The Brazilian Early Childhood Friendly Municipal Index (IMAPI) is a population‐based approach to monitor the nurturing care environment for early childhood development (ECD) using routine information system data. It is unknown whether IMAPI can be applied to document metropolitan urban territorial differences in nurturing care environments. We used Brasilia, Brazil's capital with a large metropolitan population of 2,881,854 inhabitants divided into 31 districts, as a case study to examine whether disaggregation of nurturing care data can inform a more equitable prioritization for ECD in metropolitan areas. IMAPI scores were estimated at the municipal level  and at the district level . We developed a quantitative prioritization process for indicators in each IMAPI analysis, and those selected were jointly mapped in the socioecological model for the role of indicators in relation to the enabling environment for nurturing care. Out of 28 common nurturing care indicators across IMAPI analysis, only four were prioritized in both analyses: one from the Adequate nutrition, two from the Opportunities for early learning, and one from the Responsive caregiving domains. These four indicators were mapped as enabling policies, supportive services, and caregivers’ capabilities  and Effort, Coverage, and Quality (indicator's role). In conclusion, the different levels of nurturing care data disaggregation in the IMAPI can better inform decision‐making than each one individually, especially in metropolitan areas where municipalities and districts within metropolitan areas have relative decision‐making autonomy. The Brazilian Early Childhood Friendly Municipal Index (IMAPI) is an innovative population‐based approach created to systematically measure nurturing care for early childhood development (ECD) using routine information systems data at the municipal level. Brasilia was used as a case study to evaluate whether different levels of nurturing care data disaggregation  can inform more equitable prioritization for ECD investments in metropolitan areas. The two different levels of data disaggregation of the IMAPI can better inform nurturing care‐related decisions than each individual level in metropolitan areas where municipalities and districts within metropolitan areas have relative decision‐making autonomy. The Brazilian Early Childhood Friendly Municipal Index (IMAPI) is an innovative population‐based approach created to systematically measure nurturing care for early childhood development (ECD) using routine information systems data at the municipal level.Brasilia was used as a case study to evaluate whether different levels of nurturing care data disaggregation  can inform more equitable prioritization for ECD investments in metropolitan areas.We developed a quantitative prioritization process of indicators to analyse IMAPI at the municipal and district levels, and the selected indicators were jointly mapped into the socioecological model, specifying the roles of the indicator in the enabling environment for nurturing care.The two different levels of data disaggregation of the IMAPI can better inform nurturing care‐related decisions than each individual level in metropolitan areas where municipalities and districts within metropolitan areas have relative decision‐making autonomy. This municipality located in the Central‐Western region of the country has a large population of 2,881,854 inhabitants divided into 31 districts , which provides freely available data.The overall IMAPI‐M score is composed of 30 indicators across four Nurturing Care domains: Good health n = 14), Adequate nutrition (4), Opportunities for early learning (7), and Security and Safety (5). Indicator names and definitions can be found in Supporting Information Appendix 4, Adequa2.2.2n = 14), Adequate nutrition (2), Opportunities for early learning (7), and Security and Safety (6). We extracted the information of the IMAPI‐D score and domain subscores, and the scores of nurturing care indicators for the 31 districts through the IMAPI‐D website . For this analysis, one district (SIA district) was excluded because it is a commercial nonresidential area. Therefore, the analytical unit of IMAPI‐D was 30 districts.To make IMAPI‐D comparable to the IMAPI‐M, we used the list of 30 nurturing care indicators as a basis to select the same indicators at the district level. In the process of collecting data, two indicators, not included in the IMAPI‐M due to the lack of data, were included in the Security and Safety domain of the IMAPI‐D: 1) Water system supply  and (2) Sewage system . Additionally, three indicators were excluded due to the lack of data at the district level; two in the adequate nutrition domain (severe food insecurity and Coverage of information on child food consumption) and one in the Security and Safety domain (air pollution) ; Empowered com munities (indicators that characterize the vulnerability of nurturing care within the communities); Support services (indicators related to the availability of services and actions that directly impact ECD outcomes); Caregivers’ capabilities (indicators related to caregivers’ characteristics and their ability to provide nurturing care); Family capabilities (indicators related to families’ characteristics and their ability to provide nurturing care); and Child characteristics   Figure . This al2.3.1.2Effort ; Coverage (reflects the capacity of the ECD system to meet the demand of families for programmes and services); Quality ; and Result (reflects the effectiveness of ECD‐related programmes and services)  Figure . As with2.3.22.3.2.1We conducted a descriptive analysis of nurturing care scores and domain subscores of IMAPI‐M and IMAPI‐D. Districts were characterized by population size—small (up to 20 thousand inhabitants), medium (20–100 thousand inhabitants), and large (more than 100 thousand inhabitants); territory ; and income groups—low , medium‐low , medium‐high , and high .2.3.2.21. Indicator with a low score ; 2. IMAPI domain subscores ; and 3. Analysis of prioritized indicators  . Indicators with scores worse than the mean M (indicator with low scores) were then selected. In the second step, we checked for domains with low IMAPI‐M subscores. If there were any, indicators within low IMAPI‐M subscores were prioritized. If no low IMAPI‐M subscores other than Responsive Caregiving (limited to only one indicator) were present, then all indicators with overall low scores were prioritized.2.3.2.2.2In the first step, we computed for each nurturing care indicator the mean across the 30 districts (indicator mean D). Indicators with scores worse than the mean D (indicator with low scores) were then selected. In the second step, we identified nurturing care indicators with low scores across all districts with low IMAPI‐D domain subscores , followed by Enabling policies and Child characteristics with five (16.1%) indicators in each level, and Empowered communities, Caregivers’ capabilities, and Family capabilities with four (12.9%) indicators in each level  had low IMAPI subscores for the four domains of nurturing care, and one district (Vicente Pires) had low IMAPI in three domains  and IMAPI‐D (n = 6) analyses (Table n = 3) and Efforts (n = 1). Most of the nurturing care indicators prioritized by the IMAPI‐M analysis were related to Coverage (n = 5 out of 12), while the IMAPI‐D analysis prioritized two indicators of each Effort, Coverage, and Quality.In total, 14 nurturing care indicators were prioritized across IMAPI‐M  and Effort, Coverage, and Quality (indicator's role) Table .4We documented for the first time the strengths and weaknesses of the enabling nurturing care environment at the municipal and district level using the IMAPI approach, grounded in the NCF. We found that using IMAPI at different levels of nurturing care data disaggregation, municipal versus district in this instance can jointly strengthen the ability of decision‐makers to identify nurturing care indicators with low scores that require attention. Moreover, the quantitative prioritization process of nurturing care indicators with low scores and the innovative analytical framework combining the allocation of the indicators into the socioecological model and the role of indicators in the enabling nurturing care environment proved to be a sound process to identify gaps that will require investments to improve ECD outcomes among the most vulnerable.The two analyses of IMAPI conducted in this study, at municipal and district disaggregation levels, together prioritized 14 low scoring nurturing care indicators, with only 4 common nurturing indicators being prioritized. Despite the low overlap across IMAPI analyzes on the prioritized nurturing care indicators, the framework helped to identify the nature of the gaps to strengthen the nurturing care environment for ECD  is an example of a nurturing care programme that has received major investments to become a large‐scale programme within a short timeframe; however, PCF has not had strong monitoring and accountability of its Quality, which could be hampering effective results , can improve indicators of Results, such as prematurity, low birthweight, and congenital syphilis (prioritized by IMAPI‐M)  defining the purpose of the analysis, whether it is to pursue a comparative analysis  or to use both to select common prioritized indicators as presented in this manuscript, and (ii) discussing with managers and investors the pros and cons of including or removing indicators that may be or may not be available at the district level, as this can impact the prioritization process, as noted in the current analysis for the Security and Safety domain. Despite considerations and potential limitations, we found that in the case of urban territorial inequalities, disaggregated nurturing care data at different levels can inform a meaningful prioritization process.In summary, combining findings from different levels of IMAPI analyses, municipal and district levels, provides a powerful tool to prioritize investments and monitor the nurturing care environment within metropolitan areas. This is particularly true when district levels within large metropolitan areas have the autonomy to make their own investment decisions. Future qualitative research with decision‐makers is needed to further understand the utility of disaggregating IMAPI scores at different levels of political administration.The authors declare that there are no conflict of interests.GB and MBG conceptualized the prioritization process of nurturing care indicators at different levels of government. GB analysed the data with the support of JP and JLP. SEACK and JP coordinated the efforts of data collection of indicators in Brasilia. MEB, AS, JB computed IMAPI scores. GB interpreted the data, developed the first draft of the manuscript with intellectual inputs from MBG and RPE both, revised it critically through an iterative process. All authors revised and approved the final version of the manuscript.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


