Recently, an increased trend toward non-drinking among adolescents has been observed in several countries. The aim of the present study is to evaluate a common suggestion in literature, that adolescents do not drink alcohol because they spend more time on the internet, monitored at home, by examining associations between internet activities  and non-drinking.A health questionnaire was distributed to all 9th graders (15–16 years) in a mid-sized Swedish county in 2008, 2010 and 2012. In total, 7089 students returned the questionnaire.In contrast to the suggestion, no association was found between total time spent on computers and non-drinking. Social media/chatting was robustly associated with a decreased probability of non-drinking across the three survey years. On the other hand, computer gaming during weekends only  or both on weekdays and weekends increased the probability of non-drinking  in 2012 only. However, neither social media/chatting nor computer gaming was associated with the increased trend of non-drinking from 2008 to 2012.Internet activities were in general not associated with non-drinking among adolescents aged 15–16 years in Sweden. Although, a weak positive association between computer gaming and non-drinking was found in 2012, this effect benefited the vast majority of the boys. The larger alcohol use among those with extensive social media use/chatting may indicate that these online platforms are arenas where adolescents are exposed for positive alcohol preferences and alcohol advertising without parental supervision. The suggested mechanism is that adolescents do not drink alcohol because they spend more time on the internet in domestic environments that are more easily monitored by parents.The aim of the present study is to examine whether different internet activities, including total time spent on computers, social media use and computer gaming, are associated with non-drinking in a large population-based sample of 7089 Swedish 9th graders (15–16 years). Further, differences in these associations between the years 2008 and 2012 and between boys and girls are evaluated.Data from the Survey of Adolescent Life in Vestmanland (SALVe) were used. This health survey is distributed biannually to all students in 9th grade (15–16 years) in the county of Västmanland, a mid-sized county in Sweden. This study used data from the surveys distributed in 2008, 2010 and 2012, since these surveys included detailed questions about computer and internet habits. In total, 2605 (50.4% girls) responded in 2008, 2439 (51.7% girls) responded in 2010 and 2045 (49.7% girls) responded in 2012. Missing data were attributed to participants who were absent, failed to return the questionnaire or failed to state their sex.All students in 9th grade in the county of Västmanland were asked to complete the questionnaire during class hours. Participation was anonymous and voluntary. The present study followed the Swedish guidelines of social science and humanities, in accordance with the Declaration of Helsinki. Under Swedish law , this type of anonymous study does not require ethical approval by the medical faculty. However, an advisory statement from the regional ethical board of Uppsala was requested for the surveys distributed in 2006 and 2014. The surveys were approved at both occasions.Covariates included country of birth, family substance problems, conflicts between parents, physical abuse, number of friends, status among friends, psychosomatic problems, sleeping problems, use of cannabis and use of other illicit drugs. Initially, covariates also included socio-economic position, domestic violence toward partner or sibling, psychological abuse and depressive symptoms. However, these were not associated with non-drinking and therefore excluded. Country of birth was classified as Sweden (student and both of their parents were born in Sweden), Europe (student or one of their parents was born elsewhere in Europe or North America), or Outside Europe (student or one of their parents was born outside Europe or North America). Family substance problems were coded if the student rated one family member as having a problem with substance use. Conflicts between parents and physical abuse were coded if present. More detailed information on family substance problems, conflicts between parents and physical abuse has been published elsewhere.Alcohol consumption was measured with a version of the AUDIT-C questionnaire modified for adolescents.Internet activities included total computer time, time spent on social media/chatting and time spent using computer games. Total computer time was measured with one item on the total number of hours per day spent on computers (not school related). Social media/chatting was measured with one item on how many hours per day on a five-category scale  the student usually spent chatting on the internet/social media sites. Computer gaming was measured with a summarized score from two items on how many hours per day on a five-category scale  the student usually played online computer games or other games on the internet on weekdays and on weekends, respectively. More detailed information on computer gaming has been published elsewhere.First, in order to identify relevant covariates, bivariate logistic regression analyses were conducted calculating OR and 95% CI with non-drinking (no/yes) as the dependent variable (not shown in tables). Second, the distributions of internet activities and non-drinking from 2008 to 2012 were calculated separately for boys and girls. Third, bivariate associations between internet and non-drinking were calculated separately for boys and girls (not shown in tables), to evaluate whether associations differed. Since associations were similar for boys and girls, subsequent analyses were conducted on the total sample. Fourth, multivariate logistic regression analyses were conducted calculating OR and 95% CI with non-drinking (no/yes) as dependent variable and internet activities as independent variables adjusted for covariates. Analyses were conducted for each survey year in three models, where the first model included total computer time, the second model included internet activities  and the third model divided computer gaming into weekdays and weekends. Fifth, in order to capture whether computer gaming on weekdays and/or on weekends was associated with non-drinking, computer gaming was divided into playing on weekdays only, on weekends only, and on both weekdays and weekends, in order to assess which of these activities, if any, was associated with non-drinking. Finally, differences between survey years and between boys and girls were evaluated as interaction effects. All analyses were conducted in SPSS version 22.0.The distribution of internet activities and non-drinkers among students in 9th grade during the period 2008–2012 is shown in Internet activities were associated with non-drinking in bivariate models (not shown in tables). The total computer time decreased the probability of non-drinking among both boys  and girls . Social media/chatting decreased the probability of non-drinking among both boys  and girls , whereas computer gaming increased the probability of non-drinking among both boys  and girls . Since associations were similar for boys and girls, subsequent analyses were conducted on a gender-pooled sample.Next, three multivariate models shown in n = 35.4%, girls n = 84.3%); ≥2 hours/day on weekdays only ; ≥2 hours on weekends only ; and ≥2 hours on both weekdays and weekends . Associations between these groups and non-drinking, adjusted for covariates, are shown in However, adolescents can engage in computer gaming on both weekdays and weekends. Thus, we divided them into four groups: <2 hours/day on weekdays and weekends  in a mid-sized county in Sweden, allowing us to study differences between survey years and gender differences. Another strength is that different types of internet activities were addressed. Limitations of the study include the cross-sectional design, precluding any interpretations about causality, and that data were not available for non-responders, making it difficult to interpret how non-responders influenced the investigated associations. Further, surveys distributed in 2008–2012 were used, which is a limited time period of 4 years. Thus, valid conclusions about whether computer gaming has contributed to the increased trend toward non-drinking that has occurred since 2000 cannot be done. Furthermore, the present study do not take other competing trends into consideration such as alcohol policy changes, changes in parental norms of offspring drinking,In general, total computer time is not associated with non-drinking among adolescents. Although computer gaming in particular during weekends is associated with an increased probability for non-drinking in 2012, the effect size is relatively small and therefore not likely to contribute more than marginally to the increased trend toward non-drinking. On the other hand, the proportion of boys that benefits from this small effect size is large. Further, chatting/social media use was associated with a decreased probability of non-drinking. Thus, these online platforms may constitute forums where adolescents are influenced by positive alcohol references and online alcohol marketing, without parental scrutiny.This study was funded by grants from the Swedish Research Council for Health, Working Life and Welfare (#2015-00857) and the Systembolaget’s Research Council on Alcohol (#FO2016-0073).Conflicts of interest: The present study received funding from Systembolaget’s Research Council on alcohol (#FO2016-0073). While Systembolaget is the national alcohol monopoly company in Sweden, the funders played no role in the study design, analysis or interpretation, or in the decision of where to submit the paper for publication.The present study does not confirm the suggestion that adolescents do not drink alcohol because they spend more time on internet.Computer gaming in particular during weekends was associated with non-drinking. Although a small effect size, the majority of boys benefits from this small effect size.The time spent on social media/chatting was robustly associated with decreased odds of non-drinking across the three survey years, with a stronger association among girls than aming boys.


