are many representations of values  that all dynamically compete for action selection with different weights depending on internal and external factors. As a result, the present framework proposes that there is no integration of all these value representations into a unique utility function or equivalently a common currency to be maximized as often postulated , even ifIndeed, there is very little evidence for the fusion of different reward attributes into a single utility quantity even under the oversimplified condition in which choice options consist of only two attributes, reward probability and magnitude , 80, 157More generally, the present framework suggests that there is no overarching system computing and maximizing a unique utility quantity as well as controlling and weighting the concurrent contribution of each system to behavior. Although within the actor task set, different systems/models are assumed to influence behavior with different weights, this weighting implies no superordinate controllers. Instead, the weighting relies on the reliability of internal models that within the actor task set, directly reflects the gain by which neural representations and signals dynamically encode internal models and consequently regulate their influence on behavioral choices.Accordingly, the notion of utility function and utility maximization might provide limited insights into understanding computations involved in adaptive behavior and their neural substrates in the PFC. We hope the present framework will help to move forward and better understand the PFC function and how the multiple neural systems and internal models linking stimuli, actions, outcomes and rewards along with their dynamic interactions determine learning and behavioral choices.


We are delighted to announce the launch of a new section in the Journal of Translational Medicine entitled “Translational signaling and systems biology”.Cells respond to a broad range of signals sensing transduction mechanisms that feedforward circuits involved in cell life and fate. Great progress has been recently made on how extracellular information are fine tuned into intracellular physiological and pathological outcomes. Regardless of skill to cross (i.e. steroids) or not to cross the cell membrane (i.e. growth factors), extracellular signals are transmitted to intracellular environment through a variety of transduction pathways and sensing cues. Noteworthy, innovative methods and technologies including imaging and nanotechnologies, molecular modelling, new algorithms of artificial intelligence, bioinformatics and systems biology, have better uncovered the actual role of transduction signaling on key cell fitness, from basic to translational research –4.In addition, much attention has been paid on mechanotransduction that refers to the transmission of external mechanical forces to molecular sensors mediating intracellular changes. It is now clear that the complex apparatus harbored by cell membranes not only allows the trafficking of ions and molecules, but also acts as a smart structure in the mechanosensitive processes. For instance, conformational changes and signals originating from the ECM may lead to cell–matrix interactions in peculiar sites named focal adhesions. Then, multi-protein complexes connect extracellular space to the intracellular compartment and the cytoskeleton, turning the transmission of mechanical forces into biological features .Overall, either potential opportunities or problems may be caused by messengers and factors shared by multiple signaling pathways. In this regard, a fine cross-talk among diverse transduction cascades allows a superior regulation of cell activity respect to the action of individual pathways. On the contrary, deleterious cross talk induces certain pitfalls leading to altered biological information. In order to better appreciate this intricate landscape, it appears beneficial to scrutinize the multifaceted signal transduction network by systems biology approaches, which may strongly contribute to clarify how expected or inappropriate biological responses could occur through complex molecular interactions . In thisActually, the field is dynamic and exciting due to the chance to facilitate the translation of new therapeutic breakthroughs targeting dysregulated signaling pathways into clinical settings. Therefore, we are interested in research that investigates and characterizes translational signalling, even taking advantages of systems biology.The Journal of Translational Medicine provides high standard peer-review process and represents a space for efficiently communicating up-to-date results and scientific discussions. The new section on “Translational signaling and systems biology” will guarantee high quality and competitive publications. The Editorial Board is looking forward to receiving your contributions.