Advances in experimental design and equipment have simplified the collection of maximum metabolic rate (MMR) data for a more diverse array of water‐breathing animals. However, little attention has been given to the consequences of analytical choices in the estimation of MMR. Using different analytical methods can reduce the comparability of MMR estimates across species and studies and has consequences for the burgeoning number of macroecological meta‐analyses using metabolic rate data. Two key analytical choices that require standardization are the time interval, or regression window width, over which MMR is estimated, and the method used to locate that regression window within the raw oxygen depletion trace. Here, we consider the effect of both choices by estimating MMR for two shark and two salmonid species of different activity levels using multiple regression window widths and three analytical methods: rolling regression, sequential regression, and segmented regression. Shorter regression windows yielded higher metabolic rate estimates, with a risk that the shortest windows (<1‐min) reflect more system noise than MMR signal. Rolling regression was the best candidate model and produced the highest MMR estimates. Sequential regression models consistently produced lower relative estimates than rolling regression models, while the segmented regression model was unable to produce consistent MMR estimates across individuals. The time‐point of the MMR regression window along the oxygen consumption trace varied considerably across individuals but not across models. We show that choice of analytical method, in addition to more widely understood experimental choices, profoundly affect the resultant estimates of MMR. We recommend that researchers (1) employ a rolling regression model with a reliable regression window tailored to their experimental system and (2) explicitly report their analytical methods, including publishing raw data and code. Advances in experimental design and equipment have simplified the collection of maximum metabolic rate (MMR) data for a more diverse array of water‐breathing animals. However, little attention has been paid to the consequences of statistical choices on the estimation of MMR. We show that MMR estimates are sensitive to the type of statistical analysis used to generate them and provide a framework for model selection. It is regarded as a fundamental rate of life and a key indicator of physiological performance across tissues, cells and whole organisms  , for which data were collected over a wide body size range. We sought a model that (1) was easily applied to data from a variety of species, (2) relied on the least amount of subjective decision making, and (3) produced MMR estimates with reasonably low variance across individuals. Ultimately, this work demonstrates the importance of considering analytical methods when estimating MMR and provides a framework with which to approach such analyses.Here, we first estimated MMR and its time‐point within the oxygen depletion trace for two shark and two salmonid species using each of three analytical methods: rolling regression, sequential regression, and segmented regression. This allowed us to compare how applicable each analytical method may be across a variety of life histories: an inactive benthic shark, a demersal shark of medium activity level, and two relatively high activity level pelagic salmonids. We estimated MMR using multiple window widths within both rolling and sequential regression models to test for the effect of window width on MMR estimate. Second, we compared the resulting MMR estimates from all models within and across each species. Third, because the relationship between metabolic rate and body mass is foundational to the theories behind aerobic scope and metabolic ecology , and two highly active salmonid species, the Rainbow Trout Oncorhynchus mykiss   was then used to calculate oxygen consumption (MO2) using the equation. Vr is the respirometer chamber volume in liters, and Vf is the fish volume . Additionally, we estimated the time‐point at which MMR occurred along each individual oxygen consumption trace. All statistical analyses were carried out in R (R version 3.6.3 [2020‐02‐29]) and corresponding raw data and code are available online (see Data Availability). The R2 values of all regressions used to generate MMR estimates were above 0.9, and in most cases above 0.95.We collated maximum metabolic rate (MMR) data sets from a sedentary benthic elasmobranch, the California Horn Shark, Using simulated background respiration data, we tested the effectiveness of a signal‐to‐noise ratio analysis method in determining an appropriate regression window width to use in the analysis of MMR data . The 90% detection confidence limit reported by the manufacturers of our oxygen meters was 40 s, and this was the only variance within our experimental systems we could quantify confidently. Hence, we set 45 s between breakpoints as a more conservative minimum regression window and we removed slope coefficients from our output data frame that corresponded to window widths shorter than this.2.4=a Mb, where a and b are constants calculated for each model for each species, and M is body mass)  for all models. For each individual, we then added the raw residual MMR value (positive or negative) to the predicted MMR value at the mean body mass for each species to standardize the absolute MMR to the species‐specific mean body mass. Due to the small number of individuals tested in this study (n = 4), Gray Smoothhound were not quantitatively analyzed as we were not able to mass‐standardize their estimates and we instead reported their estimates unstandardized as mass‐specific values.We tested for the effect of model on MMR estimate within each species. Each MMR estimate was standardized to mean body mass for California Horn Shark (1.95 kg), Rainbow Trout (0.073 kg), and Atlantic Salmon (0.092 kg). To do this, we calculated residual MMR values as the difference between the measured and predicted MMR value within each species according to the relationship between MMR and body mass , we fit a linear mixed effects model with standardized MMR estimate as a function of model name with individual identity as a random effect .MMR occurred more than two minutes after an individual was placed in the respirometer chamber in 77%, 86%, 63%, and 85% of the California Horn Shark, Gray Smoothhound, Rainbow Trout, and Atlantic Salmon individuals, respectively, with the latest window occurring in a California Horn Shark after 11.5 min  smaller regression windows yielded higher estimates of MMR, (2) MMR was best estimated using a rolling regression model with a 1‐ to 2‐min window, and (3) the time‐point at which MMR occurs is often at least two minutes into the oxygen depletion trace and, hence, may be missed with certain analytical methods, such as with sequential regression or too short of a postexercise monitoring period. This study highlights the necessity of including thorough and detailed analytical methods in the design of respirometry experiments and cautions against directly comparing estimates made with extremely disparate experimental and analytical methods. Here, we outline the key considerations in applying these findings in the analysis of fish respirometry data.4.1In all cases, MMR estimates were sensitive to the window width used in analysis. All regression models required at least a minimum window width be chosen in order to estimate MMR and this choice remains somewhat subjective. If too short a window is used, MMR may be overestimated due to spurious non‐oxygen consumption variance in the system. However, attempting to guard against this with too long of a window width may unnecessarily underestimate MMR without adding significant variance‐handling benefits. At a minimum, raw traces of oxygen depletion over time should be visually checked to get a sense for how reasonable each potential window width and corresponding MMR estimate may be. Spurious changes in oxygen consumption, such as the example California Horn Shark trace in Figure While we found a negative relationship between MMR estimate and widow width in all species, this effect was considerably weaker in the small salmonids than the much larger sharks , rather than as a tool for comparing between models. Specifically, it cannot be used to compare the fit of two models that each use unique data sets, such as two oxygen consumption over time regression models where one spans across a window at 2–3 min and the other spans across 4–5 min within an oxygen depletion trace.When comparing between models and window lengths, it is important to note that we chose not to use R4.2respR make it simple to implement this model on raw data output from a wide variety of oxygen sensing equipment and improve reproducibility across studies , where MMR peaked up to five hours after exhaustive exercise. To have the best chance of catching the MMR window, an effort should be made to use the longest monitoring period possible for the tested species and experimental system. For example, measurement of oxygen depletion for California Horn Shark was ended once oxygen concentration within the respirometer chamber reached 80%, after which the flush pump was turned on, allowing for a long initial monitoring period while also protecting the individual against hypoxia. However, a long monitoring period should not be sought at the expense of significantly increasing the chamber‐to‐fish volume ratio, as this will reduce the strength of the oxygen depletion signal and increase the likelihood of measurement error.Respirometry experiments are often designed to use short, 3‐ to 5‐min monitoring periods  under the assumption that individuals will be maximumly aerobic during and immediately following strenuous exercise Brett, . Longer 4.4When plotted against body mass on a log‐log scale, the MMR estimates made with each model for California Horn Shark revealed a pattern of decreasing slope coefficients with increasing regression window widths Figure . This pa4.5Despite the rise in appreciation for metabolic ecology and the experimental methods required to estimate metabolic rate, the choice of analytical method has remained largely unstandardized. The implications of the choice of analytical methods are far‐reaching, from the quality of empirical studies and theoretical models, to the comparability of results across species and metabolic ecology's potential as a predictive tool ; Data curation (lead); Formal analysis (lead); Methodology (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Yangfan Zhang: Conceptualization ; Data curation ; Methodology (supporting); Writing‐review & editing (supporting). Nicholas C. Wegner: Data curation (supporting); Funding acquisition ; Project administration (supporting); Resources ; Supervision (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Nicholas K. Dulvy: Conceptualization (supporting); Formal analysis (supporting); Funding acquisition (lead); Methodology (supporting); Project administration ; Resources ; Supervision (lead); Writing‐original draft (supporting); Writing‐review & editing (supporting).