In this paper I argue that, by combining eliminativist and fictionalist approaches toward the sub-personal representational posits of predictive processing, we arrive at an empirically robust and yet metaphysically innocuous cognitive scientific framework. I begin the paper by providing a non-representational account of the five key posits of predictive processing . Then, I motivate a fictionalist approach toward the remaining indispensable representational posits of predictive processing, and explain how representation can play an epistemologically indispensable role within predictive processing explanations without thereby requiring that representation metaphysically exists. Finally, I outline four consequences of accepting this approach and explain why they are beneficial: (1) we arrive at a victory for metaphysical eliminativism in the ‘representation wars’; (2) my account fits with extant empirical practice; (3) my account provides guidance for future research; and, (4) my account provides the beginnings of a response to Mark Sprevak’s IBE problem for fictionalist approaches toward sub-personal representation. One of the chief philosophical exponents of this framework, Andy Clark, has recently claimed that predictive processing resolves the debates surrounding the concept “representation” and its relevance to work in cognitive science. According to Clark:The paper itself is structured as follows—in Sect. action-oriented because it pertains to the perception of environmental affordances  framework, the brain is considered to be an inference machine which uses approximations of Bayesian probability theory to maintain its own viability. PP explains action and perception to be driven by one simple tenet: minimise prediction error. Prediction error-signals signal that a given prediction is false—the sensory signal received by the brain was not the sensory signal which it predicted would be received. By minimising prediction error, the brain should be led to situations which allow it to maintain itself within prescribed homeostatic boundaries. PP understands perception to be constituted by the brain’s ‘best guess’ about the ultimate causes of its current sensory states. Importantly, this ‘best guess’ is thought to be fundamentally ed Clark . Therefoposterior probability. Posterior probability is the overall probability assigned to a hypothesis (given relevant information) and it is calculated by multiplying the hypothesis’ prior probability and its likelihood, and then dividing this amount by the prior probability that the information on which these calculations are based is correct. The prior probability is the probability given to the hypothesis independently of current events. For example, the prior probability of my being perceptually presented with a cat is quite high, because there are many cats living in my street. The prior probability of my being presented with a leopard, however, is quite low. Leopards are rarely, if ever, to be found in my street. The likelihood of a hypothesis can be calculated as follows—assume the truth of the hypothesis in question, and then determine the probability of the evidence actually acquired being collected if that hypothesis were true. For example, if I encounter a cat in my street then the hypothesis “I am perceiving a cat” will be accorded a high likelihood because my sensory stimulation is highly probable in the context of this hypothesis. Similarly, if I happen to confront an escaped leopard in my street, the hypothesis “there is a leopard in front of me” would be accorded a high likelihood. In both cases, the hypothesis in question is highly likely because the sensory stimulation received is highly probable given the hypothesis.2The brain’s ‘best inference’ will be the hypothesis which has the highest overall job-description challenge. I will explain why these PP posits do not meet the job-description challenge, and so conclude that the key posits of PP can be accepted without thereby requiring adoption of representation.Having outlined the key posits of PP  I am now going to explain how each can be satisfactorily explained without invoking the concept “representation”. I will do so by drawing on arguments in this vein recently proffered by Orlandi  and explWhen it is used in cognitive science, the concept “representation” is considered to be a psychological notion which requires the invocation of properties such as “aboutness”, “truth-conditions”, and “content”. These properties are not typically thought to exist in non-cognitive physical entities, such as stones or trees. As such, they are to be considered special, higher-level properties, which are not ubiquitous in the natural world. Accordingly, we must have good reasons to describe a given cognitive mechanism or system in terms of representation.William Ramsey has recently argued that we can only ascribe “representation” to a cognitive mechanism or system if it passes “the job-description challenge” . If a meIn short, “representation” (as it is used in cognitive science) is a psychological concept which is not ubiquitous throughout physical systems in nature. If we are to maintain a robust and empirically useful notion of representation then, according to Ramsey, a given cognitive mechanism should only be described in terms of representation if it deserves to be described in such terms. He suggests that we assess whether a mechanism does deserve to be described in terms of representation by submitting it to the job-description challenge. Ramsey argues that a cognitive mechanism should be described in terms of representation only if it passes this challenge.that such-and-such is the case, whilst errors signal that such-and-such is not the case. Nico Orlandi argues, however, that closer inspection of the role these terms play in PP explanations reveals they are not representational posits  is described as performing various cognitive tasks by employing a structure that has the job of causing something to occur when and only when something else occurs, then the system is not, on the basis of this description alone, employing internal representations. .Despite its common appeal, the receptor notion of representation [Ramsey’s name for covariation based accounts] comes with a job description that, in this context, has little to do with the role of representation...When we look at the role of receptors Ramsey’s argument, in essence, is that we gain no extra explanatory purchase by treating causal covariation within the brain in terms of representation. We do not arrive at better accounts of neural processing by giving causal covariation between neurons a representational status, because treating them as such does not provide one with any extra explanatory benefits over those one would accrue by treating them as mere non-representational causal correlations. Furthermore, treating causal covariation in terms of representation violates our pre-theoretic use of the concept. Therefore, causal correlation between neural processes fails the job-description challenge and so does not deserve a representational status.Predictions are present at all levels of the perceptual hierarchy and are passed down to the level immediately below their level of origin. Error-signals, on the other hand, are passed up from their level of origin to the level immediately above it. As such, each is concerned only with The universe is stuffed with correlations and it is implausible to count them all as representations . We agree, but note that the correlations between, for example, specific brain states and color perception look to fall onto the intuitively acceptable side of such a divide.  and has proven evolutionarily beneficial , then it should be described in terms of representation.The proponent of representation is likely to object, however, along the following lines:x would be taken to represent x because they were selected for by evolution to respond to it. If the neural states happen to co-vary with y, where y is a non-natural stimulus introduced in the lab, then the neural states will misrepresent because natural selection did not select them for signalling y. Teleosemantics requires ascribing representation by determining the function of a given mechanism, with this function determined in turn by considering what the mechanism itself was selected to do by the forces of natural selection. It could therefore be argued that covariation between neural processing is representational because it has been selected for by the biological forces of natural selection.One could, for example, argue for a teleosemantic account, upon which representation is thought to occur when there is causal covariation which has been selected for by evolution because it performs a fitness enhancing role Dretske . On thisButtressing the concepts of “prediction-signal” and “error-signal” with teleosemantics, however, is not going to help these concepts pass the job-description challenge. Both prediction and error signals are concerned only with proximal conditions, and the functioning of both can therefore be adequately accounted for without invoking the concept “representation”. Consequently, applying teleosemantics to this particular example will not help with the job-description challenge, because teleosemantics is concerned primarily with the content of a given representation. Applying teleosemantics to a given mechanism involves discerning when representation and mis-representation occur. As such, teleosemantics is only applicable to scenarios in which the concept of “representation” has already been applied—the application of teleosemantics to a mechanism requires the assumption that a given instance of causal covariation is representational, and thereafter attempts to naturalise the particular content of that representation. The theory will not, therefore, help one in determining whether or not a given mechanism deserves to be described in terms of representation to begin with. In short, the theory of teleosemantics is only applicable once a given mechanism has already passed the job-description challenge. Consequently, teleosemantics cannot be used in an argument for the claim that instances of causal covariation pass the job-description challenge