Dentistry is responsible for around 10% of antibiotic prescribing across global healthcare, with up to 80% representing inappropriate use. Facilitating shared decision-making has been shown to optimise antibiotic prescribing (antibiotic stewardship) in primary medical care. Our aim was to co-develop a shared decision-making antibiotic stewardship tool for dentistry. Dentists, patients and other stakeholders prioritised factors to include in the new tool, based on previous research (a systematic review and ethnographic study) about dentists’ decision-making during urgent appointments. Candidate behaviour-change techniques were identified using the Behaviour Change Wheel and selected based on suitability for a shared decision-making approach. A ‘think aloud’ study helped fine-tune the tool design and Crystal Marking ensured clarity of messaging. The resulting paper-based worksheet for use at point-of-care incorporated various behaviour change techniques, such as: ’information about  health consequences’, ‘prompts and cues’, ‘restructuring the physical  environment’ and ‘credible sources’. The think aloud study confirmed the tool’s acceptability to dentists and patients, and resulted in the title: ‘Step-by-step guide to fixing your toothache.’ Further testing will be necessary to evaluate its efficacy at safely reducing dental antibiotic prescribing during urgent dental appointments in England and, with translation, to other dental contexts globally. Antimicrobial resistance is a significant threat to global health, wealth and well-being, and is driven by the use of antibiotics . The WorUnlike in medical settings, where many common infections are amenable to self-care , acute dCompared to medicine, few antibiotic stewardship interventions have been developed to optimise dental antibiotic prescribing . FacilitIn 2016, a dental antimicrobial stewardship toolkit was introduced in England to provide free, online access to guidelines, information and training about dental antibiotic prescribing and resistance . Large gThe purpose of this paper is to report the development of an evidence-based, behaviour theory-informed, shared decision-making tool to optimise antibiotic prescribing by dentists, for adults with acute dental pain or infection, during urgent dental appointments, initially in England. In line with the ethos of shared decision-making , a co-development approach with dentist, patients and other stakeholders was chosen. If shown to be successful at reducing dental antibiotic prescribing, this tool will be translated into other dental contexts worldwide to contribute towards global efforts to tackle antimicrobial resistance.Dentists, patients and the other stakeholders reached a consensus on prioritisation of nine factors (from thirty-one factors identified in a published ethnographic study ) for incTo underpin intervention development, the first stakeholder meeting began the process of prioritising the factors associated with the decision whether to prescribe dental antibiotics. Having also reviewed antibiotic stewardship interventions developed for use in the primary medical care context, the stakeholders recommended translation of two key aspects for the new dental antibiotic stewardship tool:(1)Engaging patient in (rather than just giving them a leaflet or telling them the treatment decision) during urgent dental appointments; and(2)The use of diagrams on a leaflet  toolkit) to nudge and assist the dentist to explain the diagnosis to the patient .Based on the mapping of each factor to domains of the Theoretical Domains Framework (TDF)  and usiA worksheet format for the tool was chosen to engage the patient in the decision-making process and included diagrams to explain the diagnosis (as advised by the stakeholders in Stage 1). A draft worksheet see  was deveDuring the think aloud study, the stakeholders provide advice about the content and structure of the worksheet which informed modification of the draft worksheet see .The think aloud study also demonstrated to the researchers that the folded design  was too complex. Printing out the leaflet and folding it in the right way was, therefore, a barrier to its use in the intended way. This informed redesign of the worksheet to a more flexible two sides of A4 which could be printed back-to-back and folded if desired. Following feedback from the dentists and dental nurses, colours for the leaflet were also chosen which appeared equally well when printed in black and white as colour, as not all dental practices have colour printers.The worksheet to facilitate shared decision-making consisted of six sections printed ‘back to back’ on a single sheet of paper. Side one consisted of three sections see  and was Each section of the worksheet was linked to the priority BCTs, as detailed in Several of the BCTs prioritised by the stakeholders for intervention development were not encompassed within the worksheet: instruction on how to perform the behaviour, verbal persuasion about capability and goal setting (behaviour). As highlighted by one of the dentist participants in the think aloud study , the denAn online, motivational training package for dentists to accompany use of the tool is being developed, therefore, as part of the shared decision-making, dental antibiotic stewardship intervention. As well as introducing the dentists to using the worksheet, it will address instruction and persuasion relating to dentists’ skills in ‘patient management’  and their beliefs about their capabilities for diagnosing, treatment planning, gaining consent and providing dental procedures during urgent appointments. It will also set the goal of urgent dental appointments to be about ‘fixing the acute dental problem’ with definitive treatment wherever possible , ratherA logic model for the shared decision-making, dental antibiotic stewardship intervention  is presented in A shared decision-making tool, which is tailored to urgent NHS dentistry in England and acceptable to patients and dentists in this context, has been developed to optimise antibiotic prescribing by dentists for adults with acute dental pain or infection. A stakeholder group of