Oxytocin (OXT) neurons of the hypothalamus are at the center of several physiological functions, including milk ejection, uterus contraction, and maternal and social behavior. In lactating females, OXT neurons show a pattern of burst firing and inter‐neuron synchronization during suckling that leads to pulsatile release of surges of OXT into the bloodstream to stimulate milk ejection. This pattern of firing and population synchronization may be facilitated in part by hypothalamic glutamatergic circuits, as has been observed in vitro using brain slices obtained from male rats and neonates. However, it remains unknown how hypothalamic glutamatergic circuits influence OXT cell activity outside the context of lactation. In this review, we summarize the in vivo and in vitro studies that describe the synchronized burst firing pattern of OXT neurons and the implication of hypothalamic glutamate in this pattern of firing. We also make note of the few studies that have traced glutamatergic afferents to the hypothalamic paraventricular and supraoptic nuclei. Finally, we discuss the genetic findings implicating several glutamatergic genes in neurodevelopmental disorders, including autism spectrum disorder, thus underscoring the need for future studies to investigate the impact of these mutations on hypothalamic glutamatergic circuits and the OXT system. Oxytocin (OXT) neurons of the hypothalamus are involved in several physiological functions, including milk ejection, uterus contraction, and maternal and social behavior. Synchronized, burst firing of oxytocin neurons in the hypothalamus during lactation leads to pulsatile release of oxytocin into the bloodstream to stimulate milk ejection. Glutamate has been shown to influence this pattern of oxytocin neural activity in both in vivo and in vitro models. Extrahypothalamic glutamatergic inputs innervate oxytocin neurons, yet their role in modulating oxytocin neural activity outside the context of lactation is not fully understood and could be of relevance for neurodevelopmental disorders (NDDs), given the recent discoveries of several NDDs‐risk genes encoding for glutamatergic synaptic components. We propose that future studies directed at dissecting the role of glutamatergic circuits in regulating the OXT system and modulating social behaviors will enhance our understanding of the mechanisms of function of the OXT system, and that investigating the impact of NDDs‐associated mutation on the OXT system has the potential to uncover new pathophysiology that could be underlying social behavior deficits. We also emphasize the need for future studies to investigate whether hypothalamic glutamatergic circuits contribute to physiological functions other than parturition and lactation in females and/or males in vivo. Furthermore, we speculate that investigating the impact of mutations in glutamatergic genes on these circuits, as well as on the OXT system, will be of significant relevance to neurodevelopmental disorders characterized by social behavior deficits, including autism spectrum disorder (ASD).2–1), whereas a small percentage of these cells display intermittent activity separated by periods of quiescence, which they described as a “phasic” pattern of discharge. Furthermore, they reported that 10–20 s prior to increases in intramammary pressure indicative of milk ejection, about half of the recorded neurosecretory cells accelerate to a high‐frequency rate of discharge (30–80 spikes s–1). This high‐frequency firing lasts for 2–4 s, occurs at regular intervals of 4–8 min with transient periods of inhibition, and is strongly influenced by the number of suckling pups. These findings led to the speculation that high‐frequency or “burst” firing underlies the release of pulses of OXT into the bloodstream during lactation, when OXT action on the mammary gland is needed for milk let‐down. This speculation was supported and further delineated by a subsequent study conducted in 1977 by Poulain and colleagues, who demonstrated that, in both the PVN and SON, there are at least two distinct sets of neurosecretory cells, one of which displays “burst” firing during milk ejection (OXT neurons) and the other that displays an asynchronous “phasic” pattern of firing in response to hemorrhage (vasopressin [AVP] neurons). The background firing rates of neurosecretory cells were also further differentiated as fast continuous (> 3 spikes s–1) or slow irregular (< 3 spikes s–1), the latter of which was more common.Much of our knowledge about the characteristics of OXT neurons is gleaned from in vivo electrophysiological experiments conducted in the 1970s and 1980s, which were performed on anesthetized lactating female rats.Following these findings, it was theorized that, for OXT to be released into the bloodstream at high levels during lactation, OXT neurons must fire bursts at the same time. To address this theory, Belin, Moos and Richard investigated the relationship of the firing patterns of multiple OXT neurons by carrying out paired extracellular recordings in bilateral SON nuclei or PVN/SON nuclei in lactating female rats.3These ground‐breaking studies performed in vivo were followed by several in vitro studies, which used acute hypothalamic slices and organotypic slice cultures to further inform the mechanisms of neuronal activity. In vitro studies offer the advantage of being able to control the extracellular environment and perform more stable intracellular recordings. Furthermore, the general pattern of activity of hypothalamic neurosecretory cells in vivo has also been observed in vitro, with the majority of cells engaging in spontaneous slow irregular firing, whereas some display fast continuous or burst firing patterns.2+, OXT and glutamate,More targeted electrophysiological recordings of hypothalamic neurosecretory cells in vitro became possible with the development of specific antibodies to identify these cell types, which could be used in conjunction with dye‐labeling of recorded neurons to differentiate between OXT and AVP neurons.4The idea of using OXT to stimulate the firing of OXT neurons was formulated based on early evidence suggesting that OXT neurons are capable of regulating their own activity and the paracrine release of OXT by a positive feedback mechanism. Intracerebroventricular administration of OXT in lactating female rats was found to cause an increase in the firing frequency and in the burst amplitude and frequency of OXT neurons.Similar mechanisms of OXT action on hypothalamic OXT neurons has also been observed in vitro. OXT administered through medium to slices of hypothalamus taken from adult male or lactating female rats led to an increase in OXT release, which could be blocked by an OXT antagonist.55.1l‐glutamate directly into the PVN of male rats also led to increased concentration of OXT in the blood, which was coupled with decreased heart rate.l‐glutamate infusion.l‐glutamate infusion, but reported no changes in central OXT levels. It is possible that the microdialysis and RIA approach was not sensitive enough to detect changes in PVN dialysate OXT, whereas changes in plasma OXT were of a high magnitude (seven‐fold increase) and could be easily detected by these methods.The excitatory amino acid neurotransmitter glutamate has been shown to play an important role in stimulating OXT activity within the hypothalamus, thereby regulating OXT release.S,3R)‐l‐amino‐ cyclopentane‐1,3‐dicarboxylic acid had no effect. However, further investigation revealed a partial role for NMDA receptors in OXT release, given that co‐application of AMPA and NMDA agonists in the SON or PVN stimulated increases in plasma OXT that were then blocked by either AMPA or NMDA antagonists .dl‐2‐amino‐5‐phosphonopentanoic acid (AP5) into the SON respectively increased or decreased the firing rate of OXT neurons and burst amplitude. However, it is critical to note that NMDA and AP5 did not affect the occurrence of bursts in non‐bursting OXT neurons. Therefore, Moos and colleagues reported that, although both NMDA and AMPA receptors are implicated in the enhancement of OXT neural activity and the stimulation of OXT release in vivo, NMDA receptors alone do not facilitate burst occurrence.In an effort to further understand the role of glutamate receptor subtypes in regulating OXT neural activity, OXT release and its physiological relevance, the effect of specific excitatory amino acid agonists and antagonists on OXT release in lactating rats was assessed. Glutamate receptors can be ionotropic (fast) or metabotropic (slow), with the former consisting of NMDA receptors and non‐NMDA receptors (AMPA and kainate).5.2d‐glutamylglycine attenuated spontaneous and evoked EPSPs.Electrophysiological studies in male hypothalamic acute slices and organotypic slice cultures have further supported a stimulatory role for glutamate. Application of glutamate and its agonists kainate and quisqualate elicited excitatory postsynaptic potentials (EPSPs) in hypothalamic neurons,Although outside the scope of this review, it should be noted that other key neurotransmitters, such as GABA,6Evidence for glutamatergic synaptic innervation of hypothalamic magnocellular neurosecretory cells has been supported by both structural and functional studies. Several ultrastructural immunocytochemistry studies conducted in male and female rodents have revealed that presynaptic axon terminals with glutamatergic immunoreactivity make contact with dendrites and cell bodies of AVP and OXT neurons. Approximately 20% of all presynaptic axon terminals on hypothalamic neurosecretory cells are glutamatergic, with many forming “shared” synapses in which terminals make contact with two or more postsynaptic cell bodies/dendrites.These discoveries coupled with observations concerning the effects of glutamate on OXT neuron activity brought the attention of the field to the potential role of local glutamatergic synaptic circuits in synchronizing the firing of OXT neurons. Activation of local circuits with glutamate microdrop application was shown to cause an increase in the frequency of EPSPs/EPSCs in some neurons,Direct evidence for synchronous synaptic inputs to OXT neurons from local glutamate circuits came from electrophysiological recordings in organotypic slice cultures of the hypothalamus. These slice cultures retain a certain number of cells intrinsic to the slice section and maintain the gross synaptic organization of the original tissue after several weeks in culture.73H]D‐aspartate coupled with autoradiography in male rats.3H]d‐aspartate, indicating that these nuclei are interconnected by glutamatergic neurons. It is important to note that, although the approach applied in these studies provided important information on the origins of excitatory inputs to the hypothalamus, it lacked specificity because it is not selective for inputs to OXT neurons; therefore, the definite identity of these inputs could not be accurately determined. Recently, Tang and colleagues applied a retrograde tracing approach in females which allowed for the detection of afferents