Understanding perceptual priors, hyperpriors and likelihoods as biases means thinking that, as a result of repeated exposure to the structure of the world in the evolutionary past She argues that theorists are tempted to explain biases in terms of representation largely because they are in the grips of the traditional cognitivist idea that perception is to be understood in terms of internal inferential transitions between premises and conclusions in some kind of language of thought , a large bowl at the bottom, and a pumping mechanism which connects the bottom bowl to the top. The pump plays a biasing role within the fountain by ensuring that the vast majority of water in the fountain stays pooled in the bottom bowl, with only a small amount being pumped back up to the fountain top at any given time. We would not be at all tempted to ascribe the concept of “representation” to the functioning of this pump, and this is presumably because the ascription of representation to this pump fails the job-description challenge—describing the pump’s biasing role in terms of representation does not provide one with any explanatory benefits over and above those one would accrue by simply treating it as a mere non-representational bias in a water fountain system.Orlandi contends that a similar conclusion should be drawn in the case of the PP concepts “prior” and “likelihood”. She argues that these concepts should be taken to refer to certain biases within a neuronal system, and that one should not treat these biases in terms of representation because there is no explanatory benefit in doing so. Consequently, Orlandi concludes that the concepts “prior” and “likelihood” are better understood as referring to mechanisms which pre-dispose brains to configure themselves into specific organisational patterns in response to environmental stimulation. Once more, the argument I am presenting here is not that biases cannot be understood in terms of representation. Rather, it is that their functioning can be understood entirely without invoking representation, and that treating them in representational terms is not explanatorily beneficial. Biases do not pass the job-description challenge, and so we have no reason to treat them in terms of representation. Therefore, I conclude that the PP concepts “prior” and “likelihood” should be taken to describe non-representational biasing processes occurring within the neural system.4[R]epresentations are only those performance-guiding structures that are de-coupled from their causes, where this fact materialises in their standing for distal or absent conditions. Orlandi , p. 133.Orlandi claims that the winning hypothesis is concerned with distal conditions because it is formulated on the basis of sensory information received by the brain  and yet is itself about things beyond brain-based sensory receptors (such as cats and leopards). She argues that the winning hypothesis is de-coupleable from its environmental causes because it can be deployed even in the absence of environmental causes. Finally, she argues that the winning hypothesis deserves to be treated in terms of representation because it is used by the brain to reason with and plan action. Consequently, according to Orlandi, although PP processing itself does not deserve a representational status the result of this processing does. I am inclined to reject Orlandi’s claim because I believe that it begs-the-question on two crucial points: (1) its presumes that cognition is concerned with distal, and not proximal, states-of-affairs; and, (2) it rests on an assumption of the problematic representation demarcation thesis.Consider, finally, the concept “posterior probability”. Although Orlandi argues for a non-representational stance toward the processes underlying PP, she concludes that the results of this processing  do deserve to be described in representational terms. Orlandi arrives at this conclusion because she thinks that the winning hypothesis fulfils the three conditions which she takes to be both necessary and sufficient for the ascription of “representation”:assumption that cognition is brain-bound: if cognition is brain-bound, then it follows that it will be concerned with distal states of affairs (events beyond the brain) and that internal cognitive states will be de-coupleable from their causes.Cognitivist theorists tend to assume that, in resolving a given cognitive task, the organism is restricted to the use of information and resources contained within the brain. Indeed, it is primarily for this reason that cognitivists invoke the concept of “representation” within their explanations at all: environmental input alone is generally considered too impoverished to explain successful cognition, and yet organisms do nevertheless successfully cognise. Cognitivists typically assert that the environmental poverty of stimulus for a given cognitive task is ameliorated via the presence of brain-based representations Chomsky . Thus, “performative and temporally extended aspects of cognition will lead one to the realisation that there is no poverty of the stimulus in most cognitive domains , one guarantees that no instance of cognition will ever be non-representational. Either we must find a representational explanation of the cognitive activity in question, or, it will not count as a cognitive activity at all. Ramsey thus rejects RDT because it requires cognitive science accept a priori constraints on its domain of study and he thinks that no serious science should accept such constraints. His second reason for rejecting RDT is that RDT undermines the empirical status of the representational theory of mind. “Representation” is proposed as a theoretical posit which is supposed to play an empirical role in providing an empirical explanation of cognition. If, however, one accepts RDT, the cognitivist research programme within cognitive science no longer looks to be empirical in nature. Representation is not being posited for empirical reasons, because it plays an important role in an empirical theory of cognition. Rather, it is proposed for conceptual reasons, because it is a priori assumed that any empirical theory of cognition must be a theory of representation. Representation thereby becomes an unfalsifiable theoretical posit, and so the cognitivist research programme loses its empirical credentials. Consequently, Ramsey’s second reason for rejecting RDT is that it requires an unscientific approach to cognitive science. Finally, Ramsey rejects RDT because it encourages a wildly deflationary understanding of representation, such that even mere causal mediation or correlation is considered to be sufficient for representation ; and, the RDT. Enactive and ecological versions of PP can accept that the ‘winning hypothesis’ forms the categorical basis of personal level cognition without thereby taking it be concerned with distal events or to be de-coupleable from its causes. Similarly, although such accounts could agree that the ‘winning hypothesis’ is causally implicated in guiding and executing action, concluding that it deserves a representational status on this basis alone is misguided, because reliant on acceptance of RDT. Therefore, the concept of “posterior probability” can satisfactorily be explained within PP accounts without thereby accepting representation.At this point, we have arrived at a non-representational version of PP. “Prediction-signal”, “error-signal”, “prior”, “likelihood”, and “posterior probability” have all been described in non-representational terms. Importantly, no explanatorily beneficial insight was lost by describing these concepts in such non-representational terms. Having explained how the key PP posits can be understood in non-representational terms, I will now turn to explaining how even PP explanations which make indispensable use of representation can be accommodated within an eliminativist framework. I will argue that such representational posits are indispensable for epistemological, and not metaphysical, reasons.requires representation—even though we can provide a non-representational construal of PP’s key posits, much of the literature on PP simply cannot be made sense of without invoking the concept “representation”. The following quote, from Andy Clark, is representative of this general attitude:Could we perhaps have told our story in entirely non-representational terms? One should always be aware of sweeping assertions about what might, one day, be explanatorily possible! But as things stand, I simply do not see how this is to be achieved. Clark , p. 293.I am willing to concede that there are some PP explanations which do indispensably require the concept of representation. However, I will argue that, even when the concept is indispensable, we are not required to renege on the eliminativist conclusion of the previous section. My reason for making this claim is that I think representation is explanatorily indispensable in some PP explanations for epistemological, and not metaphysical, reasons. In this section I am going to apply a make-believe approach to scientific models to PP, and thereby explain how representational posits could be epistemologically indispensable to working scientists and yet metaphysically non-existent. I will conclude that representation can in some instances play an epistemologically indispensable role in PP models, but I will not thereby conclude that in such cases representation metaphysically exists.PP theorists are likely to object to the argument outlined above, because much of the work carried out within the PP research paradigm Realismx exists.Fictionalismx does not exist. However, it plays a useful explanatory role and so can be treated as if it exists.Eliminativismx does not exist.Fictionalism about a given posit is often adopted by theorists when there is reason to believe the posit is ontologically suspect, and yet the posit itself is explanatorily indispensable. It is, for example, a popular position within debates about the metaphysics of modality and mathematics. In the case of mathematics, although it is difficult to envisage everyday human practice that does not make reference to mathematical objects, mathematical objects themselves are ontologically suspect . Adopting