patients and members of the dental team co-produced the worksheet and focused content on the behavioural influences which they judged as the highest priority to be tackled. Further work to develop an accompanying online motivational training package is underway. Evaluation of the intervention (both worksheet and training) during urgent dental appointments will follow.Using an evidence-based system for intervention development was an important strength of this study. The behaviour change wheel (BCW) provided the theoretical framework ,19, withFor development of this shared decision-making intervention (which is about levelling the playing field between patients and clinicians), the researchers viewed sharing responsibility and power between stakeholders and researchers as essential to the ethos of the project. Whilst patient and public involvement and engagement in research are now an expected component of research activity in the UK , the resCo-production has been defined as ‘collaboration in governance, priority-setting, conducting research and/or knowledge translation’ . TensionThink aloud studies provide a constructive way to show the acceptability of an intervention and have been hypothesised to improve the uptake and adherence thus leading to a greater change in behaviour when implemented in healthcare . Using aHigh rates of antibiotic overprescribing (not in accordance with guidelines) are known to exist across NHS medical and dental care ,27,28. PIn addition to the 31 dentist-factors associated with decision-making during urgent dental appointments, the ethnographic study feeding into this study also identified 19 patient-factors . The strSome patients in the ethnographic study exhibited well-developed negotiation/communication skills , and barOther patients in the ethnographic study reported strongly emotional feelings about dental treatment (such as anxiety or phobia) . StudiesAntibiotics do not work for dental pain which is caused by an inflammatory process (such as pulpitis) . TreatmeThe ethnographic study which underpins this intervention development also suggested that it takes longer to deliver dental procedures than antibiotics during urgent dental appointments . A scenaA limitation of this study is that the shared decision-making, dental antibiotic stewardship intervention resulting from this study focused on just 9 of the 31 factors identified as influences on dentists’ decisions whether to prescribe antibiotics. A number of the factors not addressed by this intervention link to the wider environment context for urgent NHS dental care provided in England. These healthcare system factors include the access to (availability of) routine and specialist NHS dental services required to complete definitive treatment started during urgent dental appointments . The lack of routinely collected dental prescribing data also makes it impossible to hold NHS dental contractors accountable for their dental antibiotic prescribing rates or to financially incentivise optimal urgent dental care through the NHS dental contract . The impIf shown to have the desired impact, translation of this intervention into other healthcare contexts would be straightforward. With seven of the nine factors targeted by this intervention having been identified in an international systematic review of dentists’ decision-making about whether to prescribe antibiotics to adults with acute dental pain or infection , it is likely that translation between dental contexts will be successful. Testing of the intervention in urgent dental care in NHS England and beyond will be needed to assess the efficacy of this tool as an antibiotic stewardship intervention which could contribute to global efforts to tackle antibiotic resistance.Development of the tool followed the Behaviour Change Wheel (BCW) approach , in threwww.liveillustration.co.uk made a graphic (cartoon) record of the meeting, which acted to both collate ideas and stimulate discussion among the whole group about the factors and initial feelings about relative importance of each factor. The second stakeholder workshop was organised by a theatre arts charity (www.theatreofdebate) and was designed to broaden and deepen understanding and insight among the group about the factors before then prioritising them as targets for intervention development. To achieve consensus among the group about the priorities, eight pairs of stakeholders ranked their top three priorities, which they then shared and discussed among the whole group. Finally, through discussion, the group decided on the factors which they identified as priorities to include in interventions.Understanding dentists’ antibiotic prescribing behaviour was based on 31 factors which had been identified in a previous ethnographic study about influences on treatment decisions (including but not limited to antibiotic prescribing) during urgent NHS dental appointments in England . A stakeEach factor had been mapped to the TDF in previous studies ,11. The A draft worksheet was developed incorporating these BCTs and with reference to frameworks for shared decision-making  and adviwww.plainenglish.co.uk) to confirm the clarity of the worksheet.A draft worksheet was developed by the principal researcher (WT), incorporating the BCTs and other elements identified during the stakeholder workshops. A think aloud study with key stakeholders  was undertaken through interviews to check the content, to review preferences for the mode of delivery   and to fEthical approval for the study was gained from the University of Leeds Dental Research Ethics Committee (DREC ref: 101218/WT/267 dated 18 December 2018).A shared decision-making tool, comprising a worksheet with multiple behaviour change techniques built into the text, image content, and mode of delivery, has been produced which aims to reduce antibiotic prescribing for adult patients with toothache or infection during urgent NHS dental appointments in England. The next step will be to evaluate it at point of care and to translate it into other dental contexts so that dentistry around the world can contribute to international efforts to tackle antibiotic resistance.