that specifically innervate OXT neurons. Using a viral system based on a retrograde trans‐synaptic EnvA‐pseudotyped G deletion‐mutant rabies virus in combination with a helper adeno‐associated virus expressed under the OXT promoter,Although studies conducted in vitro have greatly informed our understanding of the patterns of OXT neural activity and the release of OXT peptide in response to glutamate, the question of how sensory stimuli in the environment translate to neural signals to affect the pattern of hypothalamic OXT neurons remains of interest. Some insight on this matter emerged from studies in female rats where somatosensory information from the uterus, during parturition, or the nipples, during suckling, has been shown to trigger OXT release via the activity of A2 adrenergic afferents from the nucleus tractus solitarius of the brainstem to the PVN and SON.8Our enhanced understanding of the role that glutamate plays in regulating OXT neural activity and the presumption, based on independent observations that OXT neurons are innervated by glutamatergic afferents, raises new questions about the physiological relevance of the hypothalamic glutamate‐OXT circuits. It has been demonstrated, based on several in vivo studies, that intrahypothalamic glutamate may contribute to OXT neuron activity and release during lactation in vivo9Cntnap2, a member of the neurexin family of cell adhesion molecules, Penagarikano and colleagues found that Cntnap2‐deficient male and female mice had lower levels of PVN‐OXT neurons and reduced levels of OXT in whole‐brain extracts compared to wild‐type littermates.Cntnap2‐deficient mice and that chemogenetic activation of endogenous OXT neurons in the Cntnap2‐deficient mice similarly produced behavioral improvements. Lower levels of PVN‐OXT neurons, specifically in the medial region of the PVN, were also recently reported in a Shank3‐deficient male mice, which harbors a mutation in the Shank3 gene that encodes for a postsynaptic density scaffolding protein of the glutamatergic synapse.Shank3‐deficient male rats in which deficits in social recognition memory, attention and synaptic plasticity were ameliorated by i.c.v. administration of OXT.Grin1‐deficient mice demonstrated that subchronic intraperitoneal treatments with OXT in both males and females increased sociability in the three‐chamber test, yet the structure and function of hypothalamic OXT neurons were not assessed in these mice and it is unknown whether Grin1 mutations affect the hypothalamic glutamate‐OXT system.Glutamatergic signaling plays a pivotal role in early brain development by regulating the proliferation and differentiation of neural progenitor cells,Currently, the field is well‐poised to address these questions given recent technological advancements.10The causality between OXT and ASD has been extensively discussed in the last two decadesThe authors declare that they have no conflicts of interest.Amanda Leithead: Conceptualization; Writing – original draft; Writing – review & editing. Jeffrey G. Tasker: Supervision; Writing ‐ original draft; Writing – review & editing. Hala Harony‐Nicolas: Conceptualization; Funding acquisition; Resources; Supervision; Writing – original draft; Writing – review & editing.https://publons.com/publon/10.1111/jne.13061.The peer review history for this article is available at


Domestic livestock grazing has caused dramatic changes in plant community composition across the globe. However, the response of plant species abundance in communities subject to grazing has not often been investigated through a functional lens, especially for belowground traits. Grazing directly impacts aboveground plant tissues, but the relationships between above‐ and belowground traits, and their influence on species abundance are also not well known. We collected plant trait and species relative abundance data in the grazed and nongrazed meadow plant communities in a species‐rich subalpine ecosystem of the Qinghai–Tibet Plateau. We measured three aboveground traits  and five belowground traits . We tested for shifts in the relationship between species relative abundance and among all measured traits under grazing compared with the nongrazed meadow. We also compared the power of above‐ and belowground traits to predict species relative abundance. We observed a significant shift from a resource conservation strategy to a resource acquisition strategy. Moreover, this resource conservation versus resource acquisition trade‐off can also determine species relative abundance in the grazed and nongrazed plant communities. Specifically, abundant species in the nongrazed meadow had aboveground and belowground traits that are associated with high resource conservation, whereas aboveground and belowground traits that are correlated with high resource acquisition determined species relative abundance in the grazed meadow. However, belowground traits were found to explain more variances in species relative abundance than aboveground traits in the nongrazed meadow, while aboveground and belowground traits had comparable predictive power in the grazed meadow. We show that species relative abundance in both the grazed and the nongrazed meadows can be predicted by both aboveground traits and belowground traits associated with a resource acquisition versus conservation trade‐off. More importantly, we show that belowground traits have higher predictive power of species relative abundance than aboveground traits in the nongrazed meadow, whereas in the grazed meadows, above‐ and belowground traits had comparable high predictive power. Species abundance with and without intensive grazing can be predicted by both aboveground and belowground traits that are associated with a resource acquisition vs. resource conservation trade‐off. Intensive grazing can alter the predictive abilities of aboveground and belowground traits for species abundance. The selection of these traits was based on their strong associations with plant resource acquisition and resource conservation strategies  whether grazing and grazing removal alter plant aboveground and belowground traits so that they can shift from resource conservation to resource acquisition in Qinghai–Tibet Plateau; (b) whether the resource acquisition versus resource conservation trade‐off reflected in both aboveground and belowground traits can determine species relative abundance in the nongrazed and grazed meadows; and (c) whether aboveground and belowground traits have the comparable predictive power of above‐ and belowground traits on species relative abundance in the grazed and nongrazed meadows.22.1Elymus nutans, Kobresia humilis, and Thermopsis lanceolata, and soils are classified as alpine meadow soils  and one grazed meadow. The land‐use histories over the last 30 years for the grazed and nongrazed meadows were obtained by interviewing local farmers. Since year 2000, the Chinese government has established the fence to protect some areas of this grazed meadow, thereby resulting in a nongrazed meadow. Thus, the nongrazed meadow  had not been grazed for 18 years prior to the study. Yak is the only disturbance, and grazing by small mammals has not been reported or observed in either the grazed or the nongrazed meadow.2 quadrats in each of the grazed and nongrazed plots  and five belowground traits   Table . Measure]) Table  and Berg]) Table , and fur]) Table . Specifi2.4vegan” R package  to evaluate whether species trait values varied between the grazed and the nongrazed meadows can be significantly differentiated by these eight aboveground and belowground traits. We thirdly tested whether species relative abundance for all species in the nongrazed and grazed meadows can be well predicted by PCA axis (PC1 and PC2) using linear regression. We also examined whether species relative abundance of each plant species can be well predicted by its responding aboveground and belowground traits in the nongrazed and grazed meadows individually using linear regression. Finally, we used a variance partitioning analysis to quantify the relative contribution of aboveground and belowground traits to species relative abundance for all species in the grazed and nongrazed meadows, thereby revealing whether aboveground traits can be a good proxy for belowground traits in predicting species relative abundance for all species in the nongrazed and grazed meadows, respectively. Specifically, species relative abundance with traits as explanatory variables can be divided into four complementary components: (a) “purely aboveground traits,” variance explained by aboveground traits alone; (b) “shared aboveground and belowground traits,” variance explained by both aboveground traits and belowground traits; (c) “purely belowground traits,” variance explained by belowground traits alone; and (d) “unexplained residual variation”  than aboveground traits (31%) in predicting species relative abundance  in species relative abundance. Moreover, variance explained by both above‐ and belowground traits is very high (68%), which might be attributed to the strong correlations between aboveground and belowground traits. These results indicate that grazing may select for species with high resource acquisition strategies, possibly with a very narrow range of trait values, resulting in above‐ and belowground traits with comparable predictive power for species relative abundance. Compared with belowground traits, aboveground traits are relatively easier to measure and have been much more widely studied across global communities  than the aboveground traits (32%) in predicting species relative abundance. One possible reason is that resource competition may make resource conservation strategy prevail in the nongrazed meadows, which in turn may allow for a wider range of trait combinations, thereby leading to above‐ and belowground traits with different predictive power for species relative abundance. Indeed, our previous works have reported that competition in nitrogen uptakes was a major influence on community assembly in the nongrazed meadows in this subalpine ecosystem  in species relative abundance should have been measured and tested. Many studies on the Tibetan Plateau involving fertilization treatments on the grazed and nongrazed meadow communities have established soil‐available nitrogen and phosphorus as critical limiting nutrients with substantial impacts on community structure ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Kai Jiang: Data curation ; Formal analysis ; Methodology ; Writing‐review & editing . Weichen Hou: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Validation . Yang Zhao: Investigation ; Methodology . Xinhang Sun: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Validation ; Writing‐original draft . Lu Wang: Investigation ; Methodology . Yike Zou: Investigation ; Methodology . Zhenhao Zhu: Investigation (supporting). Hui Zhang: Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Supervision ; Validation ; Writing‐original draft ; Writing‐review & editing .Appendix S1Click here for additional data file.