fictionalism about mathematical entities enables one to keep them within one’s ontology whilst avoiding having to explain how they could causally interact with physical objects.When concerned with ontological matters, there are (roughly) three attitudes one can take toward the entity in question:6We have seen that the key posits of PP can all be explained without accepting or requiring representation. Nevertheless, it is arguable that much work on PP makes indispensable use of representational content Clark . Fiction7A number of theorists have recently applied Kendall Walton’s theory of pretence to the case of fictionalism about scientific models. They have argued that many scientific models provide theorists with prescriptions to imagine certain scenarios, and that the results of these imaginings allow for predictive and explanatory utility without committing theorists to the literal existence of each posit in their model. In this sub-section I introduce and motivate this approach by outlining Adam Toon’s work on the topic. Then I draw on the work of Arnon Levy, and explain how the application of representations to the brain (at least in the case of predictive processing) can be shown to be nothing more than metaphorical talk which is used by cognitive scientists as an instrumentally useful tool. I conclude that the indispensability of representation for PP models does not require commitment to the literal existence of representations. It is wholly compatible with the practice of PP that cognitive scientists are using representational talk only as a metaphorical tool which allows them to keep track of real causal interactions within the brain.Walton’s  ‘mimesisThe bubble machine counts as the Death Star.The bubbles generated count as escaping space-ships.The limits of the back garden, signified by the fence, count as the limits of the Death Star’s reach.From these principles a number of sub-principles obviously follow. For example, a corollary of (2) is that:The larger the bubble, the bigger the spaceship is.Bubbles which pop count as destroyed.Bubbles which float over the garden fence count as having escaped.Although the child is engaged in a game of imaginative make-believe, the things which happen in the game depend entirely on things which happen in the actual world. For example, the fate of a given space-ship depends entirely on the fate of a bubble in the actual world. Its fate cannot be decided by the whims of the child. Consequently, the child is able to accurately track events which occur in the real world. For instance, they can track how many bubbles float past the garden fence.Walton argues that fictions should be seen as analogous to the sorts of make-believe games which are played by children. These games are often based on what Walton labels “generative principles”, which are the rules upon which the imaginative game is based. Consider the example of a child playing a game with a bubble machine in their garden. In this game the bubbles are imagined to be space-ships which have escaped from the Death Star, and they must make their way home without being destroyed. Given the rules of this imaginative game, a number of generative principles follow:ibid., pp. 654–655):Directionality information is transmitted from a sender to a receiver.Connecting variation transmitted information will cause changes in the receiver .Active versus passive distinction this is a metaphor, which gets at the difference between the parts of the system which change and the parts that do not. The metaphor “active” refers to the changes of state in sender and receiver (of information), whilst the metaphor “passive” refers to the parts of the system which do not change (the information itself).Levy does note that an argument similar to his own could be given about the use of information talk in neuroscience. However, he does not himself provide such an argument. In the next sub-section I will apply Levy’s three rules to the case of predictive processing.Levy  argues tI begin my application of Levy’s informational principles to the brain by considering the simplest component of the brain  considered relevant for our ability to engage in cognitive activities—the neuron. A neuron is a cell which is composed of three parts: dendrites, soma (or cell body), and axon Fig. .Fig. 1A Neurons both receive and pass on information in the form of electrical and chemical signals. Electrical and chemical signals are passed across the synaptic cleft (the gap between the axon of one neuron and the dendrite of another) and picked up by the dendrites. These chemical and electrical signals move into the soma of the neuron, and when they reach a certain concentration (the exact concentration can vary from cell to cell) an action potential will send electrical signals down the axon and cause the neuron to fire. When a neuron fires it passes electrical and chemical signals from its axon to the dendrites of other cells.9At this neurophysiological level of description Levy’s three rules are satisfied. Directionality is satisfied because signals pass from one neuron to another (from the axon of one to the dendrites of another). Connecting variation is also satisfied, because the electrical and chemical constitution of the soma of the receiving neuron will change in response to the electrical and chemical signals received from the neuron which passed on the signal. Finally, the metaphorical distinction between active and passive is also satisfied. Although the electrical and chemical constitution of the two neurons will change, the information being transmitted remains the same. This information is constituted by informational signals and these remain unchanged whilst they pass across the synapse. At this level of description there is no need for the use of representational posits. Indeed, the only predicate in use which is remotely psychological is the term “signal”. However, this term refers to electrical and chemical properties and so can be seen as straightforwardly metaphorical. It can be translated into the language of chemistry and physics with no loss in explanatory power.In order to consider a second model of neural processing, I will now ascend a few levels of description:10This diagram depicts the mechanism through which the passing and regulation of top-down and bottom-up signals occurs between cognitive (top box) and sensory (bottom box) areas of the brain.At this higher level of description, which makes reference to groups of neurons in different regions of the brain, Levy’s rules are still satisfied. Neurons in the bottom half of each box are designated as the senders of top-down signals whilst neurons in the top half of each box receive these signals. Similarly, neurons in the top half of each box are designated as the senders of bottom-up information whilst neurons in the bottom half are designated as receivers. Consequently, directionality is satisfied. Connection variation is also satisfied because the neurons  will change in response to the signal received. The nature of this change is more complicated than the previous example of the lone neuron. This is because the exact manner in which the neurons change is now dependent on an inter-play between top-down and bottom-up signalling. However, the fact that neurons will change state in response to the signals they receive remains transparent. Finally, the active and passive metaphor is still satisfied. Deep layer neurons are associated with beta-range oscillations, and superficial layer neurons with gamma-range oscillations. As such, the oscillations they rely on to transmit information and the type of information they transmit never changes, although the states of the neurons themselves will change. Although we have ascended to the level of many neurons which are situated in spatially separated parts of the brain, this description of brain processes is clearly firmly rooted in what we can observe and understand using the tools and framework of neurophysiology. If any psychological predicates are used at this level of explanation it is clear they are being used metaphorically.I will now consider a third diagram. This diagram is also concerned with top-down and bottom-up neural interactions. However, it is pitched at a slightly higher level of abstraction than the previous diagram:This diagram is a representation of how the predictive processing paradigm conceives of the brain. R1 through to R3 represent different levels of the brain, from superficial (R1) to deep (R3). The green lines are the same top-down signals which are linked with beta-range oscillations that we encountered in Fig. Toon defines his ‘models as make-believe’ [MM] account as follows:MM: M is a model representation if and only if M functions as a prop in a game of make-believe. Toon , p. 62I have argued that the PP paradigm adheres to the ‘brain-as-representational-Bayesian-machine’ game of make-believe. In this particular case the brain is the prop upon which the game of make-believe is predicated (upon which the various ‘generative principles’ are based). I studied three diagrams of neuronal processing. Each successive diagram represented the brain at a higher level of abstraction, and as the level of abstraction increased so did the use of representational idioms. However, every single diagram satisfied Levy’s three principles in the same manner. The exact same causal changes were modelled in each diagram with the only difference being that, as the models became more abstract, the causal changes were modelled in more generality. The neuroscientist is able to ascend from the level of individual neurons, and instead model interactions between groups of neurons , by introducing representational predicates. This practice allows the scientist to model real causal processes without having to concern themselves with the minutiae of detail which consideration of the brain as a whole would require. Rather than being seen as pointing to the metaphysical indispensability of brain-based representations, the introduction of representational idioms can be seen as nothing more than a metaphorical and fictional posit which allows the neuroscientist to keep track of real causal changes occurring at the neurobiological