Malayan tapirs are listed as endangered and are bred in captivity under governmental management. The success of captive breeding programs varies and the underlying causes are unclear. Here, we investigate how tapir reproduction is affected by previous breeding experience, enclosure type/size and visitor numbers so that appropriate steps can be taken to achieve self-sustaining captive populations. Data on social and reproductive behaviors were collected from six tapirs , from different breeding centers in Peninsular Malaysia for 18 weeks. Results revealed that social and reproductive behavior of both sexes was significantly influenced by social and environmental conditions. Larger enclosure size tended to increase social and reproductive behaviors, whereas high number of visitors reduced initial interaction between males and females. No specific breeding month was confirmed; however, reproductive behaviors were highest in April. Overall, this study contributes to a better understanding of the relationships between social and reproductive behaviors, and captive environments on Malayan tapirs. In future, frequency of sexual interactions should be monitored regularly to identify animals exhibiting below-average frequency and who might, therefore, be prone to reproductive difficulties. Tapirus indicus) in captivity3 and in the wild4, there is little research on the reproductive behavior of this species that investigate what factors affect the breeding success of this species. Malayan tapirs are threatened and endangered throughout their geographic regions due to habitat destruction, illegal logging and hunting pressure. In addition, their low reproductive rate has perpetuated the decline of Malayan tapir populations5. Therefore, a better understanding of their reproductive behavior will not only aid in conservation measures, but will also greatly improve our knowledge of their ecology and evolution.Despite several long-term studies of Malayan tapir , smaller enclosure sizes are associated with reduced breeding success in males due to an increase in dominance- associated traits17. Meanwhile the increase in stress-related behaviors such as chasing, stereotypic behaviors and mouthing in female captive black rhinoceros could be due to the percentage of wall surrounding their enclosure17. The high exposure to zoo visitors within their enclosure causes fear17 and the increase in fighting between breeding partners results in correspondingly elevated mean corticoid levels25. Nevertheless, in some other species such as chimpanzees exposure to zoo visitors was found to have a neutral or even positive and enriching effect as human visitors represented the possibility to obtain food rather being a direct stressor19. In Malayan tapirs, however, enclosure characteristics have been reported to significantly affect their general behaviors and activity budget8, but the effects (including visitor-related disturbance levels) on tapir social and reproductive behaviors are currently not quantified.Although previous studies have yielded important new insights into the reproductive biologyHere, we report the social and reproductive behaviors of captive male and female Malayan tapirs, focusing on how these behaviors are affected by captive environment , month and maternal experience (parity and pregnancy).th March and 4thAug 2016, alternating between two-week observation periods at each location to achieve equal and temporally-comparable coverage because the weather in Peninsular Malaysia is quite changeable. Three pairs of adult Malayan tapirs from three different sites were observed for a total of 6 weeks per pair , olfactory cues in the air , or exact determinant of noise levels . Detailed descriptions of the layout of each enclosure are available in Arumugam et al. (2018).This study was performed at Sungai Dusun Wildlife Reserve  and two local zoos: Zoo Negara , and Zoo Melaka  in Peninsular Malaysia . This study was completed in strict accordance with relevant guidelines and regulations from the Department of Wildlife and National Parks (PERHILITAN), Zoo Negara and Zoo Melaka. Experimental protocols and applications for behavioral study were approved by the Department of Wildlife and National Parks (PERHILITAN), Zoo Negara and Zoo Melaka Committees.28 and other Perissodactyla32 was constructed by incorporating some modifications after an initial behavioral observation period in November 2015 at ZN prior to actual data collection  as low, medium or high.Outdoor weather variables (temperature and humidity) were measured using HygroThermometer Clocks , and the number of visitors in front of the viewing points of tapirs’ enclosure were counted using click counters every 35 minutes34. The weather variables were inter-correlated (i.e. if temperature (°C) increased, humidity (%) decreased; r = −0.77, p < 0.05) and generally yielded similar results. Thus, we used only temperature for our main analyses , four environmental factors  and month were included as fixed effects. The frequency of social  and reproductive behavior of male (identification and courtship) and female  specific groupings were modeled as the response variable on the Y-axis  (exp [−0.5*∆AICc]), divided by the sum of the likelihoods for all models considered35. The 95% confident intervals for model-averaged parameter estimates were calculated using the model.avg function in R. The relative importance of each fixed effect was calculated as the total ω of all plausible models. Significant fixed effects were selected if the confident interval did not overlap zero.The frequency of each behavior was summed for each observational day per 30 s observational period for the duration of the respective behavior and the number of visitors, temperature and humidity variables were standardized to a mean of zeroNone of the social behaviors varied significantly with month, indicating a non-seasonal pattern of social engagement by tapirs in captivity. Only one reproductive behavior was influenced by month Table . IdentifThe mean frequency between pairs for initiation and vocal behaviors were significantly affected by female reproductive status . Identification and courtship behaviors were followed mostly after initiation behaviors. However, as we did not analyse behavioral sequences in this study, the detailed behavioral progression of pair-bonding in Malayan tapirs need to be investigated in more detail in future studies. Identification by smelling female’s urine or vaginal area followed by a flehmen response is a common way for a male mammal to determine female estrus39 and increase the intensity of behavioral estrus signs40. Female reproductive status of being pregnant or non-pregnant influenced the initiation behavior between the paired tapirs, which is also very commonly seen in other terrestrial mammals42,. However, initiation behaviors were seen also between the partners in the pregnant pairs A and C, which likely indicates that this behavior can also function to reduce aggression between couples and/or strengthen pair-bonds as previously suggested to prevent injuries or fatality during their encounters42. Female B who did not have any maternal experience was more receptive during estrus than pregnant females A and C reflecting similar observations by Metrione, (2010) in Southern white rhinoceros (Ceratotherium simum simum). It was also proven in domestic goats Capra hircus males that engage in investigation behavior such as anogenital sniffing more often when exposed to zero maternal experienced females compared to parous or pregnant females43. Therefore, we suggest that the reason why Male B displayed higher frequency of identification and courtship behaviors throughout the study period may have been due to the presence of receptive females that act as androgen inducer44.There were several factors, which possibly affected initiation, identification and courtship behaviors. Females’ maternal experience, followed by environmental factors, was the primary reason to induce the male-female sexual receptivity27. Large enclosures also allowed the male and female to separate themselves more effectively during aggressive encounters associated with breeding17. Larger enclosures also allowed the tapirs in this study to perform courtship and pre-courtship behaviors effectively. Nevertheless, although tapirs in artificial enclosures were successfully engaged in initiation behavior showing even when in the presence of visitors, the frequencies of initiation behaviors were higher during periods of low visitor numbers (<150), and the associated reduction in visitor-generated noise levels  surrounding the enclosure. In the presence of visitors and background noises, all tapirs were found to be continuously vigilant, either in a standing, sitting or a lying down position, without any social interaction with their partner. Unfortunately, due to logistical reasons, it was impossible for us to collect data at night during the predominant natural activity period of tapirs45. Nevertheless, considering the increasing popularity of extended visitor access to zoos19 and animal displays with reversed day/night light cycle27, the potentially problematic effects of visitor-related disturbance on the reproductive behavior of this species (mirroring observations in other species of similar conservation concern24 etc.) need to be highlighted. For animal welfare as well as captive breeding success, it is thus crucial to keep also nocturnal or crepuscular animals away from visitor disturbance, and thus to allow social behaviors to take place during any time of day. Although here we could only interpret on noise levels, nocturnal mammals such as tapirs typically rely heavily on olfactory signals in intra-specific communication46, and thus smell-related disturbance from visitors also needs to be investigated in future studies.Enclosure size positively affected initiation behaviors, whereby the larger semi-natural enclosure enabled tapirs to engage in approaching and following behaviors as they would do in the wild27 to avoid physical and mental stress resulting in miscarriages in pregnant females.In addition during visiting time, it is also critical to ensure all tapirs, but particularly the females, to be kept at least one meter away from visitors as suggested in the Tapiridae Care Manual47, or in the case of the pregnant females in pairs A and C - to avoid contact with their mates, and these behaviors are thus part of the normal intra-specific behavioral communication repertoire rather than a direct reaction to visitors and/or other environmental factors.Antagonistic behaviors were found not to be influenced by any of the tested parameters. Any displayed antagonistic behaviors were likely due to attempts of individuals to increase levels of arousal of their opposite-sex partner32, or his lack in morphological height, as male B was much smaller than female B and was therefore not able to reach the female B’s vaginal area to begin thrusting. From personal communication with the zoo keeper of pair B, we were informed that this pair did not produce any offspring in the time they have lived together. Based on that, if the body sizes of the paired male and female are not compatible, we believe that there will be no possibility for successful reproduction in tapirs. Consequently, this may lead to severe reproductive problems such as cystic endometrial hyperplasia, leiomyomas , adenoma, para-ovarian cysts and/or hydromucometra48 in tapirs, particularly in those females who do not reproduce for a long period of time, making future reproduction unlikely even if paired with a more suitable mate.Although all reproductive behaviors were most frequently observed in male B during copulation events, only mounting was observed and no successful mating happened with female B. At each mating event, male B failed to show thrusting, which was likely due either to his immaturity and inexperience12 whereas others do not50. Previous studies have reported no evidence of seasonality in other tapir species52, but it is still unclear in Malayan tapirs. We observed all males performing identification behavior on their female partners during every month of the study period between March and August. However, identification behaviors were highest specifically during the month of April, likely indicating that April is the month when the female showed high stimulus value  such as changes in genital morphology or changes in olfactory cues provided in the urine, feces, and vaginal discharge53.Apart from that, many mammals in the wild have a specific breeding season or month54, and female tapirs were observed to vocalize regularly as a warning to threaten the male to stay away whereas males threatened females by using physical aggressiveness more often such as biting, kicking or pushing the female away and used vocalizations less often (unpubl. data). Males were usually heard vocalizing only during initiation behavior (following). A further study on types of vocalization associated with male and female behaviors however is highly necessary to obtain a better understanding of tapir vocal communication related to their social and reproductive needs in captivity.Although we did not record vocal behavior systematically in the framework of this present study, female tapirs appeared to prefer vocal communication; while male tapirs were more interested in physical interaction. Animals emitting vocalizations can signal the physiological and psychological status of the caller41. Different types of vocal communication were noted throughout the study at different events during the social and reproductive encounters which need further investigation.The results of this study contribute to the understanding of the relationships between reproduction and social behavior, and the influence of environmental factors on reproductive behavior in captivity. Our findings showed that, in captivity, the social and reproductive activity patterns depend mainly on the female’s maternal experience as well as environmental conditions. Being in a larger enclosure with fewer visitors and associated lower anthropogenic noise levels helped particularly non-pregnant and nulliparous females with the advancement of initiation behaviors or so-called consortship into breeding behaviors. The pregnant females and the resident male tended to alleviate aggression and dominance by initiating social interactions as a defensive strategy to protect themselves from injuries similar to maresIdentification behaviors were highest in April contributing evidence that female Malayan tapirs may have the highest sexual stimulus value in April. This potential seasonality in Malayan tapir reproductive biology needs to be investigated further in future studies. Despite showing a successful behavioral sequence up to the last stage , one male could not achieve successful mating as it was much smaller than the resident female. Consequently, it could lead to female health problems, which are potentially hazardous if females are not mated for a long period of time, annihilating also future reproductive capacity. Therefore, introducing the male or female to a novel and physically compatible mating partner could help to increase the changes of mating activity resulting in a self-sustaining population. Future studies need to investigate in more detail the effects of visitor-induced (noise-) disturbance, particularly with a focus on the role of vocal communication in tapir reproduction.Table S1.