Acute myeloid leukaemia (AML) is a clinically and molecularly heterogeneous disease characterised by uncontrolled proliferation, block in differentiation and acquired self‐renewal of hematopoietic stem and myeloid progenitor cells. This results in the clonal expansion of myeloid blasts within the bone marrow and peripheral blood. The incidence of AML increases with age, and in childhood, AML accounts for 20% of all leukaemias. Whilst there are many clinical and biological similarities between paediatric and adult AML with continuum across the age range, many characteristics of AML are associated with age of disease onset. These include chromosomal aberrations, gene mutations and differentiation lineage. Following chemotherapy, AML cells that survive and result in disease relapse exist in an altered chemoresistant state. Molecular profiling currently represents a powerful avenue of experimentation to study AML cells from adults and children pre‐ and postchemotherapy as a means of identifying prognostic biomarkers and targetable molecular vulnerabilities that may be age‐specific. This review highlights recent advances in our knowledge of the molecular profiles with a focus on transcriptomes and metabolomes, leukaemia stem cells and chemoresistant cells in adult and paediatric AML and focus on areas that hold promise for future therapies. Acute myeloid leukaemia (AML) is a clinically and molecularly heterogeneous disease characterised by uncontrolled proliferation, perturbed myeloid differentiation and acquired self‐renewal of hematopoietic stem and myeloid progenitor cells. This review highlights recent advances in our knowledge of the molecular profiles of adult and paediatric AML with a focus on transcriptomes and metabolomes, leukaemia stem cells and chemoresistant cells. AMLAcute myeloid leukaemiaAPLacute promyelocytic leukaemiaATOarsenic trioxideATRAall‐trans‐retinoic acidCHclonal haematopoiesisESenrichment scoreFAOfatty acid oxidationGSEAGene Set Enrichment AnalysisHSCshaematopoietic stem cellsITDinternal‐tandem duplicationsLSCleukaemia stem cellMDSmyelodysplastic syndromesMPNmyeloproliferative neoplasmsMRDminimal residual diseaseNGSnext‐generation sequencingpLSC6paediatric 6 gene LSC scoreTARGETTherapeutically Applicable Research to Generate Effective TreatmentsVAFvariant allele frequencies1www.cancerresearchuk.org). Incidence increases with age, and 70% of patients diagnosed with the disease still die from this disorder. The current overall survival rate in children is only 60–70% and thereafter falls progressively with age to < 5% in those aged over 65 years. Both children and adults die within 5 years from diagnosis due to a combination of relapse  is a haematological malignancy, with incidence rates having grown by 29% since the early 1990s  or myelodysplastic syndromes (MDS). Mutations acquired over time can lead to clonal enrichment in the haemopoietic compartment in the absence of any clinical manifestations termed clonal haematopoiesis (CH). Such clones may then acquire subsequent mutations transforming into malignant clones that underlie the development of AML. In children, the vast majority of patients present with de novo AML, whilst in adults, AML more commonly arises from an underlying MPN or MDS, and this characteristic increases with age . . 115]. I7Challenges remain including assessing the correlations between leukaemia‐specific markers across the different cytogenetic and mutational subgroups in adult and paediatric AML, not only in identifying the surface markers that can aid subclone identification but also MRD detection that could be used to guide patient‐specific treatment strategies, which potentially involve targeting the leukaemia‐specific transcriptional and metabolic programmes. It is clear that paediatric AML is a disease distinguishable from adult AML with regard to the gene mutation spectrum. However, current risk stratification approach only includes common mutations based on prognostic values when the total number of discovered mutations surpasses that. It may be more clinically significant to consider allelic burden profiles of AML‐associated mutations  in order to decide on targeted therapies and risk categorisation. It is also important to closely study novel mutations and transcriptional profiles post‐therapy so as to gain perspectives on clonal evolution and instrumentation to relapse. Indeed, targeted sequencing of known mutations is limiting and may not be optimal for advanced disease, therapy‐related and secondary disease or for paediatric subgroups. The ensuing interpretation of the molecular profile in AML patients across the age range and most critically at different phases of disease  can be prominent in better appreciating the disease pathogenesis and promise an improvement in patient management approaches. Moreover, conventional combination therapies to target the chimeric clonal subpopulations have a possibility of precarious effects since either pre‐existing or acquired therapy‐resistant clones could proliferate and prevail to result in a fatal relapse. Transcriptional profiling has revealed significant and targetable features of AML cells and being able to identify and specifically kill these chemoresistant clones is the current challenge. Efforts across the globe including Genomics England are endeavourering to bring together ‘omic’ profiling and clinical data to better inform on best treatments for individual patients.The authors declare no conflict of interest.


Household gender roles influence infant and young child feeding behaviours and may contribute to suboptimal complementary feeding practices through inequitable household decision‐making, intra‐household food allocation and limited paternal support for resources and caregiving. In Igabi local government area of Kaduna State, Nigeria, the Alive & Thrive (A&T) initiative implemented an intervention to improve complementary feeding practices through father engagement. This study describes household gender roles among A&T participants and how they influence maternal and paternal involvement in complementary feeding. We conducted 16 focus group discussions with mothers and fathers of children aged 6–23 months in urban and rural administrative wards and analysed them using qualitative thematic analysis methods. Most mothers and fathers have traditional roles with fathers as ‘providers’ and ‘supervisors’ and mothers as ‘caregivers’. Traditional normative roles of fathers limit their involvement in ‘hands‐on’ activities, which support feeding and caring for children. Less traditional normative roles, whereby some mothers contributed to the provision of resources and some fathers contributed to caregiving, were also described by some participants and were more salient in the urban wards. In the rural wards, more fathers expressed resistance to fathers playing less traditional roles. Fathers who participated in caregiving tasks reported respect from their children, strong family relationships and had healthy home environments. Our research findings point to the need for more context‐specific approaches that address prevalent gender normative roles in complementary feeding in a variety of settings. •Some mothers and fathers still hold traditional attitudes about their roles in feeding and caring for children.•Traditional gender normative roles of fathers limit their involvement in the ‘hands‐on’ activities that support feeding and caring for children.•Some mothers and fathers are performing ‘non‐traditional’ gender normative roles despite social disapproval.•The shifts from the traditional roles of mothers and fathers are more salient in urban compared with rural wards. Our objectives were to identify and thematically describe how household gender roles influence complementary feeding within the context of a father‐focused complementary feeding intervention.The study was conducted in six administrative wards of the Igabi local government area of Kaduna State, Nigeria. Wards are the smallest of the three levels of administrative units  in Nigeria , a local Nigerian organization. As part of the implementation, ICARE trained 13 community‐based organizations (CBOs), 18 religious and traditional leaders (14 imams and 4 pastors) and 60 community health extension workers (CHEWs) and provided them with resources to carry out complementary feeding social and behaviour change communication (SBCC) activities. The resources included complementary feeding counselling cards for CBOs, talking points and sermon guides for religious and traditional leaders to educate fathers during meetings and religious services. The CHEWs were also provided with counselling cards and feeding bowls to use during home visits to counsel and reinforce complementary feeding messages with mothers and fathers (when present). CHEWs counselled mothers during home visits on feeding key food combinations designed to increase children's consumption of vitamin A‐rich vegetables and animal source foods and provided them with a feeding bowl that showed nutritious foods and the quantity of food to feed infants and children at different ages. In addition, complementary feeding SBCC messages were broadcast through radio and TV advertisements and mothers and fathers received leaflets with the same messages.2.4After 12 months of intervention implementation, separate FGDs using specific discussion guides were conducted for mothers and fathers in rented halls. The FGDs were conducted in Hausa and English (where applicable) and moderated by Nigerian male and female research assistants and notetakers. All the research assistants and notetakers spoke Hausa, which is one of the major languages of the selected wards, lived in Kaduna State and were familiar with the terrain, culture and traditions of the people. They also had a college degree with extensive research experience (at least 5 years). They were also trained on the research study protocols and on qualitative research methodology.The discussion guides included questions on the roles of mothers and fathers in the feeding and care of children, barriers and facilitators to child feeding and care experienced by mothers, and mothers' and fathers' perceptions of and experiences with fathers' involvement in child feeding and care. The fathers' FGD guide also included a vignette to elicit their perspectives on gender norms related to child feeding and care. Vignettes are short stories without an ending designed to be used in qualitative research to assess which combination of factors could influence beliefs or social norms  or the equivalent in refreshments for their time.2.5Qualitative thematic analysis methods were used for coding and analysing the data from the FGDs. The analysis began with a thorough and repeated reading of all the transcripts to achieve ‘immersion and obtain a sense of the whole’  to code the transcripts. The coding process began with each coder independently coding one transcript from an FGD with mothers and one transcript from an FGD with fathers. The same two transcripts were coded by all the coders. The codes applied were compared and differences in code application were discussed to achieve consensus Saldaña, . ThereafNext, using Excel matrices of the relevant code queries, the spread of the codes was observed to identify patterns in the main themes across mothers' and fathers' groups and within urban and rural locations following the method described by Miles et al. . The mosthe current behaviours of the characters (descriptive norms) in contrast with what the characters should do (injunctive norms), participants' perceptions of household gender norms regarding father involvement in feeding and caring for their children were described  could help