Ziphius cavirostris, apnea duration: 137 mins and 2,992 m depths). Despite major anatomical and physiological differences between humans and marine mammals, humans present with an interesting physiological response to apnea to limit the effects of hypoxia and/or pressure increase, in which the main physiological functions such as pulmonary, cardiovascular, and nervous functions can be modulated acutely and chronically. The Research Topic focuses on these varied facets but also their trainability and their consequences on health in relation to some hypoxia-related pathologies such as sleep apnea syndrome, pulmonary edema and decompression sickness.Research into voluntary apnea is becoming increasingly popular in varied laboratories and in the field around the world. In the last 10 years, as many articles have been published than between 1954 and 2011. Breath-hold diving is truly an antique practice; for example, Alexandre Legrand employed professional breath hold divers called “Urinator” in his army, and Japanese Ama and Philippine Bajau people have been known to dive for hundreds of years to gather food. More recently, apnea diving became a high-level competitive sport with impressive performances , but although constant progress is made, human performance is still far from the prowess of marine mammals  and spearfishermen . Apnea training is therefore a relevant hypoxia training for several physical activities since it induces hypoxia and hypercapnia despite a low intensity of exercise  Grossman et al. reported how swimmers should better manage their ventilation per arm cycle during aerobic events to avoid consuming too much oxygen . Moreover, Guimard et al. demonstrated that performing dynamic apnea at 30% of maximal aerobic power is the best compromise between exercise intensity and apnea duration to induce a sufficient diving reflex in non-apnea trained participants. In fact, as long as the exercise intensity is mild, the diving reflex can overwhelm the muscle metaboreflex . Dynamic apnea can therefore be used in other physical activities to induce hypercapnia, hypoxia and increased lactatemia even during low intensity efforts . During static and dynamic apneas, the dive reflex decreases oxygen consumption through bradycardia and peripheral vasoconstriction, and preserves oxygen-dependent organs through increased cerebral and cardiac perfusion . Those adaptive responses have been described in both spearfishermen and freedivers, and are correlated with a high adenosine release which exerts powerful bradycardia and vasodilation in coronary and cerebral blood vessels . Moreover, an increase in serum heat shock protein levels in response to higher nitric oxide levels could improve cardiovascular adaptation to hypoxia in elite freedivers . Despite a low production of free radicals observed in freedivers during exercise and dynamic apnea . Joulia et al. . Despite diving reflex allows the freedivers to be more economical with oxygen, they become hypoxic depending on the duration of apnea and the intensity of exercise performed during dynamic apneas. These hypoxias can induce arrhythmias in both humans and marine mammals . Interestingly, for freedivers, decompression illness is poorly understood and unlike in scuba diving, it is only cerebral in nature . This suggests different mechanisms may occur within the spinal cord or skin . Both deep and repetitive breath-hold diving can lead to endothelial dysfunction . The mechanisms put forward appear to favor a mechanical hypothesis via increases in cerebral blood flow and disturbances in cerebral autoregulation as described in elite freedivers . Taylor et al. tested the hypothesis that individuals with documented obstructive sleep apnea (OSA) exhibit greater muscle sympathetic nerve discharge and synchronous blood oxygen level-dependent signal responses during volitional simulation of central or OSA by Mueller Maneuvers (MM) and apneas. The hemodynamic and neural responses to both apneas and MMs were in fact essentially identical in participants with and without OSA, indicating that recurring episodes of cyclical apnea during sleep does not alter cortical or peripheral neural responsiveness.Freedivers  and mechanical (via the baroreflex) stimuli to maintain its homeostasis. Breath-holding is also an excellent stimulus to generate natural hypoxia. This poikilocapnic hypoxia may be incorporated into training periodization to prepare the organism for the lack of oxygen during a trip to altitude or simply to improve performance at sea level. Apnea may also be considered as preconditioning stimulus to better withstand health-damaging hypoxic situations, for example during a heart attack or stroke. Future studies are warranted in this area to refine the exercise characteristics and hypoxic dose of such apneic training for achieving the desired physiological outcome.Past research on apnea as well as the recent advances published in the current Research Topic really highlight breath-holding as an excellent model for studying the interactions between key physiological functions. It allows understanding how our body responds and adjusts to different chemical (FL, FB, and FJ wrote together the editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The conventional science curricula generally favour educational practices that yield high scores on immediate examination, though it may not accurately predict students’ long-term academic achievement. In view of the pre-exam cramming phenomenon, this article shows the evidence of spacing effect in science education and probes into its theoretical mechanisms, effectiveness in experimental settings, and current applications in science learning. In brief, spacing works by repeatedly presenting the learning material across various temporal intervals. This paper suggests that spacing could significantly result in greater memory strength by alleviating multiple neurocognitive and behavioural properties of learning that are hampered by cramming. Together with the analysis of its relevance in science education, the spacing effect may further provide leverages for promoting long-term conceptual understanding and reflective skill development. However, there are many reasons that students and teachers may not be aware of or fully appreciate its benefits. Finally, this article discusses systemic barriers to why spaced repetition is underutilized in science curricula. Common sense seems to dictate that school grades reflect students’ knowledge and understanding of tested subjects . While gFor decades, a great deal of attention has been directed to the principle of spacing effect (or spaced repetition/spaced practice) . The spaIn a general sense, spaced repetition is an evidence-based information encoding technique that improves recall efficiency by dividing the enormous content into a series of short-piece information across temporally spaced intervals  What is the evidence, socially and cognitively, that spaced repetition improves learning and test results for science subjects?(2) What are the potential and barriers to utilizing spacing effect in science education, socially and cognitively?The impact of the spacing effect on memory and learning outcomes is predicated on complex neural and cognitive mechanisms. Understanding the interplay of spaced practices and these mechanisms can shed insight into the utility of spacing in science curriculums, and critiques on conventional instructional practices . This paBiological foundation of learningSynaptic Plasticity and Spacing EffectThe biological mechanisms of learning and memory are operated by modifying the connection strength between central nervous system neurons. Synapse is the site where two neurons join together through the transmission of nerve impulses and the release of neurotransmitters. Memory consolidation is a procedure in which neurons are created, linked, and (re)activated with their various patterns and firing rates. Repetition of specific memories can strengthen the existing synaptic connections and produce more synapses for long-term encoding, whereas unused memory gradually promotes weakening and apoptosis of synapses. This ability is called synaptic plasticity .While repetition can aid in memory recall by reinforcing synaptic connections, it does not necessarily expound on whether this effect is long-lasting and could face up to the offshoot of mental fatigue. Learning, like many neural activities, requires a refractory period that separates two successive stimuli . For exaAt the neuronal level of learning, synaptic transmission is facilitated by the production, uptake, release, and disposal of excitatory and inhibitory neurotransmitters, which activate memory traces in response to stimuli . For example, excitation of serotonin , glutamaThe impact of spaced repetition on learning rigorously follows synaptic plasticity mechanisms. Kelley and Whatson and Smolen et al. suggest that progressive repetition of stimuli separated by irregular spacing intervals can lead to a long-lasting enhancement of synaptic contact signalling - long-term potentiation (LTP) ,16. TheoCortical Response to Spaced LearningThe spacing effect delineates a good representation of brain areas associated with memory formation. Kornmeier and Sosic-Vasic epitomize that learning and memory exist in “different areas of the brain, like the amygdala, hippocampus, cerebellum, and cortex” . This opBy using EEG in memory research, researchers can observe the brain voltage potential in response to task-specific activities. N400 is an EEG waveform component that reveals the LTM retrieval strength of lexical items . SimilarCognitionSpacing Effect on Long-Term MemoryThe standard way of thinking about cognition holds that it involves complex mental processes of obtaining, understanding, retaining, and retrieving information. One of the most important information processing characteristics in cognition is memory. Learning begins with creating temporary memories that are stored in STM. Through repeated encoding and rehearsal, these specific memories can be transferred to LTM storage, making them last indefinitely ,10.Encoding study materials distributed throughout the spaced trials facilitates the transfer of materials to LTM storage, which involves the serial position effect. The underlying mechanism of the serial position effect is that the first and last component in a study list