In internationally adopted children disorders of linear growth, puberty development, thyroid function, and bone metabolism are frequently reported. It is important that these children receive careful auxological and endocrinological evaluations and follow-up.Pediatricians and other healthcare providers should be aware that auxological and endocrinological problems are common in newly arrived international adoptees. Children who are adopted internationally should be assessed for linear growth, puberty development, thyroid function, and bone metabolism disorders.A close auxological and endocrinological evaluation with follow-up is mandatory in these children.Pediatricians and other healthcare providers should be aware that auxological and endocrinological problems are common in newly arrived international adoptees.Approximately 260,000 international adoptions take place worldwide each year . The couIn recent years there have been important changes regarding the volume of adoptions, the countries involved and the characteristics of families deemed suitable to adopt . The livIn this review we evaluate the main auxological and endocrinological problems encountered in international adoptees in order to shed light on those that are most frequent. We hope to provide some useful suggestions for looking after these children.The assessment of growth is the most useful tool for defining the health of a child. By monitoring growth, we can evaluate a child’s nutritional status. The Tanner scale allows us to determine whether pubertal development is at an appropriate stage and diagnose any growth disorders with a non-nutritional basis .Internationally adopted children may present growth suppression as a consequence of institutional care in their countries of origin . FactorsNeglect during early childhood seems to slow growth rates in height and weight for children, suggesting that early childhood is a critical period for a child’s growth in later life . In factPost-placement recovery has been widely documented. In fact, most adoptees demonstrate excellent catch-up growth within six months of adoption. If growth failure persists after 6 months of appropriate caloric intake, nutrition-independent causes should be considered . HoweverHowever, some data suggest that the length of this catch-up period may depend on the children’s countries of origin and that there may be significant auxological variations. For example, children adopted from Ethiopia or Eritrea often reside for relatively long periods of time with relatives prior to institutionalization and exhibit few behavioural problems on arrival. In general, they have good growth, and less severe developmental delays . It can Children adopted from Romania and other Eastern European countries have often been placed in institutions with very low quality of care  and chilAs well as presenting stunted growth, malnourished children often present retarded bone maturity comparable to children affected by diseases such as coeliac disease, inflammatory bowel disease and hormonal deficiency. If puberty is delayed and/or growth continues into the early or mid-twenties, then an acceptable final adult height can be achieved. However, an individual’s maximum height may also be limited by genetic imprinting in very early development. This may be the case where full catch-up appears to have taken place but is followed by an advanced puberty and early cessation of growth –27.One study showed that children with rickets on arrival did not achieve the recovery levels of non-stunted children, suggesting that deficiencies in early life can only be partially recovered. Such auxological dynamics can also be associated with early pubertal development which can shorten the period of growth and therefore contribute to a reduced adult height. Mechanisms underlying early pubertal development and optimal management of nutritional rehabilitation after chronic malnutrition need to be clarified . One stuThe development and growth of internationally adopted children in the countries of their adoptive families  should bSince pathological conditions may radically alter bodily growth, accurately estimating a child’s age is important in the care of adopted children, even in cases in which a child’s age appears undisputed . The useFinally, there has been discussion about the possible role of organic pollutants on pre-adoption growth failure. The little available data seems show that exposure to endocrine disruptors, air pollution, second-hand smoke, and the mother’s lifestyle during pregnancy affect children’s growth and development , 34. SomThere is evidence that physiological adaptations to deprived environments in early childhood can increase the risk of obesity, early onset puberty in girls and metabolic syndrome and cardiovascular disease, particularly if there is a shift over time from resource-poor to resource-rich environments . ClinicaStudies of children in low and middle income (LAMI) countries show that early postnatal catch-up growth is associated with obesity and a high risk of developing metabolic syndrome later in life. The rapid catch-up growth observed in internationally adopted children could be a risk factor for later obesity and early onset puberty, both of which are risk factors for cardiovascular diseases, type-2 diabetes, and musculoskeletal disorders . HoweverThe association between precocious puberty and international adoption was first described by Swedish authors in 1981  in a casThere seem to be several causes for this association which are not fully understood. It is speculated that improved nutritional and socio-economic conditions lead to a period of catch up growth and the following early onset of puberty . A similSome authors have postulated that early puberty follows the period of catch-up growth only after fetal or fetal-postnatal undernutrition, but not after postnatal undernutrition . This isMoreover, early puberty is also observed in nondeprived migrating children . In defiThe current treatment of idiopathic precocious puberty is with gonadotrophin-releasing hormone (GnRH) agonists . Many stThyroid function is critical for the development of the central nervous system in the fetus and in children and iodine is an essential component of thyroid hormones . 28.9% oUnfortunately, the spread of neonatal screening as well as campaigns for reducing iodine deficiency have obtained variable results and large areas of the planet are still not fully covered Table . It is tSevere iodine deficiency may cause hypothyroidism that results in impaired somatic growth and motor development . Overt cIn neonates of the Democratic Republic of the Congo, a 10% rate of biochemical hypothyroidism has been found . This hyMost neonates with CH have a normal appearance and no detectable physical signs at birth; hypothyroidism in this period is often overlooked and delayed diagnosis may to lead to mental retardation . Pilot CVitamin D deficiency is not only reported in cold or temperate countries , but alsVitamin D insufficiency is common in internationally adopted children, from all ethnic groups, although rickets has been cited only rarely in articles about the health problems of children adopted internationally during the 1980s and 1990s –62. In cBecause children generally experience rapid catch-up growth in the first year with their adoptive families, they may need larger amounts of dietary or supplemental iron and other nutrients than other children of the same age. An evaluation of serum 25(OH) D levels after adoption can allow the prompt introduction of vitamin D supplementation/treatment if needed . In inteDevelopment of the hypothalamic-pituitary-adrenal axis is affected by genetics and pre- and postnatal environmental factors. A lot of studies report significant variations from normal functioning of the HPA caused by adversity early in life or even prior to conception . These eStudies about early life adversity (ELA) and HPA functioning provide discordant results . Some stSome interesting studies have evaluated the potential for recovery of ELA damaged HPA, following improvements in care. Foster care or targeted interventions appear to be able to re-modulate HPA long-term . The finIn internationally adopted children disorders of linear growth, puberty development, thyroid function, and bone metabolism have been frequently reported. A careful auxological and endocrinological evaluation with follow-up is mandatory in these children. Paediatricians and other healthcare providers should be aware that auxological and endocrinological problems are common in newly arrived international adoptees and should tailor their approach to caring for these children accordingly.


