P < 0.01) in the rate of handwashing with soap and water and a 5.6 p.p. increase (P = 0.03) in handwashing with at least water after toilet use. HiFive also led to a 10.8 p.p. (P < 0.01) increase in the number of handwashing facilities stocked with soap. The program had limited impact on the motivators targeted by the program, suggesting that the small improvements in handwashing may have been driven by increases in the availability of soap. More research is needed to understand how interventions can effectively trigger social motivators to improve handwashing behavior among schoolchildren, and whether the effectiveness of these programs can be augmented with “nudge”-based interventions from the behavioral sciences.This study reports the impact of the HiFive program, a 6-week handwashing campaign that targets social and emotional motivators to improve student handwashing in primary schools in the Philippines. We designed a clustered randomized trial to evaluate the impact of HiFive on student handwashing behavior, motivation, and access. Of the sample of 196 primary schools located in two districts, half were randomly assigned to receive the program in the 2017–2018 school year. Survey and observation data were collected 3 months after the conclusion of the campaign. In control schools, only 2.5% of students were observed washing their hands with soap and water, our primary outcome and 14.8% were observed washing their hands with at least water. HiFive led to a 3.7 percentage point (p.p.) increase ( Systematic reviews have found that handwashing with soap (HWWS) reduces diarrheal risk by 31–48%.10 Intervening in schools to promote handwashing has been a high priority to break the cycle of disease transmission and realize indirect benefits related to improved student attendance.11 Thus far, school hygiene interventions to motivate HWWS have had mixed results, ranging from no effect14 to over 40 percentage points (p.p.) increase in HWWS.15 No single approach has emerged as a consistent way to generate large-scale sustained improvements in student handwashing.A disproportionate share of the burden of infectious disease morbidity and mortality falls on primary school–aged children, and yet in low-income school settings, observed rates of HWWS have been documented to be as low as 2%.17 However, for children and adults alike, access to facilities coupled with knowledge about the health benefits of handwashing does not always translate into handwashing practice.19 More recent interventions pair improvements in access and knowledge with theory-based approaches to behavior change.22 One such approach is the evolutionary-ecological (Evo-Eco) model of the influences and drivers of human behavior.23 The Evo-Eco model classifies determinants of human behavior drawn from evolutionary theory, psychology, and neuroscience frameworks. Among these determinants, the model proposes a list of 15 fundamental motives based on humans’ evolutionary needs for reproduction and survival that drive behavior change.24 Of these 15 motives, two of the most commonly used in handwashing promotion campaigns are “affiliation” and “disgust.”25Traditional handwashing interventions have focused on addressing supply shortages by improving access to handwashing facilities stocked with soap and increasing demand by educating students on the importance of handwashing.25 Affiliation includes both empirical expectation (“I want to wash my hands because everyone else washes their hands”) and normative expectation (“I want to wash my hands because I think that other people believe that I should wash my hands”). Disgust is the tendency to avoid something unpleasant or offensive and is thought to be a genetically hardwired emotion that functions as a parasitic avoidance strategy.26 Disgust can manifest as an individual-level cue (“I want to wash my hands before I eat because if I do not it would be like I am eating feces”) and as a social-level cue .Affiliation is the tendency for humans to conform to reap the benefits of social living. The fear of social exclusion if an individual is seen to be acting in a way that is socially undesirable is a powerful driver of behavior.28 At the school-level, prior campaigns that harness disgust and affiliation as social motivators of behavior change have had mixed success in improving hand hygiene. The 4-week “School of Five” campaign in Bihar engaged students using posters, diaries, flipchart presentations, and demonstrations to teach children the importance of HWWS, remind children to practice safe hygiene, and build peer pressure around handwashing using the fear of contamination and disgust. Observation data collected 8 weeks following implementation found only a small impact on HWWS after defecation and before eating. Ineffective delivery of key messages, a lack of emotional response to messaging, low campaign intensity, and social and cultural factors were identified as possible reasons for the muted program impact.14 Conversely, the Povu Poa pilot study in Western Kenya highlighted more promising results. In addition to providing handwashing stations to public schools, this single-day intervention included activities in the form of skits, songs, and a public handwashing pledge to create a social norm around handwashing and to use disgust as an emotional driver for handwashing. Observed HWWS was found to be 26 p.p. higher in schools that had received the intervention than in schools that had not yet received the Povu Poa program.21 However, it is important to note that in this context, baseline availability