level. Treating the brain as if it is a representational prediction machine prescribes certain imaginative games, which both help and allow the neuroscientist to track real causal changes occurring within the brain at a greater level of generality.Although this model makes use of a lot more representational idioms than any of the previous diagrams, it is obvious that Levy’s rules are still being satisfied. The exact same directionality noted in Fig. In this paper I have argued that: the key posits of PP need not be described in terms of representation; and, PP accounts which make indispensable use of representation can be understood as doing so for epistemological reasons. If the forgoing is correct, within the PP framework, representation is either rendered entirely otiose or it is assigned a fictional status. This conclusion licenses an eliminativist conclusion about the metaphysical status of representation within PP. In this section, I am going to outline four benefits one arrives at by adopting the approach toward representation which I have sketched.hard problem of content .What we normally think of as representational contents—contents defined on distal objects and properties appropriate to the cognitive domain —are not in the theory; they are in the explanatory gloss that accompanies the theory, where they are According to Egan, representational explanations in cognitive science provide a user-relative cognitive gloss on neuronal mechanisms which helps scientists to understand the workings of the mechanism in question.A second benefit of this fictionalist approach to representation is that it fits with the everyday practice of cognitive science. My account is inspired by, and finds support from, the work of Dennett . DennettHowever, to optimally select the prediction errors...the brain has to estimate or encode their precision. Having done this, prediction errors can then be weighted by their precision, so that only precise information is accumulated and assimilated in high or deep hierarchical levels.  receptors and classical neuromodulatory receptors like D1 dopamine receptors.  encoding of precision in terms of the excitability of neuronal populations reporting prediction errors” [ibid. p. 3]. However, it is not clear why this reading is required. Certainly, bio-chemical neuronal activity itself does not require a representational explanation. If precision-weighting can be equated with neuromodulation, then there does not appear to be any reason to accept a representational account of precision-weighting. It is thus typically the case that, when the neural details are specified, a representational explanation is no longer necessary.The approach to sub-personal representation advocated by both Dennett and Egan is motivated by considerations about the everyday practice of cognitive science. The argument I have provided can be seen to support Dennett and Egan’s contention—if PP theorists do use the brain as a prop in the imaginative game of ‘brain-as-representational-Bayesian-machine’, then PP provides yet another example of cognitive science in which representational posits are in fact being used merely as instrumentally useful metaphors. Indeed, I think that PP provides a particularly strong example of such an instrumentalist approach toward representation. We have already seen that some theorists take PP to indispensably require representation only at the more abstract, system-wide level of explanation Orlandi , b, 2016I therefore take it to be wholly compatible with the practice of PP theorists that, when representational posits are considered indispensable, they are considered indispensable largely because theorists are engaged in a game of make-believe which treats brains as if they were representing. These games of make-believe are grounded on a number of generative principles which rely on real causal goings-on in the brain. Consequently, the metaphors themselves are not relevant to causal interactions in the brain, but rather only to our ability to understand and track these interactions. The practise of PP theorists therefore does not warrant taking the brain to be literally trafficking in representations. Rather, it warrants the weaker claim—that scientists (sometimes) use the brain as a prop in a game of pretence involving representations.12Aside from fitting well with the actual practice of theorists working on predictive processing, my account also has direct, practical implications for future research. If I am correct, then it follows that theorists need not concern themselves with finding specific neural implementations of the representational posits of their PP models. These models merely prescribe specific imaginings, and so it is no requirement of them that each representational aspect of the model be found to be isomorphic with some aspect of the world. If my fictionalist account is correct, then it will not be the case that failure to find a neural implementation of  PP means that it “fails as a distinctive empirical account” Clark , p. 200.13A common argument against all fictionalist positions is that they are explanatorily unilluminating. In a recent survey concerning fictionalism about sub-personal representation, Sprevak  highlighIBE: the brain is a representational Bayesian computer.as if it were one.Fictionalism: the brain is not a representational Bayesian computer. However, its functioning is comparable in certain important respects to that of a representational Bayesian computer, and so it can usefully be treated Speaking explanatorily, (a) and (b) are equivalent because they both allow us to explain the success of representational PP. Theorists tend to accept option (a) because an IBE provides the simplest explanation of why representational PP explanations work. However, we have seen in this paper that (a) is problematic for theoretical and philosophical reasons. Acceptance of (a) is problematic because: (1) PP posits do not require a representational explanation , (2) it invokes the extremely thorny hard problem of content . I have argued that (b) provides a better explanation of the success of representational PP precisely because it can account for the success of such explanations whilst avoiding the problems metaphysical realism about sub-personal representation brings.Compare the two competing claims:I am therefore not arguing that we should not use IBE in cognitive science. Nor am I endorsing the claim that we should be sceptical of IBE across the board. Rather, I am claiming that, in this particular instance, we have no need to use IBE. The reason for this is that a fictionalist explanation of PP representation is better than a literalist one. Applying fictionalism does not require an abandonment of IBE. It instead involves proper application of IBE, because it involves making proper evaluations of what a best explanation actually entails.14I began this paper by mentioning Andy Clark’s recent claim that the concept of “representation” used within the predictive processing framework requires an end to the ‘representation wars’. According to Clark, the concept is so far removed from traditional conceptions of representation that it dissolves the problems upon which the ‘representation wars’ were predicated. I have argued that Clark is correct to think that PP requires an end to the ‘representation wars’. However, I have concluded that PP requires a cessation of hostilities because it entails an eliminativist victory. In Sect.


The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg In this case, the change in the relative orientation of the CBS motifs appears to be triggered by the electrostatic attraction between the surfaces of the two interacting proteins . The aut2+ atoms to the CBS module of the bacterial channel MgtE [2+ ions, the interfacial helices from complementary CBS2 motifs  remain distant from each other due to the electrostatic repulsion exerted by acidic residues located at their interfacial α-helices  the presence of a transmembrane domain potentially capable of passing metal ions through the cell membranes; (2) the presence of a regulatory Bateman domain directly connected to the TMD in a similar fashion to other transport proteins such as MgtE, CorC, or the CLC family of chloride channels; (3) the ATP- and Mg2+-binding capacity of the Bateman module, and the conformational changes associated to the interaction with these ligands, formerly observed in other channels; (4) the presence of a C-terminal CNBH domain, whose removal impairs Mg2+ extrusion; (5) the structural role exerted by hypomagnesemia causing mutations, such as CNNM2 T568I, which impair Mg2+ extrusion at the DCT; and (6) the lack of identified interacting transporters that could potentially be regulated by CNNMS, thus positioning the latter as simple modulators of their activity. Evidence not supporting the activity of CNNMs as direct transporters includes (1) the scarce number of Mg2+ binding sites located in the regulatory Bateman module, (2) the moderate number of transmembrane α-helices in the DUF21 domain, and (3) the low oligomerization degree (a dimer) in comparison with other channels and transporters.This review summarized the current three-dimensional knowledge about the CNNM family of magnesium homeostatic factors. Their exact role remains unclear ,23,25,822+-binding mode. MgtE binds Mg2+ in an ATP-dependent manner, whereas CNNM2 only interacts with ATP at a certain (mM) Mg2+ concentration. These striking features make it tempting to speculate that these opposing affinities for the two ligands could be related to the inverse abilities of these homeostatic factors to transport magnesium ions in opposite directions across cell membranes, thus supporting the direct role of CNNMs in Mg2+ extrusion. The behavior of the MgtE channel and CNNM2 are opposite in terms of ATP- and MgThe recent structural advances have contributed to our early understanding of the mechanisms underlying the CNNMs function, as well as their interaction with different molecules. Answering the still significant number of unanswered questions will help our ability to modulate the activity of these relevant machineries and use them as promising targets to treat Mg-related disorders and several types of cancer.