mothers with household chores rather than fathers. These perspectives on what Yusuf ‘should do’ created a picture of the injunctive normative gender roles of fathers as sole providers and supervisors and mothers as sole caregivers without support from either of them in fulfilling those responsibilities.On the other hand, some fathers, on describing what Yusuf and Aisha do each day, acknowledged that sometimes mothers and fathers play roles that do not align with the socioculturally expected behaviours. These fathers mentioned mothers and fathers jointly providing resources and supporting caregiving as examples of the roles mothers and fathers are playing. The fathers went on to admit that they also helped with household chores from time to time in their own homes.3.5There were themes that were more salient in the FGDs of the mothers and fathers from rural wards compared with urban wards Figure . The detIn contrast, mothers and fathers from urban wards communicated a less traditional approach to their roles in ensuring children are fed diverse complementary foods. Both mothers and fathers explained that their roles were based on mutual agreement rather than on defined traditional normative responsibilities. Some fathers spoke about consultative discussions and decision‐making processes with mothers about what needed to be done at home for their children. Other urban fathers also spoke about more hands‐on approaches including participation in household tasks in addition to providing the recommended foods for not only their children but also for mothers as well. Another difference was that, unlike the rural wards, there were mothers who spoke positively about what fathers were doing to support feeding their children diverse complementary foods. These mothers indicated that the fathers were enthusiastic about being involved in feeding and caring for their children.4This study sought to identify and thematically describe how household gender roles influence feeding children diverse foods after the implementation of a programme that engaged fathers to support complementary feeding of their young children. Our findings revealed that the most salient theme in the FGDs was the father as ‘provider’ and mother as ‘caregiver’. This indicates that mothers and fathers hold traditional attitudes about their roles in feeding children diverse complementary foods, but with some differences when comparing urban and rural wards. Traditional gender roles norms are often prescriptive about mothers and fathers' roles in the home, particularly as they relate to child feeding and care ; hence, a total of 16 focus groups were planned and conducted. While the potential for lack of data saturation is a concern for qualitative studies, the FGD participant responses in early and later FGDs were similar, indicating that we likely achieved saturation of results in later FGDs for our study.5Our research showed that the traditional roles of fathers as providers and mothers as caregivers, which are guided by injunctive gender norms, are common in Kaduna State. Despite this, injunctive and descriptive normative roles sometimes differed, such as when mothers were sole providers or joint providers, or fathers played caregiving roles in their homes. More often in urban wards compared with rural wards, mothers and fathers discussed and agreed on their roles at home with mothers and fathers jointly providing and caring for their children. In rural wards, fathers were sole providers and supervisors of mothers and mothers were sole caregivers. Our research findings point to the need for more context‐specific approaches that address prevalent gender normative roles in complementary feeding in a variety of settings.SK and SB are employed by Alive & Thrive. They were involved in the implementation and evaluation of the intervention in Nigeria, but not in data collection or analysis for this study. All other authors report no conflict of interests.SLM, DA and VLF conceptualized this study. SLM, VLF, SK and SB contributed to study design and implementation. AI collected the data. DA, SLM, BW, KK, VLF and MEB contributed to the analysis. DA drafted the paper. All authors contributed to critically revising and approving the final version of this manuscript.


Neuroimaging and transcranial direct current stimulation (tDCS) research has revealed that generating novel ideas is associated with both reductions and increases in prefrontal cortex (PFC) activity, and engagement of posterior occipital cortex, among other regions. However, there is substantial variability in the robustness of these tDCS‐induced effects due to heterogeneous sample sizes, different creativity measures, and methodological diversity in the application of tDCS across laboratories. To address these shortcomings, we used twelve different montages within a standardized tDCS protocol to investigate how altering activity in frontotemporal and occipital cortex impacts creative thinking. Across four experiments, 246 participants generated either the common or an uncommon use for 60 object pictures while undergoing tDCS. Participants also completed a control short-term memory task. We applied active tDCS for 20 min at 1.5 mA through two 5 cm × 5 cm electrodes over left or right ventrolateral prefrontal  or occipital  cortex, concurrent bilateral stimulation of these regions across polarities, or sham stimulation. Cathodal stimulation of the left, but not right, ventrolateral PFC improved fluency in creative idea generation, but had no effects on originality, as approximated by measures of semantic distance. No effects were obtained for the control tasks. Concurrent bilateral stimulation of the ventrolateral PFC regardless of polarity direction, and excitatory stimulation of occipital cortex did not alter task performance. Highlighting the importance of cross-experimental methodological consistency, these results extend our past findings and contribute to our understanding of the role of left PFC in creative thinking. A recent explosion in cognitive neuroscience research on creativity has elicited important findings toward our understanding of the neural bases of creative thinking from studies employing various neuroscience methodologies, including structural and functional magnetic resonance imaging (fMRI), neuropsychological, and noninvasive brain stimulation experiments2. These investigations have generally endorsed a two-stage model of creative thinking according to which creativity entails an initial process of idea generation, followed by a process of selection, elaboration, and refinement of the ideas produced and their evaluation in context. An emerging pattern of results across these studies suggests that cognitive control—the set of regulatory operations supported by prefrontal cortex (PFC)—is critical for each stage of the creative processes through the application of flexible filtering of task-relevant information during creative response generation7.Creativity is the ability to generate ideas that are both novel and useful9. Conventionally, relative to placebo or ‘sham’ stimulation, during and immediately following the application of tDCS, anodal stimulation is thought to elicit cortical excitation in the area underling the electrode due to neuron soma membrane depolarization, whereas cathodal stimulation is thought to elicit cortical inhibition in the area underling the electrode due to neuron soma membrane hyperpolarization10, although these effects might be tDCS-intensity dependent11 and substantially more complex neurophysiologically14.A growing literature within creativity neuroscience has employed transcranial direct current stimulation (tDCS), typically over PFC regulatory regions, to augment creative performance. TDCS is a noninvasive brain stimulation methodology that alters cortical excitability through the application of small electric currents (1–2 mA) via electrodes on the scalp16). As a result, despite the potential of tDCS for the augmentation of creative cognition, comparisons across studies are difficult. Further, an evaluation of this literature suggests that the differences in robustness of tDCS‐induced cognitive enhancements for creativity may be attributed to heterogeneous sample sizes and substantial methodological diversity in the application of tDCS interventions across laboratories, including differences in devices, electrode sizes, strength of stimulation current, stimulation duration, and use of various control tasks20.The use of tDCS for the enhancement of creative cognition has elicited diverse results that point to both the potential but also the substantial variability in the success of tDCS interventions depending on the type of stimulation used , the neural target of tDCS , and the particular requirements of the creativity task employed. Critically, interactions among a creativity task’s reliance on generative or selective processes and tDCS application parameters  can determine the presence of positive effects of tDCS  study, participants in a between-subjects design were tasked with generating either the common or an uncommon use for everyday objects , along with a perceptual baseline task, while undergoing fMRI21. The results revealed a dissociation between left ventrolateral PFC that was more involved in common use generation relative to baseline, and lateral occipitotemporal cortex that was more involved in uncommon use generation relative to baseline, a finding suggestive of a potential tradeoff between cognitive control and posterior brain systems during creative thinking23. We have further shown that inhibiting the left inferior lateral PFC using tDCS elicited considerable increases in the speed and fluency in which participants generated ideas for uncommon, but not common, uses on this task. In contrast, inhibiting the right PFC or sham stimulation did not influence performance on either task24—although other studies have suggested the possible involvement of right frontotemporal regions in creative thinking, including for insight27 and other problem solving29 tasks.To address these issues in past literature, here, we aimed to examine the impact of tDCS on creative ideation using a consistent methodological paradigm across four experiments employing twelve different tDCS montages that were informed directly by our prior fMRI and tDCS work on creative thinking. The selection of tDCS targets was guided by past neuroimaging findings24); (b) excitation of the left or right ventrolateral PFC or sham stimulation ; (c) inhibition of the left with concurrent excitation of the right ventrolateral PFC, the reverse montage of inhibition of the right with concurrent excitation of the left ventrolateral PFC, or sham stimulation ; and (d) excitation of the left or right occipital cortex or sham stimulation . Electrical field modeling was used as a confirmatory tool and verified the specificity of the stimulation montages over ventrolateral PFC or occipital cortex for all experiments (cf.24). Sample size was determined based on the medium-to-large effect size observed in our past tDCS study using this task24, but with the aim to improve power by increasing the number of subjects per condition relative to this past experiment by approximately 20%. To provide continuity with our past studies that served as the foundation for our predictions, we used the version of the Alternative Uses task employed in our past work24 that presents participants with 60 images of everyday objects and asks them to generate either a single common or a single uncommon use for each. This version of the task requires a single response per item to satisfy experimental neuroscience research constraints, while presenting participants with 4–5 times the