of soap and water was nonexistent, and it is unclear how much of the change in handwashing was attributable to increased student motivation based on social messaging as opposed to the provision of handwashing stations.Findings from three community-level campaigns targeted at caregivers of children in India, Nepal, and Zambia suggest that using social motivators from the Evo-Eco theory may be an effective strategy to change behavior if the intervention is focused on a single health behavior.In this study, we report the results of a large-scale, school-based campaign that triggers feelings of disgust and affiliation to motivate student handwashing. At the beginning of the 2017–2018 and 2018–2019 school years, the Philippine Department of Education (DepEd), in collaboration with UNICEF and the International WaterCentre (IWC), implemented a 6-week behavior change campaign, known as the HiFive for Hysan (Hygiene and Sanitation) program, in primary schools across two provinces in the Philippines. We randomly assigned schools to receive the program in either 2017–2018 or 2018–2019 and exploit this phased-in design to measure the impact of the campaign on rates of student handwashing.29 Using data from student surveys, observations, and interviews, the IWC found that handwashing rates after toilet use and before eating were low, although most students demonstrated an awareness of the health benefits of hand hygiene and had access to a handwashing station with running water . Students expressed strong visceral reactions to pictures and vignettes depicting poor hygiene practices of a student in different contexts and demonstrated a strong desire to conform to peer norms. These results suggested that disgust and affiliation could be powerful motives for handwashing in this context.The HiFive intervention was designed to promote student handwashing after toilet use and before eating. The program was intended to be a supplement to the Philippine water, sanitation, and hygiene (WASH) in schools (WinS) program, which incorporates skills-based learning on hygiene through daily group handwashing and daily group toothbrushing in elementary schools. HiFive was developed following a research study by the IWC that explored handwashing determinants and barriers in eight primary schools in the Philippines.1.Be based on Evo-Eco motives for behavior change, primarily disgust and peer affiliation.2.Include messaging on germ transmission to strengthen the effect of emotional and social motives.3.Leverage student demand for HWWS to improve soap availability and handwashing access.4.Include fun and engaging activities that can be easily integrated into existing curricula.This formative research informed the following set of design principles for a student handwashing intervention:Based on these design principles, the IWC and UNICEF collaborated to create the HiFive intervention. HiFive was a 6-week school campaign where teachers used a set of HiFive tools  to conduTo test proof of concept, the DepEd conducted a 2-year pilot study of the HiFive program. Across the 2017–2018 and 2018–2019 school years, HiFive was introduced to a group of primary schools in the school districts of Camarines Norte, in Region V of the Philippines, and Puerto Princesa, located in the island of Palawan in Region IVB. These pilot study locations were selected on account of being UNICEF program areas. Implementation of the first phase of the pilot study took place during the start of the 2017–2018 school year and lasted for 6 weeks from July to August.1.student handwashing with at least water after toilet use, as observed during classroom observations,2.student handwashing before eating, as reported in student surveys,3.student handwashing motivations and attitudes, as reported in student surveys,4.the availability of functional handwashing facilities with soap, as observed during facility spot checksWe designed a cluster randomized controlled trial to measure the effects of the HiFive intervention on our primary outcome: student HWWS after toilet use, as observed during classroom observations. We also measured the effects of the HiFive intervention on four secondary outcomes:To construct the evaluation sample of primary schools taking part in the HiFive pilot study, we imposed a set of eligibility criteria based on basic WASH infrastructure requirements . This enOf the 196 schools meeting the eligibility criteria, half were randomly assigned to receive the HiFive program in the first phase of the pilot study during the 2017–2018 school year (the “treatment group”), and the other half were assigned to receive the intervention in the second phase during the 2018–2019 school year (the “control group”). This evaluation tests the intent-to-treat impact of receiving the HiFive intervention during the 2017–2018 school year.Randomization was stratified by province and school WASH index to ensure that schools with similar WASH characteristics within Camarines Norte and Puerto Princesa were allocated equally among treatment and control groups. This index included 11 indicators relating to school WASH infrastructure and WASH practices, such as the ratio of students to toilets and implementation of WinS programming. Each school was assigned a point value ranging from 0 (low baseline WASH infrastructure and practices) to 11 (high baseline WASH infrastructure and practices), resulting in 24 province-WASH strata.We implemented school randomization in Stata/IC version 14.0 . Within each province-WASH stratum, schools were sorted in increasing order on a random number