will have a more robust mental representation of memory. Specifically, the primacy effect reveals that the earlier items in a study list are more likely to be converted into LTM, just as how important the first impression is in a long-term social interaction .The primacy effect can lead to a rapid accumulation of LTM with spaced learning. Kelley and Whatson suggest that memory performance after just an hour of spaced repetition can hold up to four months of massed instruction, showing a high level of LTM encoding . TheoretSpacing Effect on Short-Term MemoryThe capacity of LTM to store prior information is infinite; STM, on the other hand, stores recent memory traces with little or no consolidation and can disappear spontaneously . The traShail explains that “in the recency effect, items are stored as short-term… declines with the passage of time and by the presentation of additional information” . This arContrary to momentary benefits of massed learning in STM retrieval capacity, Zhao et al. used EEG to show that LTM retrieval of lexical knowledge might be hampered under the massed teaching condition by observing reduced activities of N400 and LPC waveform signals . Using aSpacing and MotivationMotivation plays a crucial role in facilitating LTM retention. It may derive from external incentives  or/and intrinsic incentives  . During Spacing and AttentionIn addition to the impact on memory, it is also predictable that motivation might help direct attention, leading to less mind wandering . Mind-wandering may occur frequently in lengthy and difficult sessions due to spontaneous distractions that govern STM capacity . HoweverOverall, it is evident that while spaced repetition may not show immediate benefits on classroom examinations, its effects on learners’ long-term retention, motivation, and attention are long-lasting. It also implies that current educational practices and policy initiatives are often short-sighted and profit-oriented, undermining incremental progress for learners.MetacognitionJudgements of Spaced LearningLearning involves reflective components that regulate the basic information processing in cognition, helping learners inspect their learning progress and respond to challenges. This skill is often known as metacognition . ConventMetacognition can be assessed by judgements of learning (JOL), which investigates people’s self-awareness on how well they remember or understand the given materials. Typically, in the cued-recall test consisting of multiple word pairs , making JOLs after the presentation of each word pair facilitates “the associative relationship” and meaning-making “between the cue and target”, leading to improved recall performance of STM and LTM compared to many who did not . In partCohen et al. and Logan et al. suggest that students’ decision to make JOLs under two repetition conditions depend on cognitive bias and complex issues ,35. TheiSince the memory encoding process during the initial presentation of items is weak, preference for massed learning may also contribute to the need to increase perceptual retrieval strength to avoid forgetting . Cohen eSimilarly, Kornell and Bjork conducted a study in which participants were asked to pair artwork with the artist’s name through recognition and recall tests, after studying artworks from a particular artist continuously (massed) or mixing them with others’ artworks (spaced) . AlthougAffective Processing in Spaced LearningAs mentioned earlier, repetition priming (massed conditioning) tends to reduce the effort of LTM encoding since the initial representation of items may create ease of processing at their next occurrence . Under tThe association of these factors generates a negative feedback loop on the memory consolidation of study items, which has real-life implications. Students tend to take the study content for granted (priming) while focusing their attention only on its physical property, undermining its intrinsic affective/semantic quality, which in turn impairs both immediate behavioural feedback and extended response (spacing effect) to materials they once could easily process . In respSpacing and Critical ThinkingBased on critical reflection about factual content through different lenses, students may be more equipped to judge how they would gain conscious control of these “lenses”. Currently, not much research has been conducted on the relationship between spacing and critical thinking. In a study conducted by Foot-Seymour et al., students were taught to provide a critical website evaluation within a few days (massed) or weeks (spaced), with a set of given guidelines . Not surEducational relevance in science educationEffectiveness of Spacing in Science EducationThe (meta)cognitive advantages associated with spaced repetition are demonstrated in the experimental context. However, more research is needed to expand the theoretical basis for applying spaced learning in complex educational settings, especially in science learning. This paper incorporates mathematics (problem-solving) and biology  to represent science curricula.First, Kelley and Whatson suggest that the application of spaced learning allows biology courses to deliver concepts at an