number of items included in a typical AUT paradigm for a thorough evaluation of response fluency and originality. Thus, we operationalized creative performance according to two objective metrics of ideational fluency: voice-onset reaction times (RTs) and number of omissions out of the total number of items presented. These metrics provide two unbiased ways to measure the ease of idea generation and for multiple object stimuli, which further increases the task’s ecological validity. We also operationalized originality objectively as captured by semantic distance. Semantic distance is a measure of response originality that is founded on the associative theory of creativity30, according to which responses that are farther in semantic space are reflective of a novel, task-appropriate recombination of ideas in semantic memory31. Although not a typical measure of response originality, semantic distance captures conceptual remoteness of the ideas generated, thus, serving as an objective proxy for more traditional originality assessments. We chose to focus exclusively on these objective measures of creative performance to avoid problems with subjective creativity assessments that have been noted to impact significantly neuroscience studies on creativity33. Based on our past findings, we predicted that inhibition of the left but not right ventral PFC or sham stimulation would facilitate creative performance for the unusual use generation task as captured by our objective creativity measures of fluency, but have no impact on the common use generation task, replicating prior work24. We further predicted that increases in originality, as captured by semantic distance, would also be observed—although past work using subjective measures has not consistently reported originality effects following tDCS15. If inhibition of left relative to right ventrolateral PFC were to benefit creative performance, we anticipated that excitation of the same regions might lead to the opposite effects. In line with past investigations of tDCS for creative thinking using bilateral montages34, we further hypothesized that the beneficial effect of left ventrolateral PFC inhibition might be amplified with the concurrent excitation of the right ventrolateral PFC for the generation of uncommon (but not common) uses. Lastly, we anticipated that excitation of either the left or the right occipital cortex might benefit performance on this task, given the increased engagement of these regions during creative ideation in our past neuroimaging findings21. We used the forward digit span (FDS), a brief working memory assessment, as our negative control task because it engages more dorsal regions of the left PFC than the ones stimulated in the present experiment35. Accordingly, if tDCS indeed elicits effects that are regionally specific and does not induce global  cognitive changes, it should not lead to any measurable consequences for FDS performance across our montages. Thus, no effects of tDCS were expected for the FDS task.Based on these findings, the aim of the present study was to examine the impact of different tDCS interventions to alter activity in the ventrolateral PFC and occipital brain regions shown to be important for creative idea generation in this version of the Alternative Uses task. Our objective was to examine the causal influence of increases or decreases in activity in these regions for creative performance using a comprehensive methodological paradigm across experiments. Contrary to all past tDCS research on creativity, and to allow valid comparisons across our experiments, we maintained equivalent sample sizes across conditions that were recruited from the same participant population, as well as retained the same stimulation device, electrodes, current strength, current duration, and experimental and control tasks across studies. Our selection of montages per experiment was guided by our past work and included: (a) inhibition of the left or right ventrolateral PFC or sham stimulation  right-handed, native English speakers  participated in the study for course credit after providing informed consent. Across all experiments, participants were excluded if they met criteria for contraindications for tDCS . The studies were approved by the University of Kansas Institutional Review Board. All methods were carried out in accordance with relevant guidelines and regulations.Sixty  and they are asked to generate verbally either the common (CU) or an uncommon use (UU) for each. Sixty greyscale pictures (448 × 336 ppi) of everyday objects were randomly presented on a gray background for 9000 ms each with 3000 ms interstimulus interval via E-Prime software on a Mac computer running Windows. Voice-onset RTs were recorded with a microphone integrated with E-Prime. Participant responses were also recorded via Audacity software on a Mac laptop computer for later assessment of task compliance and response originality. The FDS, a brief working memory assessment, was used as a negative control task. In the FDS, participants are presented with 16 strings of numbers of increasing length and they are asked to repeat each of these strings back to the experimenter, who is positioned outside of the participants’ field of view. Performance on the task is measured as total number of correct trials on the task.We used a modified computerized version of the Alternative Uses Task from Chrysikou et al.Participants were randomly assigned to perform either the UU or the CU task under one of three stimulation conditions: (1) cathodal tDCS over left ventrolateral PFC ; (2) cathodal stimulation over right ventrolateral PFC (area F8 in the 10/20 EEG system), or (3) sham stimulation. Task order was counterbalanced with the FDS task. Participants in the CU task were asked to generate the common, everyday use for each object , whereas participants in the UU task were asked to generate a novel use for each object . Participants were instructed to remain silent if unable to generate a response for a particular object. For the FDS control task participants were asked to repeat each string of numbers in the order that were read to them. Before the experiment began, participants underwent a brief training to become familiar with the tasks.http://www.neuroconn.de/dc-stimulator_en/) at 1.5 mA via two 5 cm × 5 cm electrodes (current density = 0.06 mA/cm2) for a maximum of 20 min, including 10 s ramp-up and 10 s ramp-down time. The electrodes were placed into saline-soaked sponges  and held in place with rubber straps. The stimulation target was guided by prior work24 to be in the ventrolateral PFC and was determined as either area F7  or F8 36 depending on the participant’s condition; the anode  was placed over the contralateral mastoid. The stimulation site was identified with a BraiNet 10/20 Placement cap  and was marked on the participant’s scalp with a marker. In the active conditions, stimulation began for 90 s prior to the experimental or control task while participants viewed a blank screen to allow for tDCS to create the hyperpolarizing changes to the underlying cortex prior to task onset. Participants in the sham condition  were stimulated over F7 or F8  under the above parameters for 90 s with the same ramp rates to create the sensation of receiving tDCS as in the experimental conditions upon which stimulation was interrupted unbeknownst to the subjects; 90 s of stimulation was considered short enough time such that there would be no significant lasting changes in excitability of the underlying neuronal population that would elicit reliable task effects.TDCS was administered in a single-blind design using a NeuroConn DC-Stimulator Plus  to compute the semantic distance between each item and each participant response across all participants and conditions, separately for each experiment. We used all five spaces incorporated in SemDis for the semantic distance calculations; according to this method, the cosine angle between the word vectors represents semantic similarity; semantic distance is then computed by subtracting this similarity from 137. Latent variable modeling was then used to extract the common variance from the five semantic models, which has been shown to benefit the reliability and generalizability of the results relative to single model approaches37. We then calculated an average semantic distance score across all valid responses for the experimental stimuli for each participant in each experimental condition, which was then used for the statistical analysis. Finally, for the FDS task the number of correct responses  was collected for each participant and averaged across subjects for all studies.Across all 4 experiments, participant responses were transcribed and manually inspected for compliance with task instructions per condition, with less than 1% of erroneous responses removed. There were no task order effects, hence, all results are reported collapsed across task order for all experiments. For the UU and CU tasks, median voice-onset reaction times for each participant were collected and averaged across subjects in each condition for all experiments. Similarly, the number of omissions for the UU and CU tasks was collected for each participant and averaged across subjects in each condition for all experiments. Response originality was scored according to semantic distance, a computational method that employs natural language processing to quantify the semantic relatedness of texts. Specifically, we followed the SemDis approach that relies on a latent semantic distance factor—comprised of the common variance from five semantic models—and which has been shown to correlate with subjective judgments of novelty in creativity tasksF = 49.61, p = 0.02, η2 = 0.96) but no main effect of stimulation condition . Post hoc Tukey’s Honestly Significant Difference (HSD) tests showed that for the UU task participants who received cathodal stimulation over left ventrolateral PFC (L-) generated responses significantly faster relative to participants who received cathodal stimulation over right ventrolateral PFC  or sham stimulation (p < 0.001), who did not differ from each other (p = 0.99). Post hoc comparisons for the CU task were not significant .For Experiment 1, there was a significant main effect of task  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD tests showed that for the UU task participants who received cathodal stimulation over left ventrolateral PFC (L-) generated significantly more responses relative to participants who received sham stimulation (p = 0.04) but not relative to those who received cathodal stimulation over right ventrolateral PFC , who did not differ from each other (p = 0.06). Post hoc comparisons for the CU task were not significant .There was a significant difference in number of omitted responses between UU and CU tasks  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD tests comparisons for either the UU or the CU task were not significant .Verbal response data for the UU task from three subjects (one per stimulation condition) were lost due to a recording error, leaving 57 participants for analysis. There was a significant difference in the semantic distance of the generated responses between UU and CU tasks  = 0.23, p = 0.82; η2 = 0.19; Fig. ps > 0.97).As predicted, there were no significant differences in FDS performance among the stimulation conditions  right-handed, native English speakers  participated in the study for course credit after providing informed consent. None of the subjects had taken part in Experiment Sixty ; (2) excitation of the right ventrolateral PFC ; or (3) sham stimulation using the active conditions electrode configuration for Experiment 2, counterbalanced