variable, and the top 50% of schools in each stratum were assigned to treatment and the remainder to control. If a stratum had an odd number of schools, then the last school in this randomized order was alternately assigned to treatment and control, resulting in an equal number of treatment and control schools in the sample overall.We conducted power calculations to estimate the required sample size to detect a 10 p.p. improvement in HWWS, which we identified in collaboration with UNICEF as a relevant minimum detectable effect size for informing program recommendations. Assuming 80% power, a two-sided alpha of 0.05, an intra-class correlation of 0.30, and 196 clusters, we estimated that we would need to observe at least 20 handwashing opportunities per school, or 3,920 in total. We assumed that enumerators would need to spend approximately 2 hours of observation per classroom per school to achieve this sample; however, in actuality, this assumption was overly conservative.classroom observations of student handwashing behavior after toilet use,student surveys of self-reported handwashing behavior before eating and after toilet use as well as student motivation and perception toward handwashing, andfacility inspections of toilet and handwashing station availability and facility characteristics.Baseline data were not collected because the HiFive program was implemented at the beginning of the school year and it was not possible to observe student handwashing before the start of school. Endline data were collected between November and December 2017, approximately 3 months after the completion of the HiFive program. We selected 3 months after the completion of the program to collect data because we hypothesized that collecting data right at the end of the program, when the messaging was most salient, could overestimate the medium-term effects of the program. Sixty local enumerators with prior training in data collection, but who were not involved in program implementation, were recruited and trained on our data collection instruments. Each enumerator conducted the following data collection activities:All classrooms in grades one to six were eligible for observation, unless the classroom did not have a functioning toilet visible from inside the classroom (2.6% of classrooms) or the classroom had a handwashing facility inside the toilet but did not have a handwashing facility outside the toilet (8.5% of classrooms). Enumerators used SurveyCTO digital data collection software  and a complete listing of eligible classrooms to randomly select one classroom in each grade for observation (excluding classrooms that did not have observable handwashing stations). During each observation, enumerators spent 2 hours recording students’ handwashing behavior. While the teacher and students were aware of enumerators’ presence, enumerators explained that they were there to observe “normal classroom activities” and made no mention of handwashing or sanitation. Enumerators chose a discrete spot in the back of the classroom from which to observe without drawing attention to themselves. Each time a student used the toilet, the enumerator noted whether the student washed his or her hands at a handwashing facility immediately after exiting the toilet and whether the student used soap. All students whose handwashing behavior was observed became part of the classroom observation sample.Because students ate lunch at different times and different locations, including at their homes, we were unable to observe whether students washed their hands before they ate lunch. As a result, we relied on surveys of students in grades four to six to measure student self-reported rates of handwashing before eating; younger students in grades one to three were not surveyed because of difficulties in understanding survey questions and remaining focused for the duration of the survey. In each school, two classroom sections per grade were randomly selected for surveying. Enumerators handed out consent forms to students in selected classrooms during their first school visit. Students brought the consent forms home to get a parent or guardian’s signature. Approximately 1 week later, enumerators returned to the school, collected signed consent forms, and randomly sampled eight students per grade, stratified by gender, from selected classrooms using the list of students who returned consent forms.30 In list randomization, surveyed students received a list of yes/no questions and were asked to respond with the total number of questions (not which ones) that they would respond “yes” to. Thirty percent of the students in our sample (across both treatment and control groups) were randomly selected to receive a four-item question list without the sensitive question asking about handwashing behavior. The other 70% received the same four-item list in addition to the sensitive question on handwashing behavior . The list randomization impact estimate is the double difference of the average number of items between the treatment group receiving the five-item list and the treatment group receiving the four-item list, and the control group receiving the five-item list and the control group receiving the four-item list. The third and final method that we used to measure handwashing before eating lunch was to directly ask students whether they had done so (direct response).To obtain the most credible possible estimates from student surveys, we attempted to triangulate handwashing before eating using three survey techniques. The first method was a script recall question to test