The ability to recognize oneself in a mirror is known as self-recognition, whereas delayed self-recognition is the ability to recognize the relationship between current self and past actions. While 3-year-old human children have self-recognition without the ability for delayed self-recognition, 4-year-old human children demonstrate the capability for both. Chimpanzees, the most closely related species to humans, have displayed the ability for delayed self-recognition. However, little is known about whether this ability is shared among all hominid species. In this study, we examined whether orangutans, the most distantly related species to humans within the hominid group, could recognize their own past actions using the preferential-looking paradigm. Our results demonstrated that orangutans were able to discriminate between a delayed video of themselves presented after a 2-s delay and a recorded video of the day prior. This suggests that orangutans have the ability to relate their own past actions to current actions, although we found no evidence of self-directed behaviour. We believe these findings will contribute to our growing understanding of hominid self-recognition. This was previously investigated with the mirror self-recognition (MSR) task, in which the subject's face is marked with a spot and then the subject is presented with a mirror. When subjects possess the ability for self-recognition, they will inspect the spot on their face upon seeing their reflection –3. By abOlder children possess a more mature concept of self-through-time, which serves to connect the present and past selves. Povinelli suggested that there are two types of self : ‘presenFrom the viewpoint of comparative cognitive studies, there have been many investigations on self-recognition ability in nonhuman animals. In a landmark study, Gallup demonstrated that chimpanzees have the capacity for self-recognition using mirrors . Gallup Other animal species may also have the ability for self-recognition, but previous research has primarily focused on the animal's reaction to the mirror. Few studies have examined whether nonhuman animals possess a temporally extended sense of self. For example, a recent study demonstrated that chimpanzees could recognize and react to a past self-action after a delay of 4 s. Several circular stickers were placed on the face and head of chimpanzees, who were then presented videos after 0- to 4-s delays . Three oet al. [This study examined whether orangutans, a great ape distantly related to humans and chimpanzees, can discriminate between a delayed video of themselves and a recorded one with a temporal delay and exhibit implicit recognition. We conducted two experiments regarding self-recognition. In experiment 1, as the preliminary study, we examined the reactions of orangutans to live self-videos and recorded videos on two monitors to confirm whether the orangutans could distinguish between synchronized and non-synchronized self-actions with the preferential-looking method, i.e. the paradigm of simultaneously presenting two images. We assessed whether the preferential-looking method is valid in the investigation of orangutan self-recognition. If the experiment only presented delayed and recorded self-video to orangutans and our results found that orangutans did not possess the ability for delayed self-recognition, we would not be able to differentiate whether it was truly an inability to recognize delayed self-video, or if the problem was based on the experimental design and we were not able to properly examine the delayed self-recognition of orangutans. We predicted that orangutans would prefer to watch a real-time video of themselves, rather than a recorded one in experiment 1. In experiment 2, we examined whether orangutans are able to discriminate between videos of themselves after a 2-s delay  from re2.2.1.Three Bornean orangutans housed at the Tama Zoological Park in Tokyo, Japan, were studied: two adult females  and one adult male (28-year-old Borneo). Gypsy was born in the wild and has lived at the Tama Zoological Park for over 50 years. Julie was born and raised in Tama Zoological Park, and Borneo was born in Singapore Zoo and moved to the Tama Zoological Park in 1998. Gypsy and Julie were housed socially and had direct and visual contact with other orangutans. Borneo also had the opportunity to see other individuals but was housed separately. All individuals were fed fruit and vegetables, and water was available ad libitum. These orangutans have previously participated in other cognitive experiments .2.2.The orangutans were tested in their resting room (1.8 m wide × 2.6 m deep × 2 m high), which is surrounded by a mesh fence (each hole in the mesh measures 5 × 5 cm). The stimuli were presented using two 17-inch liquid crystal monitors  positioned diagonally to each other (150° angle) and controlled by a laptop computer . A Web camera  was placed on each monitor to record and display the face and body of the orangutans. The distance between the participant and the monitors was approximately 60 cm. A video camera  was placed on a tripod between the monitors and a laptop was used to record the gaze of each orangutan. To create real-time and delayed self-videos, we used a device  that can display images of individuals after a delay of 0–20 s in the same left–right orientation on the monitor as seen in a mirror. We used GOM Player  to display the recorded video. All stimuli were presented in full-screen format.2.3.We prepared three types of stimuli that differed in temporal properties: a real-time self-video presented with no temporal delay, a delayed self-video presented after a 2-s delay and a self-video that was recorded the day before the experiment. During the experiment, participants were simultaneously presented either a real-time self-video and a recorded self-video, or a 2-s delayed self-video and a recorded self-video.2.4.Experiment 1 started when an orangutan spontaneously moved in front of the apparatus. The experimenter initiated the experiment as soon as the orangutan began paying attention to the monitor. All orangutans were tested under the same experimental conditions, which comprised a 5-min recording phase and a 5-min test phase. These two phases were separated by 1 day to avoid habituation of the orangutans to the stimuli. During the recording phase, the Web camera on the left monitor recorded the actions of the orangutan for 5 min. One day later, the Web camera on the right monitor was used to record the actions of the orangutan, also for 5 min. During the recording phase, a real-time self-video was presented on the monitor to attract the orangutan's attention to the monitor . These videos constituted the recorded self-videos. During the test phase, one monitor displayed a real-time self-video, and the other a recorded self-video, to each orangutan. A session ended when the experimenter switched off the monitors and two sessions were conducted per month to prevent the orangutans from habituating to the stimuli. The position of the stimuli (left or right side) was counterbalanced between sessions. Orangutans were not subject to any restrictions on their behaviour during the recording and test phases. The total viewing time for each monitor was recorded using the video camera, which was positioned on a tripod behind the two monitors. Experiment 2 was the same as experiment 1, except that the 2-s delayed self-video was used instead of the real-time self-video. To avoid interfering with the behaviours of the orangutans being assessed, the experimenter and other individuals remained out of view during the experiment.2.5.κ = 0.88). There were 53 trials available for analysis for Gypsy, 34 for Julie and 21 for Borneo in experiment 1, and 9, 54 and 28, respectively, in experiment 2. This study used the row data of looking time, rather than reporting the percentage of time subjects looked at the stimulus because a recent study suggested that data transformation could have negative consequences and that raw data should be used to maintain the ratio scale [Each recorded video of orangutan looking behaviour was divided into 10-s parts, each of which constituted a trial. There were two sessions (300 s per session) in each experiment, and each session was divided into 30 segments (10 s each). This method, which divides sessions into several segments, previously confirmed the validity for analysing the perceptive abilities of human newborns  and coulio scale . Moreovep-value of 0.005 was considered statistically significant, based on previous recommendations [To compare the looking time of the orangutans between real-time self-videos and recorded self-videos in experiment 1, and between the delayed self-videos and recorded self-videos in experiment 2, we ran a generalized linear mixed model (GLMM), which enabled us to estimate the parameters for both fixed effects  and random effects . GLMM can also accommodate the non-normal distributions typical of behavioural data without directly transforming the raw data, thus maintaining the ratio scale. Given that the responsible