across subjects .The design and procedures were identical to Experiment 1 with the exception that the participants were randomly assigned to perform either the CU or the UU tasks under the following stimulation conditions: (1) excitation of the left ventrolateral PFC  and no task × stimulation condition interaction . Post hoc Tukey’s HSD comparisons did not reveal any significant pairwise comparisons neither for the UU nor for the CU task .For Experiment 2, there was a significant main effect of task  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD comparisons did not reveal any significant pairwise comparisons neither for the UU nor for the CU task .There was a significant difference in number of omitted responses between UU and CU tasks  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD tests comparisons for either the UU or the CU task were not significant .There was a significant difference in the semantic distance of the generated responses between UU and CU tasks  = 1.16, p = 0.32; η2 = 0.04; Fig. ps > 0.31).As predicted, there were no differences in FDS performance among the stimulation conditions  uses.The aim of Experiment 3 was to extend the findings of the prior two studies by testing whether inhibition of the left with concurrent excitation of the right ventrolateral PFC would benefit AUT performance relative to the reverse montage of inhibition of the right with concurrent excitation of the left ventrolateral PFC, or sham stimulation. Consistent with past research that has explored the impact of bilateral montages for creative thinkingN = 60) right-handed, native English speakers  participated in the study for course credit after providing informed consent. None of the subjects had taken part in Experiments 1 or 2.Sixty ; (2) inhibition of the right ventrolateral PFC with concurrent excitation of the left ventrolateral PFC ; or (3) sham stimulation using the active conditions electrode configuration for Experiment 3, counterbalanced across subjects .The design and procedures were identical to Experiment 1 with the exception that the participants were randomly assigned to perform either the CU or the UU tasks under the following stimulation conditions: (1) inhibition of the left ventrolateral PFC with concurrent excitation of the right ventrolateral PFC , and no task × stimulation condition interaction . Post hoc Tukey’s HSD comparisons did not reveal any significant pairwise comparisons neither for the UU nor for the CU task .For Experiment 3, there was a significant main effect of task , but there was no main effect of stimulation condition  or a significant task × stimulation condition interaction .There was a significant main effect of task  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD tests comparisons for either the UU or the CU task were not significant .Verbal response data for the UU task from one subject in the L + R- condition were lost due to a recording error, leaving 59 participants for analysis. There was a significant difference in the semantic distance of the generated responses between UU and CU tasks  = 0.65, p = 0.61; η2 = 0.39; Fig. ps > 0.26).As hypothesized, there were no differences in FDS performance among the stimulation conditions  uses during the AUT. Guided by the results of that study, Experiment 4 aimed to examine whether excitation of the left or right occipital cortex with tDCS would lead to benefits for fluency and originality relative to sham stimulation. We anticipated that excitation of either left or right occipital cortex might benefit performance on this task, given the increased engagement of these regions during creative ideation in our past neuroimaging findings21.Our past neuroimaging workN = 66) right-handed, native English speakers  participated in the study for course credit after providing informed consent. None of the subjects had taken part in Experiments 1–3.Sixty-six ; (2) excitation of the right lateral occipital cortex ; or (3) sham stimulation using the active conditions electrode configuration for Experiment 4, counterbalanced across subjects .The design and procedures were identical to Experiment 1 with the exception that the participants were randomly assigned to perform either the CU or the UU tasks under the following stimulation conditions: (1) excitation of the left lateral occipital cortex , and no task × stimulation condition interaction . Post hoc Tukey’s HSD comparisons did not reveal any significant pairwise comparisons neither for the UU nor for the CU task .For Experiment 4, there was a significant main effect of task  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD comparisons did not reveal any significant pairwise comparisons neither for the UU  nor for the CU task .Similar to RTs, there was a significant main effect of task  or a significant task × stimulation condition interaction . Post hoc Tukey’s HSD tests comparisons for either the UU or the CU task were not significant .There was a significant difference in the semantic distance of the generated responses between UU and CU tasks  = 0.08, p = 0.92; η2 = 0.08; Fig. ps > 0.26).There were no significant differences in FDS performance among the stimulation conditions (21 and focused on areas of maximal fMRI activation in the left ventrolateral PFC and the lateral occipitotemporal cortex. Our approach differs from all past tDCS research on creativity in that we maintained equivalent sample sizes across conditions that were recruited from the same participant population, and retained the same stimulation device, electrode sizes, current strength and duration, and experimental and control tasks across all 4 studies. To our knowledge, this is the first rigorous multi-study investigation of the effects of tDCS on creative thinking across a large sample of subjects with this level of methodological consistency that can allow for valid comparisons across experiments.Studies using tDCS for the enhancement of creative performance have reported positive effects of both anodal and cathodal stimulation over PFC regions for creativity. On the other hand, the significant variability in the results across studies—partially attributed to substantial methodological diversity in the application of tDCS across investigations—has made it difficult to reach concrete conclusions regarding the effectiveness of tDCS for the augmentation of creative cognition. To address these concerns, here, we examined the impact of multiple tDCS montages across 4 experiments on a multiple-item, single-response version of the Alternative Uses Task—a classic measure of creative thinking—adapted for experimental neuroscience research. Montage selection was guided by past fMRI work using the same task24, as well as other neuroimaging and neurostimulation findings that have reported benefits of left hemisphere hypofrontality for the generative aspects of creativity41. Although past work34 has suggested benefits of increased activity in right PFC for creative ideation, neither unilateral excitation of the right PFC, nor excitation of the right with inhibition of the left PFC elicited significant effects for performance in our studies. We attribute the deviation of our results from this past literature to the differences in the location and size of the tDCS electrodes, as well as differences in the particular creativity measures used15. Lastly, despite the robust engagement of lateral occipitotemporal cortex in the uncommon uses task in our fMRI study21, enhancing activity in these regions with anodal tDCS did not elicit any measurable benefits to ideational fluency or originality.Our results showed that only unilateral cathodal inhibition of the left ventrolateral PFC can improve ideational fluency as measured by participants’ voice-onset RTs, whereas similar—but weaker—results were observed for the number of responses produced. No effects of stimulation were reported for originality, as captured by mean semantic distance. These results replicate our past findings with the same task and stimulation parameters in a smaller sample34 and flexibility34, but not originality34. Although fluency has been argued to be a more objective measure of creativity than originality42, the semantic distance approach we employed in this set of experiments to index originality37 circumvents the vulnerability of this construct to subjectivity bias. On the other hand, originality can be confounded by fluency43. Indeed, the high correlation among fluency, originality, and flexibility further speaks to the view that all three constructs measure similar but distinct aspects of creative thinking45. It is, thus, likely that different brain regions might contribute differently to each of these aspects of creative thought, with fluency associated with ventrolateral PFC involvement, whereas originality associated with ventral temporal cortex41. Additional research exploring both neuroanatomically-specific effects and neural oscillations is required for a comprehensive assessment of this proposition.The dissociation between the facilitatory effects of left ventrolateral PFC inhibition for ideational fluency and the lack of effects for originality in the version of the AUT employed in this study replicates prior tDCS research that has shown measurable increases in fluency41. A reduction in the selection mechanisms that determine the salience of generated responses would restrain the dominance of certain ideas relative to others, thus, increasing fluency in the ideas reported. However, such increases in fluency are not necessarily paired with originality gains: A reduction in the evaluative processes involved in creative thinking may render more ideas as possible candidates for generation, but need not increase the semantic distance of the generated responses—an account consistent with prior work on the role of hypofrontality for creative thinking47.Alternatively, the benefits of left ventrolateral PFC inhibition for the fluency of creative ideas, but not semantic distance we observed in Experiment 34. Further, the use of a between-subjects design for the present experiments limits our ability to capture with precision the effects of different montages for each participant. Although within-subjects, multi-session investigations are challenging, they can provide a powerful way to solidify the effects we report here. Lastly, despite the use of current flow models confirming the specificity of our montages, targeting the brain with high-definition tDCS holds significant potential to elicit more robust results in future experiments. Notwithstanding these limitations, our study is the first methodologically rigorous examination of the effectiveness of multiple tDCS montages for creative cognition that supports the potential benefits of transient reductions in ventrolateral PFC activity for creative thinking.We note that although our sample size was guided by prior effect sizes and it is increased relative to past studies, the relatively small number of participants by condition is a limitation— though one contextualized by the large number of participants, overall. To allow for continuity with our prior work and to examine the specific involvement of the ventrolateral PFC in creativity idea generation, we employed a multiple-item, single-response measure of the Alternative Uses Task. Because of this choice, we were only able to evaluate response fluency and originality, but not flexibility—a typical aspect of AUT scoring that is predicated on multiple responses per item. Although we are unable to evaluate flexibility in the present set of experiments, additional experimental work is required to determine the impact of changes in ventrolateral PFC excitability for flexibility in creativity tasks