Cryptococcus neoformans can cause life-threatening infections in immune compromised individuals. This pathogen is typically acquired via inhalation, and enters the respiratory tract. Innate immune cells such as macrophages and dendritic cells (DCs) are the first host cells that encounter C. neoformans, and the interactions between Cryptococcus and innate immune cells play a critical role in the progression of disease. Cryptococcus possesses several virulence factors and evasion strategies to prevent its killing and destruction by pulmonary phagocytes, but these phagocytic cells can also contribute to anti-cryptococcal responses. This review will focus on the interactions between Cryptococcus and primary macrophages and dendritic cells (DCs), dealing specifically with the cryptococcal/pulmonary cell interface.The fungal pathogen  Cryptococcus neoformans is a ubiquitous environmental fungal organism that primarily inhabits soil and bird excrement, and this organism can cause opportunistic infections in humans , and particularly those from the pulmonary tissues   allow intracellular growth will be important in understanding their role in either prevention of cryptococcal dissemination or enhancement cryptococcal dissemination of to the central nervous system (CNS).In mice, classically activated macrophages produce large amounts of nitric oxide, a main effector mechanism contributing to anti-cryptococcal activity , while those from mice infected with the low-uptake strains had increased expression of M1-associated genes   play an important role in controlling + myeloid DCs, plasmacytoid DCs (pDCs) and CD103+ DCs  within the lysosome , BDCA1− CD14+ macrophages, and BDCA1− CD14− macrophages  can be differentiated into macrophages in vitro. While not tissue resident lung immune cells, PBMC-derived monocytes and macrophages provide an approximation of pulmonary macrophage responses to a cryptococcal challenge. Some of the first work with human PBMC-derived macrophages and C. neoformans was in 1973 by Diamond and Bennet who showed that macrophages cultured from PBMCs were able to phagocytose the fungus but were unable to kill it effectively , both pathogen and host appear morphologically normal and continue to function  are present throughout the human lung, they are difficult to study due the problems associated with acquiring them from healthy donors , were also tested for DC maturation  and FcγRIII (CD16)  and pulmonary macrophages, and DCs. Differences in the origin of cell types and even environmental difference in specific organs/tissues are far too great to ignore. Future studies will need to focus on subsets of pulmonary phagocytic cells in order to better understand the interactions between pulmonary phagocytes and the fungal pathogen BN, AH, and KW contributed to the writing, editing, and revision of the manuscript.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.


Antimicrobial resistance (AMR) is an emerging global health crisis that is undermining advances in modern medicine and, if unmitigated, threatens to kill 10 million people per year worldwide by 2050. Research over the last decade has demonstrated that the differences between genetically identical cells in the same environment can lead to drug resistance. Fluctuations in gene expression, modulated by gene regulatory networks, can lead to non-genetic heterogeneity that results in the fractional killing of microbial populations causing drug therapies to fail; this non-genetic drug resistance can enhance the probability of acquiring genetic drug resistance mutations. Mathematical models of gene networks can elucidate general principles underlying drug resistance, predict the evolution of resistance, and guide drug resistance experiments in the laboratory. Cells genetically engineered to carry synthetic gene networks regulating drug resistance genes allow for controlled, quantitative experiments on the role of non-genetic heterogeneity in the development of drug resistance. In this perspective article, we emphasize the contributions that mathematical, computational, and synthetic gene network models play in advancing our understanding of AMR to discover effective therapies against drug-resistant infections. Antimicrobial resistance (AMR) is an emerging health crisis that is undermining modern medicine . AMR ariCandida glabrata  against drug-resistant pathogens and cancers to be investigated experimentally.Mathematical models have been used to investigate the effect gene network structures or motifs have on AMR. For instance, it was shown computationally that gene network motifs can enhance drug resistance by modulating gene expression noise within a cell population . CharlebE. coli, depended on the rate of stress induction; fits to data allowed the fitness benefit that the pumps conferred under different stress induction rates to be accurately predicted by mathematical models. In another study, it was predicted that incorporating negative feedback via drug efflux pumps into synthetic gene networks can increase the response of the gene network at low antibiotic inducer concentrations  cells with a recombinase-mediated integration site known as a “Flp-In” landing pad , FarquhaDNA sequencing of the gene circuits after adaptation to puromycin and monitoring expression and survival after temporary removal of drug revealed adaptation mechanisms . The selA new interdisciplinary field of research is emerging that combines multi-scale quantitative models with synthetic biology to rationally design gene networks using engineering principles for AMR research. One important goal is to use these models to predict the effects of non-genetic drug resistance on the evolution of genetic drug resistance. Another important goal is to advance pharmaceutical and clinical AMR research by investigating new “resistance proof” antimicrobial compounds and novel therapeutic treatment strategies.Moving forward, a challenge that must be addressed is how to adapt the mathematical models and translate the experimental discoveries made using synthetic systems to pathogens with complex and highly interconnected gene regulatory networks. More research on pathogenic micro-organisms and mammalian cells is needed to elucidate the underpinnings of non-genetic resistance at the molecular and single-cell levels. Specifically, capturing the complexity of native resistance mechanisms, which are not completely understood, with synthetic gene networks presents both an obstacle and an opportunity. Quantifying known drug resistance effects in genetically engineered organisms may elucidate native resistance mechanisms in pathogens. One possible approach involves adding additional regulatory interactions iteratively to a well-understood synthetic gene network controlling a drug resistance gene and making predictions for their impact on drug resistance. Eventually, the networks become complex enough to mimic the phenotypes caused by native resistance mechanisms. In the cases of cancer subtypes, introducing synthetic gene circuits controlling a gene with specific mutations associated with chemotherapy resistance will be challenging, with genomic instability possibly corrupting the gene circuit. Targeted genomic integration in various cancer cell lines and primary cell strains will also differ in efficiency, making comparisons between cell types difficult. To address these challenges, a better understanding of the gene regulatory networks, mutations, and signal transduction pathways associated with chemoresistance in specific cancers is needed.M. tuberculosis persisters to reduce treatment times (Research incorporating quantitative modeling and genetically engineered networks will be critical to fully understand how non-genetic and genetic mechanisms interact in the development of drug resistance, and to discover effective strategies that target acute non-genetic drug resistance to alleviate the development of permanent genetic drug resistance in infectious diseases and cancers. Several promising approaches include synergistically using noise-enhancing compounds to reactivate latent HIV to increase sensitivity to existing antiviral drugs , using cnt times , eliminant times , and thent times .Overall, combining mathematical models and synthetic gene networks is leading to new quantitative model systems for drug resistance research, which are desperately needed to advance our fundamental understanding of the multi-stage drug resistance process. Ultimately it remains to be seen how discoveries made using these quantitative model systems will translate to pathogens and cancer. However, the potential of this new area of research to help mitigate the socio-economic costs of drug resistance warrants its relentless pursuit.DC conceptualized and supervised the study. KF, HF, and DC contributed to the literature review. HF and DC developed the figures. KF and DC wrote the manuscript. All authors contributed to the article and approved the submitted version.KF was employed by the company Precision for Medicine. The remaining 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.


Therapeutic psychedelic administration and contact with nature have beenassociated with the same psychological mechanisms: decreasedrumination and negative affect, enhanced psychological connectednessand mindfulness-related capacities, and heightened states of awe andtranscendent experiences, all processes linked to improvements inmental health amongst clinical and healthy populations. Nature-basedsettings can have inherently psychologically soothing properties whichmay complement all stages of psychedelic therapy (mainly preparationand integration) whilst potentiating increases in nature relatedness,with associated psychological benefits. Maximising enhancement ofnature relatedness through therapeutic psychedelic administration mayconstitute an independent and complementary pathway towardsimprovements in mental health that can be elicited bypsychedelics. Nature relatedness has been associated with a broad range of benefits topsychological health and well-being. It is a measurable, trait-likeconstruct of one’s self-identification with nature, defined as a sense of‘oneness with the natural world’  or a‘su2A receptor) can fosteceptor) and appreceptor). Furthereceptor). An increceptor). Thatpseceptor) is a notIncreased nature relatedness is just one of many psychological benefits thatcan be occasioned through therapeutic administration of psychedelics to bothhealthy and clinical populations. In clinical populations, psychedelicsubstances are currently being investigated for the treatment of majordepressive disorder and existential anxiety secondary to a terminal cancerdiagnosis , addictiThe mechanisms by which psychedelics confer benefits to well-being shareoverlap with how nature relatedness and nature contact yield benefits.Psychedelics have been associated with eliciting sustained increases inmindfulness-related capacities , facilitIn this review, we lay out the argument for utilising nature-based settings andpractices for some stages of psychedelic therapy (mainly preparation