extraordinary speed and accelerate learning efficiency without compromising students’ attention and cognitive capacity . This isSuch curriculum design can improve learning efficiency during memory encoding of advanced concepts. Still, consolidation and maintenance of these memories in spaced repetition could be more time-consuming as students need to regularly revisit these difficult concepts. Kerfoot discovered a parallel relationship between study time and learning efficiency in biomedical education . CompareIn mathematics education, it is no surprise that the benefit of spaced learning is resistant to forgetting. Despite its many advantages, there grows up a controversial issue on whether massed mathematical practice must be replaced entirely by spaced repetition or not ,5,41. InInterleaved PracticeBased on the above suggestions, educators and students in mathematics education must be cautious not to rely exclusively on the overarching principle of spaced repetition, thereby disregarding alternative education approaches that might be more appropriate for specific subjects. Interleaved practice is a derivative principle of spaced repetition, a strategy for rearranging problem sets designed for one topic into multiple topics in each course material/assignment  It juxtaposes “different kinds of problems in an interleaved order” for each assignment so that “spacing intervals between consecutive problems of the same kind might expand” . Hence, Micro-LearningAnother derivative practice embedded in spaced repetition mechanisms is micro-learning, which operates by breaking a large chunk of information into smaller learning units with the knowledge required  to prevent mental fatigue and memory interference . LearnerThrough curriculum design and mobile device assistance, a few derivative spaced practices inspired by the spacing effect have been demonstrated in the real pedagogical application, to strengthen memory storage of conceptual knowledge and comprehensive skill modelling. Additionally, due to the growing popularity of educational technology, it would be ideal for further research to take an open-source application and change the spaced timing structures based on psychological evidence or academic recommendations made by educator-researchers, then conduct experiments to evaluate its effectiveness. This would allow for both within- or between-subjects designs.Barriers of Spaced Practices in EducationFor researchers, the advantages of the spacing effect are becoming increasingly evident; however, many overlook its practical values in the curricula . This phThe foundation for the cramming phenomenon is embedded in the curriculum design dominating conventional curricula, which illuminates the impact of massed practices on classroom issues such as the amount of homework, test frequency, teacher feedback, and level of mastery . When deThrough the implementation of a review session, it can be seen that some educators hold a certain degree of understanding of the spacing effect, regardless of whether they may like it. However, lack of institutional support and resources hinders their desire to further integrate spaced practices in the pedagogy. Lindsey et al. argue that providing and planning for spaced practices in the classroom “is beyond what any teacher or student can reasonably arrange” . In mostNowadays, the validity of textbooks with spaced repetition is still debated. For example, the learning objectives of Saxon texts, which primarily use interleaved practice, are criticized by many educators on their learning objectives, with one research report determining that Saxon texts do not certainly enhance test performance . This isFuture Application and Research of Spacing Effect in Science CurriculaOverall, even though the spacing effect reveals its important theoretical and practical values in learning, its utility in conventional education and teacher practices has not become evident. It would also be a waste of effort to completely disprove the functionality of massed practice and is against present research findings suggesting that some degree of massing is necessary for learning biology and mathematics. So this paper requests conventional science curricula to establish smart goals for the stepwise development of spaced practices. The first suggestion is for educators to revisit the content repeatedly over the duration of the course, so each consecutive repetition will increase the depth of knowledge and allow new perspectives to emerge. Next, the paper calls for the incorporation of multimodal educational resources in the curricula, be it digital tools such as mobile and computer devices with adaptive algorithms, MOOCs, websites/applications with customized “flashcards” features based on prior recall accuracy, and interleaved textbooks with rearranged assignment questions (more research is needed). Ultimately, these resources could be an excellent asset for spacing to operate in a friendly and cost-effective way, potentially reducing pressure on teachers and making spacing a more rewarding experience.Future research is needed to discover and evaluate potential strategies to illustrate the spaced repetition principle in science learning. It