variable was the looking time, we set a Poisson distribution. The statistical model fittings were performed using R v. 3.5.1  equippedndations .3.To test the ability of orangutans to integrate temporal information, we devised two experiments based on the preferential-looking paradigm. In experiment 1, three orangutans were given the opportunity to view real-time videos of themselves presented with no temporal delay and similar videos of themselves recorded the day before the experiment. We monitored the looking times of the orangutans to ascertain whether they could distinguish between synchronized and non-synchronized self-actions. In experiment 2, we used self-videos presented after a 2-s delay instead of real-time videos.z = 4.530, p < 0.0001). We found no significant effect on the number of trials  in experiment 1, suggesting that orangutans spent more time looking at the real-time video of themselves than looking at the recorded one. z = 5.948, p < 0.0001), while the number of trials was not significant  in experiment 2. This suggests that orangutans spent more time looking at the delayed video of themselves than looking at the recorded one, although the number of trials might be significant when the significance level was p = 0.05. No testing behaviour was observed in either experiment.4.Experiment 1 confirmed that orangutans could discriminate between real-time and recorded videos of themselves, in turn suggesting that they can relate self-generated actions to contingent visual feedback. These results are consistent with previous studies in chimpanzees , althougIn experiment 2, the orangutans spent more time looking at the delayed video of themselves than at the recorded one, suggesting that orangutans possess the ability to relate past and current self-actions. Although the differences in looking time between the delayed and recorded self-videos could be a weak indicator for self-recognition, stronger evidence was needed to demonstrate delayed self-recognition. Our results suggest the possibility that orangutans could potentially have the ability to recognize the relationship between past and current self-action. In the previous study, 3-year-old children who showed self-recognition with live and 1-s delayed feedback could not identify themselves when the video had a delay of longer than 2 s . These rPongo species [The limitations of this study included its small sample size, a lack of variation in sex and age and the total number of sessions. However, it should be noted that despite the small sample size, these results do reveal the depth of capability of the  species . The issFinally, future comparative cognitive studies should examine animals' self-recognition ability using experiments that assess temporal extensions in self-recognition. Although some researchers have argued that the ability to conceive of the past self is restricted to humans , the fin


Slc12a2 gene RNA expression level was found to be significantly decreased only in gonad-intact and in castrated DCA-treated males. The authors discuss the gender-related DCA effects on the thymus.The aim was to investigate the effect of dichloroacetate (DCA) on thymus weight, Hassall's corpuscle number (HCs), and NKCC1 RNA expression in Wistar rats aged 4–5 weeks. They were investigated in the controls and DCA-treated gonad-intact and castrated males and females. The treatment lasted 4 weeks with DCA 200 mg/kg/day. At the end of the experiment, rat thymus was weighted, and its lobe was taken for the expression of NKCC1 RNA determined by the PCR method and of Hassall's corpuscles by immunohistochemistry. DCA caused a thymus weight decrease in DCA-treated gonad-intact rats of both genders as compared with their controls (p < 0.05), and no such impact was found in castrated DCA-treated males and females. DCA caused an increase of the HCs in gonad-intact males (p < 0.05), and no such increase in the DCA-treated gonad-intact females was found. There was gender-related difference in the HCs when comparing DCA-treated gonad-intact males and females: males showed significantly higher HCs (p < 0.05); no gender-related differences were found in the castrated DCA-treated groups. The Sodium dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDHK) . DCA is ɑ (PDHA1), thus inactivating it; the mechanism for PDC inhibition is related with the posttranscriptional upregulation of one or more PDHK isoforms, leading to phosphorylation of the E1ɑ subunit of PDC and maintaining the glycolytic profile of proliferating cells [The DCA principal target is the pyruvate dehydrogenase complex (PDC). It inhibits all isoforms of PDHK, keeping PDC in the catalytically active form facilitating the oxidative removal of pyruvate. PDHK isoforms can phosphorylate E1ng cells , 6.The PDHA1 gene is located on the X chromosome, and such gene location has different consequences for males and females with PDHA1 congenital deficiency; gender-related clinical problems depend mainly on the residual PDHA1 enzyme activity; PDC congenital or acquired deficiency may be a cause of lactic acidosis –9.Pyruvate dehydrogenase is present in normal and in cancer tissues, and the PDC/PDHK axis has been suggested as a specific target in cancer treatment , 11. DCA0–G1 phase as well as thymocyte accumulation in the G2–M phase, but no significant effect was found on thymus weight or thymocyte cell cycle in DCA-treated castrated males, indicating that DCA works synergistically with gonad hormones in males [Thymus could be a valuable model for investigating the effect of medicinal products on thymocyte proliferation. Following castration, hyperplasia of thymus and gender-related increase in the Hassall's corpuscles number (HCs) in thymus appear in rats . HCs arein males .+/K+/2Cl− cotransporter (NKCC) by DCA in male rats: (1) a single dose significantly increased 24-hour urinary output as well as Cl−, Na+, K+, Ca2+, and Mg2+ excretion; (2) changes following 4-week treatment included increase in the size of the Henle loop's thick ascending limb's epithelial cells (an effect related to NKCC2 inhibition) and (3) significant decrease in RNA NKCC1 expression in thymus of male rats [Slc12a2). This gene was found to be broadly expressed in thymus [Recently, we reported inhibition of Naale rats . The intale rats , 23. Then thymus . NKCC1 pn thymus .J. A. Clayton (2014) noted that female and male cells differ in their response to chemical agents but preclinical research often neglects association of medicines effects with gender and sex hormones . We did The effect of the DCA treatment on the thymus was investigated in the following 8 groups of age-matched Wistar rats of both genders: gonad-intact and castrated male and female controls and in respective male and female DCA-treated groups. ad libitum. The experiments were performed in compliance with the relevant laws and institutional guidelines for animal care in order to avoid any unnecessary animal distress.The permission was obtained from the State Food and Veterinary Service of Lithuania to use experimental animals for research (2015-05-18 No. G2-28). The animals were purchased from the Animals Facility of the Veterinary Academy at the Lithuanian University of Health Sciences . The experiment was carried out at the Animal Research Center at the Lithuanian University of Health Sciences . The animals were housed in standard colony cages with free access to food, in the conditions of constant temperature (21 ± 1°C), humidity, and the light /dark cycle (12 h / 12 h). A commercial pellet diet was providedFor the experiment, 4–5-week aged Wistar rats were selected with the same animal number (n = 6) in the groups; there was no difference in rat weight among the formed groups. In the animal groups selected for castration, the male orchidectomy and female ovariectomy operations were performed. The castration was performed at the age of 28 ± 2 days . The accommodation period after the castration was one week. After the accommodation period, the treatment of gonad-intact and castrated animals was started. At the end of the experiment, one castrated DCA-treated female was eliminated from the study due to a fistula formed after the operation and significant weight loss. ad libitum. The treatment duration was 4 weeks. The DCA dosage, administration in drinking water, and thymus preparation methods were described as previously [Treatment with DCA aqueous solutions (200 mg/kg/day) in drinking water was used. The only source of drinking was the DCA solution for treated groups, and fresh tap water was provided for the control groups; DCA solution and water were offered to animals2 camera. To minimize the thymus contamination with red blood cells, the carotid arteries and the aorta were cut and the animals exsanguinated. Upon killing the animals, their thymus was harvested and the contaminating blood was removed by rinsing with RPMI-1640 . The weight of the thymus was evaluated, and the left rat thymus lobe samples of the study groups after thymus surrounding connective tissue were removed and the thymus lobe was stored in the RNAlaterRNA stabilization