andintegration). The current model for psychedelic therapy requires thepsychedelic sessions themselves take place in a secure clinical setting forthe safety and predictability this provides. However, it may be thatincorporating some elements of nature contact and connection into thepreparation and integration phases could support the therapy model,potentially by amplifying some of the key therapeutic mechanisms, whilstalso increasing the likelihood that a patient will use nature as an ongoingresource. The potential of psychedelics to increase nature relatedness maywarrant the development of a new model of psychedelic therapy specificallyfocused on increasing nature relatedness for the sake of the well-being thiscan bring to the individual, and the nature-protective potential this couldbring to our communities. Such a model could be used with a healthy population, with whom the possibility of conducting thepsychedelic session itself in a secure and sheltered natural environmentcould be explored.The inherent healing power of nature has been recognised for centuries. Nature When it comes to mental health, contact with nature is associated withreductions in mental distress ,anxietyContact with nature is a strong predictor of psychological well-being, elevatirelatedness and psychological health andeudaimonic well-being, that is, subjective experiences linked toliving a life of virtue in pursuit of human excellence, associatedwith experiences of self-actualisation, vitality and personalexpressiveness  unitiveexperience may be central to the experiences of increasedinterconnectedness that can occur. Ego-dissolution hasbeen described as ‘a disruption of ego-boundaries, whichresults in a blurring of the distinction betweenself-representation and object-representation’  and is sNature contact itself can lead to greater connectedness,acting in a similar way to psychedelics. Just asego-dissolution under psychedelics is associated with thedissolution of self-referential boundaries , connectThe capacity of psychedelics and nature contact to increase asense of connectedness is notable, as a sense of‘disconnection’, alienation or isolation has beenimplicated with a broad range of mental illnessesincluding eating disorders , bipolarFeelings of interconnectedness are a core facet of the peakor mystical-type experiences that psilocybin can occasion. Other cThe experience of awe has been linked to enhanced well-being, life saPsychedelics may elicit strong emotional states during theacute experience, and challenging emotions such as fear orgrief are not uncommon . HoweverMajor depression diagnoses are characterised by high levelsof negative affect, with concurrent attenuated levels ofpositive affect , this beMindfulness has been defined as ‘being attentive to and awareof what is taking place in the present’ : 822. PsContact with natural settings can yield meditative,reflective mind states  and percPsychedelics have been shown to increase personality trait opennessto experience in an enduring way . OpennesPsilocybin was found to increase trait absorption  for at least a month post experience in healthyvolunteers . AbsorptClassical psychedelics such as psilocybin are currently designated asSchedule 1 drugs in the UK and USA, imposing onerous and highlyrestrictive regulations around their use in a research and therapeuticcontext , withclThe set  and setting framingpsychedelic usage is known to be a key determinant of experientialoutcomes . In clinPart of the efficacy of psychedelics when utilised in a therapeuticcontext appears to be through their capacity to act as catalysts oramplifiers of psychotherapeutic practices and processes . Nature There are many reasons to consider that therapeutic psilocybin sessions,despite the usual ‘inward focus’ could, for some participants, bebetter supported by being held in a natural setting than in a hospitalroom. Given that anxiety is a predictor of challenging or anxietyreactions to psychedelics , the impGiven that our species has spent almost its entire existence in naturalenvironments, it is likely we have an innate preference for them. The perHowever, despite all the benefits of calm, beauty and a sense ofinterconnectedness with all life that a natural setting couldpotentially bring, there are major barriers to attempting to holdpsychedelic sessions in nature. There may be issues with disturbances,privacy, inclement weather to name but a few . Indoor In situations where a clinical room is needed, bringing some naturalelements into the clinical space can be beneficial, for example plants(including those that belong to the session participant), nature-basedphotography and art and a nature-based backdrop. Screens depictingwoodland scenes were incorporated into the clinical protocol of thePhase II psilocybin for major depression treatment room at ImperialCollege London see .Even better than this would be the use of a hybrid indoor/outdoor secure,sheltered structure incorporating biophilic design elements  in a natEven when psychedelic sessions take place in a clinical environment,elements of nature contact and connection can be incorporated intosome aspects of the preparation and integration sessions (before andafter the psychedelic session). Preparation and integration sessionsfor individuals and groups of people undergoing psychedelic therapycould include some elements of nature immersion. The purpose ofpsychedelic preparation sessions is to establish trust between theperson who will be having the psilocybin experience and the therapistswho will be supporting them through the psychedelic session.Preparation sessions usually take place the day before, and includepsychoeducation about the likely effects of psilocybin, discussion ofthe participant’s intentions for the upcoming session, and some timefor establishing therapeutic rapport .HorticuOther nature immersion exercises could enhance two other key aspects ofpreparation and integration sessions: mindfulness training and talkingtherapy. The practice of Shinrin-Yoku (forest bathing), a Japaneseform of nature therapy and active mindfulness practice containsParts of both preparation and integration sessions contain standardtalking therapy elements: discussion of challenges in life, reflectingon traumatic experiences. Although some participants may feel morecomfortable having these discussions with their therapist in aclinical room, others may feel more comfortable ‘walking and talking’with their therapist in woodlands or gardens adjacent to the clinic which coAnother benefit of linking preparation and integration to an outdoornature-based setting is the possibility of establishing a connectionto the outdoors as a ‘therapy room’ one can later return to bythemselves to self-sooth .EstabliThe practice of journaling with an emotional focus has been used toeffectively supplement therapeutic psychedelic sessions . Nature Spending time in nature may be one of the most effective practices formaintaining the benefits of psychedelic sessions. However even ifpeople are not able to access beautiful nature on a regular basis,nature can still teach via metaphor. Two core metaphors offered bynature are of interconnectedness and seasonal change.Interconnectedness – recognition of being a small part of a greaterwhole, for example, demonstrable in many different ways, and seasonalchange – with the recognition that humans, like all of nature, gothrough cycles of light and dark; death and rebirth can betherapeutically beneficial. Just as nature goes through spring,summer, autumn and winter, so do human beings, and it can be helpfulto remember that dark times play their part in the cycle, and that‘this too shall pass’. Our culture is not synchronised with therhythms of nature: the valuing of productivity, ‘doing’ and happiness,and devaluing of retreat, sadness and loss, may be a contributingfactor in the current mental health crisis. Nature can teach humanshow to accept darkness as a fundamental part of life, and one thatprecedes growth. Recognition of this can be life changing for peoplewho suffer from depression. Metaphors about nature have beenincorporated into the ACE model for psilocybin for depression , an upcoAs well as incorporating nature contact into preparation and integrationsessions where the specified intention is to use the psychedelicsession for the improvement of mental health, a more specific model ofnature-focused psychedelic work could be developed, with the specifiedintention of enhancement of nature relatedness. In the former context,an internal focus is required, whereas in the latter context a moreoutwardly focused session could be facilitated. This would add to thearray of different therapeutic options in psychedelic work. As therapymodels expand and more is learned about optimising the experience,there is a need for a variety of options. Just as with mindfulnessmeditation practices there are exercises for inner and outer focusedmindfulness, this is also applicable to psychedelic usage. Whereas theinner focused clinical session might encourage connectedness to self,the outer focused session in nature could encourage interconnectednesswith the environment. There could be a therapeutic model developedwhich starts with inner-focussed work in an indoor clinical settingbefore then graduating onto an outdoor nature-based setting oncepeople are more experienced with psychedelic effects.Aside from their intrinsically psychologically restorative and soothingqualities, nature-based settings could enhance some aspects of thepreparation and integration phases of psychedelic therapy, and could undercertain circumstances be used for psychedelic sessions themselves, withoutany neglect of vital safety concerns regarding safeguarding vulnerablepeople under the influence of psychedelics. Such settings have the potentialto reduce anxiety and rumination, increase mindfulness, and elicittranscendent experiences and feelings of awe and connectedness. Furthermore,given the numerous demonstrated benefits to mental health associated withincreasing nature relatedness, maximising its enhancement in combinationwith psychedelic therapy could constitute an independent and complimentarypathway by which psychedelics can lead to improvements in mental health,with nature contact undervalued and heavily underutilised as ahealth-promoting resource .Future studies should seek to investigate the benefits of natural settings andhow they may complement (or supplement) clinical or indoor settings ingreater detail, employing fine-grained assessments of the settings inquestion, with thorough attention to potential risks. In addition, futurestudies should incorporate nature relatedness measures such as the NR-6, or the The chemist and inventor of LSD and discoverer of psilocybin Albert Hofmanncame to view the capacity of psychedelics to reconnect our increasinglynature-alienated species to the natural world as perhaps their mostimportant fundamental property. He recalled that among his most satisfyingexperiences were hearing people say things like ‘I grew up in the city, butonce I first took LSD, I returned to the forest’ : 4.