is the work of curriculum design research to help prepare classrooms that appropriately reflect the utilization of the spacing effect. A simpler suggestion and easier-to-implement experimental design would be to structure a course module or modules of what we know about the spacing effect and compare student performance at the designated time with that of a standard blocked approach, which would take minimal effort and provide significant data and evidence.Future research in spaced repetition also needs to generate a complete knowledge of the diverse student groups’ learning experiences. The research could proceed with an extensive qualitative analysis beyond the quantitative methodologies. A mixed-method design might be of particular interest. These approaches may provide a thorough contextualized investigation of the current spaced practices, accounting for the needs of students who require additional support based on their narratives. Also, mixed-method research would help create better metrics for assessing the qualities of spaced practices in science education.Also, research of spacing effect was predominantly conducted by controlling for students’ baseline knowledge proficiency to differentiate the effectiveness of spaced and massed practice in learning. It rarely assesses individual capabilities for those with learning difficulties and challenges. Under the circumstances, a standardized spaced educational approach - preserving the same volume of course load and examinations for each student - might be less effective than scaffolding. More research needs to tackle spaced repetition in a personalized domain, such as adaptive spaced schedules.Furthermore, research in spaced repetition should incorporate multiple behavioural and sociopolitical determinants to its efficacy on learning and memory. Intervening variables such as post-learning mental activities, sleep functions and circadian rhythms, and use of technology could be examined as the independent variables in examining spaced learning outcomes, as retention intervals loaded with intensive cognitive activities will significantly increase the likelihood of forgetting. It is also of utmost importance to connect research findings on spaced learning to education policymaking. Yet, it might require a macro-level and constructivist interpretation of societies and ideologies.Spaced repetition is a fruitful tool for facilitating conceptual and reflective learning skills. Yet, its benefit is mainly exemplified in experimental settings and self-regulated learning. The paper demonstrates that the spacing effect can significantly improve learning, but not the judgement of learning. Together, the paper highlights some important insights. First, the spacing effect promotes LTM consolidation. Next, spaced practices result in a more robust enhancement of LTM encoding, recall, and recollection through both direct and indirect effects of memory processing characteristics. Contrastingly, massed practices increase instantaneous (STM) retrieval strength but reduce LTM encoding. Additionally, there are clear advantages to using spaced instruction in science curricula. However, as the cramming phenomenon becomes a norm, negative judgement of the credibility of spaced practices continues to prevail among many learners and educators, even though they might have somehow engaged in it through unconventional forms of learning, such as electronic technology, with or without realizing its advantages. However, this paper does not attempt to advocate that blocked practices should be replaced by pure spacing in science curricula, due to the nature of science learning and the complexity in the interplay between theory, practice, policy, and administration. Consequently, this paper suggests many collaborative research opportunities in a multitude of research areas, including cognitive and computer science, socio-political studies, and educational studies. The ultimate goal is not to speak against a certain traditional practice, but to promote a greater societal awareness of long-term learning growth and break down the obstacles to equitable science education for all students.This paper is not without limitations. First, its scope is restricted in a theoretical account since it does not identify complexities in education policymaking and teacher training for spaced practice, as the spacing effect may not apply in every situation. Besides, the criteria for denoting cognitive processing characteristics unique for science education should be extended to different mental measurements beyond memory strength, since memory is also essential to other areas of education such as language, art, and humanities. Additional research findings should also explore the interplay between the spacing effect and interpretative learning skills, rather than the “memory consolidation” of interpretative skills. Finally, spaced learning may not always be suitable in every condition in science education, thereby lumping biology and mathematics together as science education that uses different sets of reasoning skills, while claiming the effectiveness of spacing effect underestimates the complexity of learning each subject in real life.