reagent  at -80°C until the further RNA extraction and RNA analysis. The right lobe of the thymus was taken for the histomorphometric evaluation.Completing the experiment, the animals were killed in a 70% COμm sections, and stained with hematoxylin and eosin (H-E). For immunohistochemical examination, slices were placed on poly-L-lysinecoated glass slides. After deparafinization in xylene and rehydration, the sections were pretreated with the antigen-retrieval solution  in a pressure-cooker and then incubated with cytokeratin monoclonal antibodies  for the identification of high molecular weight cytokeratins (HMW CK). Antibodies detection was performed with the EnVisionPlus-HRP kit . Sections were counter-stained in weak Mayer's hematoxylin. The histological and immunohistochemical evaluation of the samples was performed with an OLYMPUS BX40F4  microscope using the CellSens Dimention 1.9 Digital Imaging Software for Research Applications . Histological sections were selected from the middle portion of the right thymus lobe. The total area of the thymus lobe and the area of the medulla were counted. The presence of Hassall's corpuscules, which are heterocellular, consisting of thymic epithelial cells (major cellular component), macrophages, interdigitating dendritic cells, myoid cells, and, occasionally, mast cells and lymphocytes [2 of thymus medulla in each group. The methodology of the HCs histological examination was as described previously [The right lobe of the thymus was fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned in 3 phocytes  was evaleviously .laterRNA stabilization reagent  at -80°C until further RNA extraction. The frozen tissue was ground in liquid nitrogen. Total RNA was extracted using the TRIzol™ Plus RNA Purification Kit  according to the manufacturer's instruction. The RNA quality was assessed using a NanoDrop2000 spectrophotometer  using the A260/280 ratio. The extracted RNA samples were stored at -80°C until further analysis. The methodology of RNA extraction and the evaluation of the NKCC1 expression in thymus were used analogically as described [Rat thymus samples of all study groups were stored in RNAescribed . Slc12a2 (Rn00582505_m1) and Glpdh (Rn01775763_g1) genes. High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor  was used for reverse transcription reaction in 20 μl reaction volume containing 50 ng of total RNA incubated at 25°C for 10 min, transcripted at 37°C for 120 min, and terminated by heating at 85°C for 5 min using Biometra TAdvanced thermocycler . The synthesized cDNA was stored at 4°C until use or at -20°C for longer time. The Real-time PCR was run in triplicate with 4 μl of cDNA template in a 20 μl reaction volume , 1 μl of TaqMan Gene Expression Assay 20x , and 5 μl of Nuclease-Free Water  with the program running at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. the reaction was performed using an Applied Biosystems 7900 Fast Real-Time PCR System .RNA expression assay was performed for U test. The one-way ANOVA analysis was used to determine significance among the groups, and post hoc tests with Fisher's least significant difference were used for comparison among the individual groups. To investigate the NKCC1 (Slc12a2) RNA expression changes in the DCA-treated group, the threshold cycle (CT) values were normalized with the control Glpdh gene; for the gene expression study, the delta delta threshold cycle (2-ΔΔCT) method was used to calculate the expression ratio between the DCA-treated (test) and control conditions of the target gene as compared with the reference gene. The Spearman's rank correlation coefficient (r) was used to assess the relationship among thymus weight, HCs and ∆CT. The Fisher's transformation which changes r to a Z-score and the Steiger's Z-test for comparison of correlations (z) within the population were used. Differences at the value of p < 0.05 were considered significant.The statistical analysis was performed by using the Statistical Package for the Social Sciences, version 22.0 for Windows . The normality assumption was verified by the Kolmogorov–Smirnov test. The animal weight data are expressed as the mean ± SD values. The thymus weight data are presented as the mean and the 95% confidence interval (95% CI). When the normality assumptions are not met, data are expressed as a median and a range . Differences between two independent groups were evaluated using the nonparametric the Mann–WhitneyThe data of rat thymus weight of the study groups after the treatment and their control groups are summarized in At the end of the experiment, the gonad-intact males had a significantly higher body weight as compared with the gonad-intact female control , and there was no significant rat body weight difference when comparing the gonad-intact male control and the gonad-intact DCA-treated males (p > 0.05); no difference was found between the respective female groups (p > 0.05).The castrated DCA-treated rat groups of both genders showed a statistically significant body weight decrease as compared with the castrated control groups: the body weight of castrated DCA-treated males was by 15.07% lower than that of the control  and by 16.76% lower in the DCA-treated females as compared with their control .The data on the effect of DCA treatment on thymus weight are shown in p = 0.02) and females (p = 0.001) control groups, because castration is related with thymus hyperplasia. There was no gender-related difference in animal thymus weight when comparing gonad-intact male and female as well as castrated rats  and DCA-treated gonad-intact females (p = 0.001) as compared with their controls. No significant treatment impact was found in castrated DCA-treated male and female groups when comparing with their castrated controls  and females (p = 0.009). The treatment of rats with DCA caused a statistically significant increase of HCs in the male as compared with the gonad-intact control group . A significant difference in the HCs was found between DCA-treated gonad-intact males and females (p = 0.04), and males showed a significantly higher Hassall's corpuscle number. However, none of such differences were statistically significant in the castrated DCA-treated groups of both genders when comparing with the castrated control groups . The correlation between thymus weight and HCs in gonad-intact and castrated DCA-treated male groups was significant . The difference between the ΔCT of target and reference genes as expressed by the ΔΔCT is shown in -ΔΔCT) in the gonad-intact DCA-treated male was 0.103-fold change lower than in the control. This means the 90% downregulation of expression as the expression level is decreased by 90% to the level of 10% under control conditions. Also, there was a significant Slc12a2 gene expression change in the castrated male DCA-treated group as compared with the control: its expression level was found to be decreased by 76% (Expression difference in thed by 76% . HoweverThe correlation of thymus weight with ∆CT in the controls and in the DCA-treated gonad-intact and castrated groups of both genders was insignificant (p > 0.05); no significant differences comparing correlation coefficients of the control with the DCA-treated, respectively, groups were found  in the DCA-treated gonad-intact and control males as well as in the castrated male groups. The Slc12a2 gene RNA expression level  in the gonad-intact DCA-treated males was decreased by 90% under control conditions and significantly decreased (by 76%) in the castrated males. No changes in the tested NKCC1 RNA gene expression level were determined in the studied gonad-intact and castrated female DCA-treated groups as compared with the control of both gonad-intact and castrated female rats.The study shows that the DCA could target the NKCC1 protein where a gender-related difference of the DCA effect on the NKCC1 gene expression in rat thymus was determined. A significant difference was found between the ∆CT values . Elimination of DCA including glutathione- (GSH-) dependent oxygenation to glyoxylic acid mainly depends on GSTζ-catalyzed dechlorination by mitochondrial or cytosolic enzymes [ζ, the AppKm for GSH obtained in female rats is 2.5–3.2-fold higher than males; the higher AppKm of GSH is associated with lower access or binding to GSH [DCA acts as inhibitor of glutathione S-transferase-zeta1 (GST enzymes –67. Regag to GSH . The preg to GSH –74. Datag to GSH ; the prig to GSH ; e.g., gThe DCA treatment decreases the thymus weight of gonad-intact rats of both genders, but such impact is absent in castrated rat groups, indicating the synergistic pharmacological mechanism of DCA and gonad hormones.The different mechanisms are responsible for changes in thymus epithelial cells transformation into Hassall's corpuscles under the DCA effect in gonad-intact and castrated animals.The DCA treatment decreases the NKCC1 gene RNA expression in the gonad-intact and castrated males, and no such effect was determined in females.The investigation of the DCA treatment efficacy should be evaluated with regard to gender and the exposition of gonad hormones.