Background and Objectives: An essential part of occupational stress management is identifying target groups and developing a wellbeing program that tailors interventions to the specific needs of the target groups. This study aims to explore whether psychosocial risk determinants and organizational intervention objects differ across employees’ groups based on sociodemographic factors in a Lithuanian public primary healthcare institution. Methods: All 690 health workers of the institution were invited to participate (response rate 68%) in a cross-sectional survey between February and March 2017. The questionnaire contained items related to sociodemographic factors , 14 psychosocial risk determinants, and 10 organisational intervention objects. Results: The results of the study showed that differences by gender were not statistically significant except for one organisational intervention object . Only a few organisational intervention objects  had mean rank scores differing statistically across age and job seniority groups. Five organisational intervention objects  had mean rank scores differing statistically across education groups, and all organisational intervention objects (except stress management training) had mean rank scores differing statistically across occupational groups. Regarding psychosocial risk determinants, excessive work pace had mean rank scores differing statistically across age and job seniority groups. Four  and six psychosocial risk determinants  had mean scores differing statistically across education and occupational groups, respectively. Statistical significance was considered with p-value < 0.05 and 95% confidence interval. Conclusions: The findings showed that different psychosocial risk determinants and organizational interventional objects were emphasized by different sociodemographic groups in the institution, but they did not impact groups in the same measure. Therefore, it is crucial to start by determining the risk group’s specific needs before developing and implementing stress management programs. Patients have high expectations of the speed and quality of healthcare, so demand and time pressures are increasing. The digital transformation of health services and fast changing work environment are additional challenges for health workers. Problems observed in a number of healthcare institutions include increased staff shortage, staff turnover, sickness absenteeism, high workload, and work demands. In addition, the health workforce itself is ageing and there are problems of retention due to demanding working conditions and relatively low pay in some countries. Thus, it comes as no surprise that the health workforce is an occupational group whose wellbeing and job performance are adversely affected by job stressors. Many researchers have investigated the relationship between job stressors and job performance. Many studies also showed consistently negative relationships between job stressors and job performance ,2. PolicHowever, there is no simple solution to managing job stressors and to improving employees’ health and wellbeing at work. At an EU level, it is the employers’ obligation to improve the working environment by dealing with all types of risk, including psychosocial risks . Over thThis study is a cross-sectional study designed to examine health workers’ attitudes toward the psychosocial risk determinants and organizational intervention objects using a complex quantitative tool based on sociodemographic factors. All data was collected by paper questionnaires from February to March 2017. The sample consisted of 467 health workers employed in one of the largest primary healthcare institutions  in Lithuania. The institution employed 690 health workers in 2017. All health workers were invited to participate in the study. At the start of the study, health workers were provided with information about the study and 690 paper questionnaires were distributed; 468 questionnaires were returned compiled, one of which was damaged; 467 questionnaires (response rate 68%) were suitable for the analysis of the research results.Participation in the survey was voluntary with guaranteed anonymity and confidentiality under Lithuanian law which does not require ethical approval for this type of study. The study was authorised by the administration of the institution.The self-administrated questionnaire was used as an instrument for data collection. The instrument contained items adopted from an established questionnaire that was used for complex stress management study at Lithuanian automation and electrotechnical companies . The que-Fourteen items for psychosocial risk diagnosis: hazardous working conditions; work overload; excessive work pace; overtime; tight deadlines; unclear role; conflicting roles; being under-skilled for a job; responsibility for decision making and actions; lack of control over work pace; lack of control over work method; and interpersonal relationships . Cronbach’s alpha = 0.702;-Twenty-seven items for organisational intervention object ascertainment: social support , organisational support , participation in decision making ; communication ; justice of reward ; manager feedback ; stress management training (1 item); work–life balance (1 item); skills/abilities matching to the job demands (1 item); and variety of tasks (1 item);-Five groups on the basis of sociodemographic factors: gender , age , job seniority , educational level , and occupational status ).never or strongly disagree) to 5 . Prior to analyses, some items of the questionnaire were reversed so that higher scores showed greater negative impact of psychosocial risk determinant and more relevant to organisational intervention object.In total, the questionnaire consisted of 46 items, which were rated on a 5-point frequency scale ranging from 1 . A descriptive analysis was carried out to examine the sociodemographic groups of employees in the institution. Nonparametric tests for comparisons of the groups  were used then.Data were analyzed using the statistical software package IBM SPSS Statistics . In all analyses, statistical significance was considered with Gender. The observed differences by gender in the institution was not statistically significant except work–life balance. Women showed a higher average work–life balance score than men  and five organisational intervention objects  had mean rank scores differing statistically across education groups. Health workers who hold university degrees gave the highest scores for overtime, health workers who hold higher school degrees gave the highest scores for being under-skilled, health workers with other levels of education gave the highest scores for unclear role and conflicting roles  and all organisational intervention objects (except stress management training) had mean rank scores differing statistically across occupational groups. Doctors reported the most negative perception of workload, overtime, and tight deadlines; nurses notified the most negative perception of being under-skilled; other health workers declared the most negative perception of unclear role; and heads of units marked the most negative perception of responsibility (sibility . The higsibility .The study aimed to explore whether psychosocial risk determinants and organizational intervention objects differ across employees’ groups based on sociodemographic factors in a public primary healthcare institution. The findings of the study confirmed that different sociodemographic groups of health workers emphasized different psychosocial risk determinants and organisational intervention objects.In fact, there was no difference between gender groups. Only one organisational intervention object, work–life balance, was more relevant to women than to men. This may be due to gender imbalances in the institution—as is the case in all Lithuanian primary healthcare institutions. However, previous research findings provide contradictory information on gender differences in wellbeing at work . The stuIn summary, the results are in accordance with the studies indicating that occupational groups are the key factor that should be considered when managing psychosocial risks at organisation ,29,30,31Nevertheless, this study also has limitations. First, the study is a cross-sectional study and cannot make conclusions regarding causality. Second, the findings of this study are restricted to the public healthcare institution. Third, it did not include individual intervention objects that focus on helping individual employees to develop skills to manage, cope with, and reduce stress at work, whereas organisation-level interventions address the health and wellbeing of relatively large groups of workers in a uniform way . Fourth,The findings suggest that systematic assessment of risk groups on the basis of sociodemographic factors, especially occupational status, could facilitate psychosocial risk management in the public primary healthcare institution. It should be noted that the results of this study were based on a specific sample and that the method that was used to make internal comparisons of attitudes of different sociodemographic groups towards the psychosocial risk management inherently challenges generalizability and replicability of the results. Nevertheless, the findings might be generalized to countries with tax-financed universal healthcare system, considering the sociocultural context.The study has proposed a way of diagnosing psychosocial risks and of tailoring interventions to all health workers of the public primary healthcare institution by using a simple and robust tool. For future research, it would be useful to examine attitudes of different sociodemographic groups towards the psychosocial risks and organizational intervention objects in a private primary healthcare institution. In addition, the study and the findings can be a basis for developing stress management programs in public healthcare institutions and an incentive for new theories of qualitative studies.


While these statements offer an effective way of verifying programs handling basic data types and simple structured types, they are inappropriate as a mechanism for nondeterministic generation of pointers, favoring the use of insertion routines to produce dynamic data structures when verifying, via model checking, programs handling such data types.Software model checkers are able to exhaustively explore different bounded program executions arising from various sources of non-determinism. These tools provide statements to produce non-deterministic values for certain variables, thus forcing the corresponding model checker to consider relational bounds, that disregard values deemed invalid by the structure’s type invariant, thus reducing the state space to be explored by the model checker. Precomputing the relational bounds is a challenging costly task too, for which we also present an efficient algorithm, based on incremental SAT solving.We present a technique to improve model checking of programs handling heap-allocated data types, by taming the explosion of candidate structures that can be built when non-deterministically initializing heap object fields. The technique exploits precomputed We implement our approach on top of the CBMC bounded model checker, and show that, for a number of data structures implementations, we can handle significantly larger input structures and detect faults that CBMC is unable to detect.


This review compares and contrasts models of how these foundations are linked, focusing on a recent report in mammals that shows tonic horizontal cell release of GABA activating Cl− and HCO3− permeable GABA autoreceptors. The membrane potential of horizontal cells provides the driving force for GABAR-mediated HCO3− efflux, alkalinizing the cleft when horizontal cells are hyperpolarized by light or adding to their depolarization in darkness and contributing to cleft acidification via NHE-mediated H+ efflux. This model challenges interpretations of earlier studies that were considered to rule out a role for GABA in feedback to cones.How neurons in the eye feed signals back to photoreceptors to optimize sensitivity to patterns of light appears to be mediated by one or more unconventional mechanisms. Via these mechanisms, horizontal cells control photoreceptor synaptic gain and enhance key aspects of temporal and spatial center-surround receptive field antagonism. After the transduction of light energy into an electrical signal in photoreceptors, the next key task in visual processing is the transmission of an optimized signal to the follower neurons in the retina. For this to happen, the release of the excitatory neurotransmitter glutamate from photoreceptors is carefully regulated via horizontal cell feedback, which acts as a thermostat to keep the synaptic transmission in an optimal range during changes to light patterns and intensities. Novel findings of a recently described model that casts a classical neurotransmitter system together with ion transport mechanisms to adjust the alkaline milieu outside the synapse are reviewed. This novel inter-neuronal messaging system carries feedback signals using two separate, but interwoven regulated systems. The complex interplay between these two signaling modalities, creating synaptic modulation-at-a-distance, has obscured it’s being defined. The foundations of our understanding of the feedback mechanism from horizontal cells to photoreceptors have been long established: Horizontal cells have broad receptive fields, suitable for providing surround inhibition, their membrane potential, a function of stimulus intensity and size, regulates inhibition of photoreceptor voltage-gated Ca Output signaling from photoreceptors takes place at synaptic complexes comprising the photoreceptor terminal, horizontal cell synaptic processes, and bipolar cell dendrites, where visual information transfer and processing is initiated  channels in the photoreceptor synaptic terminal  to hyperpolarize the cell, typically from about −40 mV to as much as −60 mV. However, in response to a large spot of light, an identical hyperpolarization occurs initially but this is followed by inhibitory feedback from strongly hyperpolarized horizontal cells that produce a delayed depolarizing phase in the cone response. Confusing as it may seem, this depolarization is what was originally referred to as inhibitory feedback (since it was an inhibition of the hyperpolarizing response to light), but we currently recognize that the underlying mechanism is a disinhibition of the cone CaV channels.Partial inhibition of cone CaV channels, and we refer to the terms “inhibition” and “disinhibition” in the context of horizontal cell modulation of photoreceptor voltage-gated CaV channels. For example, inhibition of cone CaV channels occurs when horizontal cells are depolarized during steady-state conditions of darkness or low light. In earlier reports, before the role of cone CaV channels in feedback was established by Verweij et al.  the hyperpolarizing response to focal stimulation of a cone with light , interstitial current-induced external voltage changes, or “ephaptic coupling,” and pH-mediated surface charge screening effects. While similar descriptions of feedback from horizontal cells to photoreceptors have been reported in the retinas of non-mammalian vertebrates, the fewer reports in mammalian species have to account for significant underlying mechanistic differences from non-mammalian species  Non-mammalian vertebrates have not been proven to have a GABA vesicular release mechanism in horizontal cells similar to that in mammals. Instead, early discoveries in several non-mammalian vertebrates concluded that reversed GABA-uptake transporters in horizontal cells mediate the release of GABA , cones may be reprogrammed and respond to GABA application  horizontal cell tips. Analysis of pannexins in mouse horizontal cells shows sparse localization away from the invaginating tips of the horizontal cells  fluctuates in healthy brain tissues. Assessment of pH homeostasis in the vertebrate retina showed significant disparities from the earlier conception that pHo was one of the best-regulated homeostatic variables necessary for brain function -exchangers of the outer segment, and replenishment of K+ that effluxes via these and voltage-gated Kx channels of the inner segment, and pumping of Ca2+ from the synaptic terminal. ATP use in mouse rods is increased by a factor of 5 in the dark compared to bright light, with most of this due to increased Na+/K+-ATPase and Ca2+-ATPase activity , HCO3− transporters , V-ATPases, monocarboxylic acid transporters (MCTs) and carbonic anhydrase  alters the electric field in the membrane acting upon the channel voltage sensor moves, producing the same action that a more negative membrane potential does, i.e., decreasing the probability of channel opening and moving the measured half-maximal activation voltage to more positive potentials. The relation between the pH and activation midpoint shift is on the order of a 1 pH unit decrease causing a 10 mV negative shift of V½  relations made from these currents under the two conditions. When the I–V relations were divided by the driving force for Ca2+ and fit with a Boltzmann function to define the CaV channel activation curves, it was found that the half-maximal activation voltage shifted in mice, rats, and guinea pigs to a ~5 mV more negative voltage. A shift of the channel activation curve to more negative potentials in picrotoxin represents the disinhibition of cone CaV channel currents as channel open probability increases at physiological membrane potentials. These results imply that there is tonic GABA inhibition of cone CaV channels under mesopic conditions in these species.First, to what extent are GABARs involved in the steady-state inhibition of cone CaV channel activation curves is consistent with earlier proposals  appear to play the central role in the modulation of the cone CaV channel currents. The ρ-subunit-containing GABAR inhibitor TPMPA, the GABAAR inhibitor gabazine, and the glycine receptor inhibitor strychnine were tested for their ability to produce CaV channel activation curve shifts in cones, and only TPMPA produced activation shifts. This result identified ρ-subunit-containing GABA receptors as the mediators of this action of GABA. V channel activation curve with V½ decreasing by about 10 mV. TPMPA had a similar action on mouse cones, shifting the activation curve negative by about 6 mV. Neither gabazine nor strychnine produced activation curve shifts in rodents.GABA receptors containing ρ-subunits  the subsequent buffering of cleft pH, modulating photoreceptor transmitter release via surface charge effects on presynaptic cone CaV channels , a value typically in the range of −10 to −20 mV  GABA acting on horizontal cell GABARs autaptically and tonically , that move Cl− into cells electroneutrally using the Na+ and K+ gradients . This modest disinhibitory action induced by bumetanide could follow from the block of NKCC1 in horizontal cells, which would reduce intracellular [Cl−] to sufficiently low levels that GABAR activation no longer depolarizes them and might even produce hyperpolarization.Relatively positive equilibrium potential for ClRussell, . A speciRussell,  and woulRussell,  was showon curve . This ou−transporter in horizontal cells suggests that the removal of the depolarizing influence of GABA allowed other actions, such as increased HCO3− efflux, to become dominant and alkalinize the synaptic cleft, leading to the pH-dependent disinhibition of cone CaV channels. With tonic, the autaptic release of GABA by horizontal cells appearing to inhibit cone CaV channels in a manner due in part to its depolarizing effect on horizontal cells, the question remaining was what other cleft acidifying processes are initiated or increased when horizontal cells depolarize.The disinhibitory action that followed the block of the inward Cli) and internal calcium levels and both of these stimuli increase with depolarization , suggesting that the inhibitory effects of GABARs on cone CaV channel activation is due to conditions that NHE activity produced  and the membrane potential of horizontal cells. This means that cone CaV channel disinhibition is greatest in the most negative range of horizontal cell membrane potentials, such as during stimulation with bright light. In contrast, the −30 mV reversal potential of GABAR can add to depolarization of the cell in a manner that increases cleft acidification, which is amplified by the non-linear H+ efflux via NHEs responding to intracellular metabolically driven acidification.Another mechanism involving the efflux of a pH buffer from horizontal cells, the GABA-pH model reviewed here in the 3− permeability of the GABARs should be biophysically conserved in all GABARs, requiring no species-specific claim. The pH-dependence of feedback is reported throughout studies in vertebrate retinas, and any change to the retinal environment that alters pH buffering or changes the pH is well-recognized as being capable of modulating the voltage-dependence of photoreceptor CaV channel activation. Hepes (and other pH buffers) block feedback in all models, suggesting a broadly consistent mechanism, albeit mediated by different mechanisms , but given a cytoplasm-acidifying alibi in the case of hemichannel ephaptic feedback (Vroman et al., The results reported in Grove et al.  and revi3− flux in GABAR channels, presumably in the millisecond range. Most of the experiments reviewed from Grove et al. (Feedback is considered to consist of several components (Warren et al., e et al.  studied 3− semi-permeable membrane allows the pH-influencing effect in the cleft to be the fast component of feedback in this model, albeit it is somewhat slower than the truly instantaneous ephaptic coupling. It may be under-appreciated that the GABA-pH hybrid model attributes the tonic presence of GABA in the outer retina, experimentally confirmed in many of the figures in this review, as a gate for the presence of feedback. Once given this tonic GABA presence, changes to cleft pH are temporally linked to the speed of membrane potential change, as changes in horizontal cell membrane potential immediately change the driving force on HCO3−, followed by ion flux but not channel gating.The speed of feedback is a very important issue and it has been argued effectively that there are fast and slow components in zebrafish. In zebrafish, the fast component is accounted for by the virtually instantaneous hemichannel-mediated ephaptic coupling, and the much slower component is caused by pannexin-mediated ATP release and resultant pH buffering action (Cenedese et al., Some features of the different models are less amenable to interspecies conservation, a good example being the requirement for hemichannels in ephaptic feedback and their absence in mammalian retinas, suggesting that there is not hemichannel mediated feedback in mammalian retinas. This does not exclude AMPARs playing the traditional electric feedback role (Byzov and Shura-Bura, +/K+-ATPases, which utilize 1 ATP for every 3 Na+ pumped out in exchange for 2 K+ brought into the cell (Stahl, + extrusion by Ca2+-ATPases. The chemiosmotic theory holds that the synthesis of each ATP catalyzed by the H+-ATP synthase is coupled to the translocation of three to five protons depending on the type of synthase, from the internal mitochondrial compartment to the cytoplasm (Petersen et al., +-extruder NHE is itself exponentially related to pHi (Aronson et al., i (Aronson et al., + efflux to increase exponentially with horizontal cell depolarization.The origin of dark-induced acidification in the OPL  lies witl Stahl, , and to V channels. Since the role of the horizontal cell is to modulate photoreceptor output, horizontal cells limit this acidification by also introducing a pH buffer to the cleft pH as a function of their membrane potential. Cleft pH can rapidly increase during horizontal cell hyperpolarization due to voltage-driven efflux of HCO3− via tonically active GABARs.It is likely that, in low low-light conditions, depolarized photoreceptors, OFF bipolar cells, and horizontal cells contribute to this tonic acidifying influence that inhibits photoreceptor Cav, allowing glutamate release. The horizontal cell synaptic tips responding to the glutamate would be depolarized in these experiments. According to the GABA-pH hybrid model, GABA should decrease pHo under these conditions. This is indeed what was found to occur; superfusion of GABA decreased feedback, while picrotoxin increased it. Surround-evoked inward currents persisted in picrotoxin and GABA, albeit with magnitudes that follow the shifted Cav activation curves shown here in cones voltage-clamped at −40 mV, and treated with picrotoxin and muscimol. Verweij et al.’s (if interpreted in the context that the GABARs would be on cones, not horizontal cells. The direct action of GABARs at the cone would have led to a block of surround-induced inward current changes when they applied either picrotoxin or GABA. Instead, the result presented is consistent with the autaptic GABA-pH feedback mechanism. Such an action is not predicted by any other of the existing models proposed for feedback and appears to reflect the full extent of the actions of feedback from horizontal cells to voltage-clamped cones, underscoring the differences seen in non-mammalian vertebrates, where cones express GABARs (Wu, Voltage clamp recordings of inward current induced by “pure” surround illumination upon the already standing spot response in mammals (Verweij et al., et al.’s  results BARs Wu, .The inconsistent effects of picrotoxin on horizontal cell feedback have been an additional confound in some earlier feedback models testing GABA as a potential transmitter. Studies that failed to find an effect with picrotoxin may have suffered from a late-emerging oversight. Due to the addition of a methionine residue in some species’ ρ2 subunits, their GABARs may be less sensitive to picrotoxin block (Zhang et al., Changes in extracellular acidity in response to GABAergic activity have been described throughout the brain Chesler, . These aIn the olfactory bulb, signal processing requirements differ greatly from those in the eye. Synapses formed by olfactory receptors with periglomerular interneurons utilize GABAergic presynaptic feedback to modulate presynaptic Ca channels, albeit in a different manner than at photoreceptor synapses McGann, . This prV currents in cones and probed via the net effects of pharmacological manipulations, provide insight into steady-state levels of feedback. While all interventions that modulate the activation of the CaV current are not necessarily related to feedback from horizontal cells to photoreceptors, the dissection of steady-state conditions that effect feedback clarify in a stepwise manner the pathway that accounts for feedback. To fully appreciate this synaptic mechanism, all of these aspects should be considered simultaneously. A greater understanding of this novel feedback mechanism will be achieved with further testing using non-steady-state light conditions.The mechanisms reviewed here presented as changes to voltage-clamped CaAll authors: data curation, investigation, visualization, writing—review and editing. 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.


Background and Objectives: Neurodegenerative diseases that typically affect the elderly such as Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia are typically characterised by significant cognitive impairment that worsens significantly over time. To date, viable pharmacological options for the cognitive symptoms in these clinical conditions are lacking. In recent years, various studies have employed neuromodulation techniques to try and contrast patients’ decay. Materials and Methods: We conducted an in-depth literature review of the state-of-the-art of the contribution of these techniques across these neurodegenerative diseases. Results: The present review reports that neuromodulation techniques targeting cognitive impairment do not allow to draw yet any definitive conclusion about their clinical efficacy although preliminary evidence is very encouraging. Conclusions: Further and more robust studies should evaluate the potentialities and limitations of the application of these promising therapeutic tools to neurodegenerative diseases. Neurodegenerative diseases targeting the elderly are a public health priority throughout the world with significant medical, psychological and economic repercussions. The most common disorders are Alzheimer’s disease (AD), frontotemporal dementia (FTD) and Parkinson’s disease (PD). Their prevalence and incidence had dramatically increased with age over the last decades, and they are expected to continue to grow due to the continuous increase in the average length of life in most countries. Neurodegenerative diseases are not homogeneous in their clinical profiles and underlying pathophysiology, although they are typically characterised by significant cognitive impairment. Time and accuracy of diagnosis are crucial factors, as they would allow the planning of timely and appropriate clinical management. As no effective pharmacological treatments for cognitive and motor symptoms are currently available, in recent years various studies had started to investigate the potential contribution of neuromodulation techniques  in contrasting patients’ decay. After a presentation of the most prominent epidemiological and clinical features of each disorder, the present in-depth review reports the state-of-the-art neuromodulation techniques studies targeting cognitive impairment in neurodegenerative diseases. Our twofold aim is to show the preliminary evidence currently available in the field, and to suggest that further research should evaluate the potentialities and limitations of these promising therapeutic options.Alzheimer’s disease (AD) is a pervasive neurodegenerative disorder that represents more than 60% of dementia diagnoses among the elderly . The neuFronto-temporal dementia (FTD) is a neurodegenerative disorder which shares different commonalities with AD but, differently from AD does not involve hippocampal deterioration, thus preserving episodic and autobiographical memories ,18. ClinParkinson’s disease (PD) is classically characterised by a series of motor impairments that includes tremor, akinesia, rigidity and postural instability. It has also been extensively demonstrated that cognitive decline is a major, and often even more debilitating, symptom of PD . In manyTechnological achievements have recently made available potentially useful innovative tools to researchers and clinicians. For the aim of the present review, we will now focus on neurostimulation techniques. Neuroplasticity is one of the main targets of different cognitive, physical, pharmacological and neurostimulation protocols . NeuroplTranscranial magnetic stimulation (TMS) is a technique based on the perturbation of neurophysiological activity inducing a current through a non-invasive magnetic pulse over the skull . TMS canAnother way of stimulating brain plasticity is via the “transcranial electrical stimulation” which refers to transcranial direct current stimulation (tDCS) and transcranial alternating-current stimulation (tACS). These two techniques are based on the same principle: current flow from an electrode to another inducing electrophysiological modulation in the targeted brain region . The difAn EBSCO-, Google Scholar- and PubMed-based literature review on neuromodulation studies targeting neurodegenerative diseases was conducted. Combinations of keywords entered for enquiries were: “transcranial magnetic stimulation” OR “transcranial direct current stimulation” OR “brain stimulation” AND ‘‘Alzheimer’s disease’’ OR ‘‘Fronto-temporal dementia’’ OR ‘‘Parkinson’s disease’’. The review was further extended by considering all of the relevant articles reported in the references of each paper. Analysis has been primarily focused on methods regarding brain stimulation, patients’ characteristics, presence/absence of cognitive symptoms, study design and experimental protocols, quantification of stimulation parameters of interest and brain imaging data, where available. We excluded research on healthy subjects only and/or conducted in non-human animals. As this field of applied clinical research is innovative and thus one cannot expect to find a number of large randomized controlled clinical trials to be definitely assessed in terms of their efficacy, we also included in our review well-conducted small-scale studies that represent the majority of studies conducted so far.Considering its incidence and prevalence, AD is one of the most studied neurodegenerative disorder. A vast literature addressing possible therapeutic options to reduce patients’ cognitive impairment is thus available.Single-pulse TMS (spTMS) is commonly used to probe the circuit or understand plasticity and physiological response. For clinical purposes, it has been mostly used for the early detection of AD , early dRepetitive TMS (rTMS) is a protocol in which trains of multiple magnetic pulses are delivered at specific frequencies and time delays to produce long-lasting perturbation of cerebral activity . Theta bSince most of the cognitive functions impaired in AD are related to memory recall, problem-solving, reasoning and emotional control, prefrontal regions are the main targets of NIBS. Turriziani et al.  stimulatA recent systematic review  showed tMost studies have been conducted using an eight-shape coil, which can only stimulate the superficial part of the cerebral cortex, while other coils can stimulate brain regions located deeper by a factor of three . This kiThe DLPFC is the main but not the only target region for rTMS. Precuneus is a ventral superior parietal region involved in episodic memory, visuospatial processing and global state of consciousness since it is a functional core of the default mode network . ConnecSome studies explored the effect of rTMS on different locations that are thought to underlie cognitive functions involved in AD symptomatology. Lee et al.  stimulatIn a recent study, the stimulation of similar regions on the different daily session has been adopted by Sabbagh et al.  in a larFinally, the long-term effects of rTMS have been explored by Nguyen et al. . In theiThe most used transcranial electrical stimulation technique in AD treatment is the tDCS in its anodal configuration. However, most of these studies are single-case or pilot studies that show encouraging but necessarily preliminary results ,77.Some of the first evidence for the potential therapeutic benefits of tDCS comes from a study employing anodal tDCS on bilateral temporal regions for 30 min daily for five days . In thisHowever, the causal effect of tDCS has not been confirmed across all studies. For example, Cotelli et al.  studied Another study adopting six sessions of anodal tDCS on the left DLPFC for 20 min in two weeks failed to observe significant differences between real tDCS and sham in apathy scores, neuropsychiatric inventory (NPI), ADAS-Cog and Cornell depressive scale . AuthorsDespite some encouraging evidence, results of tDCS-based treatments are not always consistent across studies, highlighting the need for a larger sample size, integration with precise neurophysiological measures to better define the target of the treatments, and more coherence in experimental designs in terms of the duration and number of stimulation sessions and uniformity of clinical outcomes to obtain a clearer picture of tDCS efficacy in AD ,89. TablAlthough tACS showed potential in entraining specific frequency bands resulting in the modulation of cognitive functions in healthy subjects, only recently, a few efforts have been employed in the administration of this technique to AD patients. It is known that gamma oscillatory activity is abnormal in AD patients . Gamma aSome optogenetic studies suggested that externally driven gamma activity could reduce Aβ depositions and p-Tau levels . In a reBesides TMS as mentioned above and tDCS and tACS, there are other less diffused novel NIBS techniques that can directly or indirectly modulate cerebral activity such as the radio-electric asymmetry conveyer (REAC), focused ultrasound (FU) and transcranial pulse stimulation (TPS) with ultrasounds. The radio-electric asymmetry conveyer (REAC) is a biomedical device that allows the induction of a small current in a portion of biological tissue through the emission of very weak microwaves in the Wi-Fi frequency range . These mOne of the most limiting factors of pharmacological treatments is that most of the chemical particles in the blood flow are not able to pass the brain–blood barrier, a regulatory interface that determines the entrance of substances in the brain . FocusedNIBS techniques have only recently been applied to PPA treatments. The increasing evidence about the efficacy of neurostimulation techniques in treating neurodegenerative disorders can be inferred by the growing number of studies, reviews and meta-analyses published in recent years ,116. HowOne of the first studies on the effect of TMS with PPA has been performed by Finocchiaro et al. . This stAn approach similar to Finocchiaro et al. has been more recently adopted by Bereau et al. . A patieAnother study  employedThese single case studies explored the effect of rTMS over left prefrontal regions showing language-specific improvements in PPA patients despite stimulation not being accompanied by specific language treatments. However, a few recent studies have combined TMS with language-related training. The administration of rTMS over the left and right DLPFC in 10 nfvPPA patients seemed to facilitate online performance in an action naming task . In anotStimulation of bilateral DLPFC in patients with different subtypes of FTD has been adopted in another recent open-label pilot study . After tIn general, rTMS studies yielded promising results about the online enhancement of linguistic and cognitive abilities in PPA and a few bvFTD patients, even if their offline duration after stimulation is still a matter of debate and further studies are required. Stimulation of the DLPFC seems to support linguistic abilities and lexical retrieval, especially in patients whose semantic knowledge is not degraded, such as in nfvPPA patients. tDCS has been classically applied in a post-stroke aphasic patient , and morCotelli et al.  applied Also, the inferior frontal gyrus (IFG) has been selected as a target for tDCS protocols. Tsapkini et al.  applied Since one of the most significant limitations of the observed studies is the small sample size, the same group recently performed a study adopting the same experimental design involving a total of 36 PPA patients diagnosed with lvPPA, nfvPPA and svPPA . BenefitIn another cross-over sham-controlled study involving seven PPA  and three AD patients showing linguistic impairments, the inferior temporoparietal regions were targeted by tDCS . In thisMost of the studies here described adopted specific linguistic training designed to affect single features of language processing such as spelling or noun retrieval. Gervits et al.  adopted Interesting results have been observed in a protocol adopting tDCS alone without linguistic or cognitive training. Teichmann et al.  performeAnother very recent large sampled double-blind study recently explored the effect of anodal tDCS over the left prefrontal cortex without the contextual administration of linguistic training on clinical measures and intracortical connectivity measures such as intracortical facilitation (ICF) and short-interval intracortical inhibition (SICI) . These iRegarding bvFTD, a recent randomized, double-blind, placebo-controlled study  tested tSince PD and its cognitive correlates have a significant impact on the health care costs as well as on the quality of life (QoL) of both patients and their caregivers, it is urgent to identify intervention strategies to slow down cognitive deterioration. To this end, pharmacological treatments have failed at specifically addressing and ameliorating cognitive symptoms in patients with PD , while aEarly investigations were focused on the possibility of ameliorating PD symptoms using non-invasive brain stimulation. These results were initially summarised in a first meta-analysis by Elahi and co-workers  in whichIn particular, Benninger et al.  in a ranIn contrast, Brys et al.,  found thMore recently, repetitive deep transcranial magnetic stimulation (rDTMS) was used in patients with PD using the H5 coil for the low-frequency stimulation of the primary motor cortex, followed by the high-frequency rDTMS of the prefrontal cortex . The maiFinally, Fricke and co-workers  hypothesVarious studies investigated the effects of tDCS on cognition in PD patients. It was generally observed that anodal stimulation of the dorsolateral prefrontal cortex (DLPFC) resulted in significant improvements in WM , phonemiA promising approach considers the possibility to integrate cognitive training and brain stimulation. To this aim, in a recent study by Lawrence et al., the authors examined the different effects on cognitive function and functional outcomes in PD patients with MCI, of standard cognitive training (1), tailored cognitive training (2), tDCS stimulation (3), standard cognitive training in association with tDCS (4), or tailored cognitive training in association with tDCS (5). In all cases, tDCS consisted of anodal stimulation of the left DLPFC. All interventions lasted four weeks, with cognitive and functional outcomes measured at baseline, post-intervention, and follow-up. Results showed that, when compared to the control group, all of the five intervention groups demonstrated variable statistically significant improvement across executive function, attention/working memory, memory, language, activities of daily living (ADL), and QoL. Most importantly, it was shown that combining tDCS with tailored/standard cognitive training provided greater therapeutic effects . Similarn = 10); or group 2, placebo tDCS plus physical therapy (n = 10). The treatment, lasting two weeks, consisted of daily direct current stimulation application for 25 min during physical therapy. The long-term effects of the treatment were evaluated on clinical, neuropsychological, and motor task performance at the three-month follow-up. The authors pointed out an improvement in motor abilities and a reduction of depressive symptoms in both groups after the end of treatment and at the three-month follow-up. However, the Parkinson’s Disease Cognitive Rating Scale and verbal fluency test performances increased only in the anodal direct current stimulation group with a stable effect at follow-up.Finally, another study by the same group of researchers  investigTaken together, all of these studies showed that tDCS could produce a series of significant improvements in motor and non-motor symptoms in PD and that this may be a relevant tool to improve cognitive abilities in PD, providing a novel therapeutic strategy for patients with mild cognitive impairment. Neurodegenerative diseases are heterogeneous in their clinical profiles and underlying pathophysiology. In most cases, they share the presence of significant cognitive impairment, depending on the diseases themselves and their clinical stage. Due to the absence of effective pharmacological treatments for their most prominent cognitive symptoms, researchers and clinicians are in urgent need of valid tools to contrast patients’ decay. Non-invasive brain stimulation techniques such as TMS and tDCS have been shown to be safe and effective methods for improving cognitive and affective functions in neuropsychiatric disorders such as depression, anxiety and PTSD symptoms. As reviewed in the present paper, neuromodulation techniques may represent a promising tool for treating the cognitive symptoms in neurodegenerative conditions in the elderly, as the preliminary evidence provided by the pilot studies published so far is encouraging. However, as current research in the field has not reached a mature level already and thus its results should be considered necessarily as preliminary, our review points out the need for further and more robust studies including larger samples of patients and a more efficient integration of neuromodulation techniques and cognitive tools. A better definition of treatments’ targets and more coherence in experimental design and clinical outcomes will generate a clearer picture of neuromodulation techniques’ efficacy in these neurodegenerative conditions.To conclude, at this point in time, given the absence of large and robust studies able to provide strong evidence in favor of the use of these techniques with these clinical targets, one cannot draw any definitive conclusion about their efficacy although preliminary evidence is encouraging (please refer to


The multi-scale approach to conserving forest biodiversity has been used in Sweden since the 1980s, a period defined by increased reserve area and conservation actions within production forests. However, two thousand forest-associated species remain on Sweden’s red-list, and Sweden’s 2020 goals for sustainable forests are not being met. We argue that ongoing changes in the production forest matrix require more consideration, and that multi-scale conservation must be adapted to, and integrated with, production forest development. To make this case, we summarize trends in habitat provision by Sweden’s protected and production forests, and the variety of ways silviculture can affect biodiversity. We discuss how different forestry trajectories affect the type and extent of conservation approaches needed to secure biodiversity, and suggest leverage points for aiding the adoption of diversified silviculture. Sweden’s long-term experience with multi-scale conservation and intensive forestry provides insights for other countries trying to conserve species within production landscapes. The ongoing global loss of species and ecosystems  continues to be managed intensively using even-aged approaches for the production of timber, pulp and bioenergy. Within this production forest matrix, Sweden has increased both the spatial extent of protected forest areas and voluntary set-asides  and Scots pine (Pinus sylvestris), which comprise 80% of standing volume , and the majority of productive forest area is used for forestry. Despite only being the world’s 55th largest country, Sweden has the fifth largest total planted forest area  that is neither protected  nor available for commercial forestry  forest has increased from 2 to 4% in the last 20 years , likely resulting in part from the 1993 allowance to count birch as production stems when meeting regeneration requirements  is the sixth most common tree species by volume . The tree ages included in this “old” category are only a fraction of potential tree lifespans  reviews of conservation measures versus species’ habitat requirements (Johansson et al. Despite advancements to its multi-scale conservation efforts, the long-term viability of Sweden’s forest biodiversity has yet to be secured. We suggest that closing the remaining gap between the habitat requirements of forest species, and the habitat provided by Sweden’s forests, is more likely to occur if the multi-scale conservation approach is integrated with changes in the production forest matrix. To disregard this interdependence is to increase the risk that habitat gains in the protected aspects of the system are insufficient to compensate for habitat losses elsewhere Fig. . NotablyIn Sweden and other countries where the majority of forest land is used for wood production, the widespread adoption of more intensive forestry practices would decrease the diversity of forest habitats in those areas, and alter the effectiveness of conservation actions (Prugh et al. The successive adoption of more intensive forestry practices could thereby limit the capacity of multi-scale conservation to meet species habitat requirements Fig. . These hWhat is potentially a more direct path to achieving biodiversity goals Fig.  that bet2 emissions achieved (Heck et al. The degree to which intensive versus diversified forestry approaches are embraced over coming decades will depend on the extent that values, such as biodiversity conservation Table , influenIf Sweden’s forest future is to involve more diversified forestry practices, this may require the identification of suitable ‘leverage points’ to instigate change (sensu Meadows We suggest that achieving forest biodiversity conservation goals in Sweden, and in similar contexts internationally, will largely be determined by how well multi-scale conservation is adapted to, and integrated with, ongoing changes in the production forest matrix. Specifically, the degree to which forestry intensification versus diversification trajectories are embraced will determine the types and extent of conservation measures needed to conserve forest biodiversity over coming decades. As long as the status of forest biodiversity remains insecure, and forest habitat remains quick to lose but slow to recover, the use of more intensive forestry should raise concerns regarding resultant habitat loss and fragmentation, and the continued effectiveness of past conservation strategies. In contrast, the use of diversified approaches should be easier to integrate with multi-scale conservation, and adds to the ‘tool-box’ of means by which biodiversity targets can be hit. Teasing out and addressing the associated knowledge gaps will be a complicated but essential part to charting the most feasible course towards securing the status of forest species. Furthermore, because the issues we raise involve social-ecological systems, finding suitable solutions to balancing production, climate change and biodiversity goals will demand insights from a wide range of academic disciplines and stakeholders. More generally, as the drivers of intensified forestry appear to be replicated wherever industrial-scale production forestry is practised (Puettmann et al.