whether students mentioned HWWS when asked to recount the events that took place between the start of their lunch break and the time they started eating lunch. The second method was a list randomization question as a way for students to report handwashing behavior without allowing the enumerator to identify individual survey responses.Using a structured checklist, enumerators conducted inspections of all unlocked student-accessible handwashing facilities within and outside classrooms, checking whether facilities had running water and soap available at the time of inspection.31 As a sensitivity check, we have compared linear probability model results with estimated average marginal effects from logistic regressions and found that results do not deviate meaningfully between the two models.The analytical model was defined before data collection and posted along with the evaluation preregistration on the Registry for International Development Impact Evaluations (study ID: RIDIE-STUDY-ID-5a12613323ff9). Impact estimates reported in the following section are from regression specifications, as described in our pre-analysis plan. Continuous outcomes are modeled using ordinary least squares. Binary outcomes are modeled using linear probability models, which we favored over nonlinear binary choice models, given the coefficient’s direct interpretation as the mean marginal effect of the HiFive intervention. In the context of experimental evaluations, linear probability models provide unbiased estimates of program impact and correctly estimate standard errors.Supplementary Appendix, point estimates are comparable without controls and with equal sample weighting (Section A-2).All regressions include school district, WASH index, and strata-level fixed effects, in addition to other control variables specific to the model to reduce variance in the outcome. Standard errors are clustered at the school level, the level of treatment assignment. Sample weights equal to the inverse probability of a classroom section being selected for observation are included for outcomes from classroom observations. For outcomes from student surveys, we include sample weights equal to the inverse of the joint probability of a classroom section being selected for student interviews and a student being selected out of those who returned signed parental consent forms. As shown in the P-values from regressions are corrected to account for the family-wise error rate using the Holm–Bonferroni method of multiple hypothesis corrections.32 Although the pre-analysis plan specified a correction for multiple hypothesis, we deviated from the pre-defined process after further methods research. Specifically, to avoid overcorrection of P-values, we separated out the primary outcome  from secondary outcomes and subgroup analyses in the family-wise adjustments. Furthermore, outcome families with fewer than four outcomes were not corrected because the correction involved a relatively marginal adjustment that would not affect interpretation of results. We include a completed consolidated standards of reporting trials (CONSORT) checklist for cluster randomized trials for this article in the Supplemental Appendix Section A-3.Before observing classrooms, we received verbal consent from the school principal and teacher. For student surveys, we obtained signed consent from parents and verbal consent from respondents. The stated purpose of our research communicated during the consent process was to observe and learn about student health. Our evaluation was granted ethical clearance from a research ethics committee accredited by the Philippine Health Research Ethics Board—St. Cabrini Medical Center and the Asian Eye Institute—in addition to research clearance from the DepEd.Although we were unable to collect student-level data before HiFive implementation, we used pre-program school-level monitoring data on WASH indicators collected by the DepEd to assess baseline balance across our sample of 196 primary schools. As expected, given the stratified randomization performed on an index of WASH characteristics, baseline WASH infrastructure and school hygiene practices did not differ significantly across treatment and control schools .Of the 196 schools, 12 schools were unreachable for student observations and four schools were unreachable at the time of student surveying. This was on account of unexpected school closures due to holidays, school events, and severe weather. The observation and survey samples remained balanced on baseline school characteristics.Despite reaching a fewer number of schools, we were able to observe more handwashing opportunities than the required sample size numbers we had estimated. In total, enumerators observed 5,296 handwashing opportunities  from 840 classroom observations (438 in treatment and 402 in control). Overall, 4,346 students aged nine to 12 years were selected for surveying from grades four to six; however, 11 students did not provide verbal consent and 40 student surveys were thrown out because of unusually short durations leaving a final sample of 4,295 surveys . Finally, 4,187 handwashing facilities were inspected .At the time of data collection, we administered a short survey to teachers and interviewed school administrators to examine the extent that HiFive was implemented with fidelity. Over 90% of teachers surveyed reported attending the 1-day training on HiFive conducted by the DepEd although many cited insufficient training time as a barrier to fully understanding instruction. As a result, HiFive implementation was hindered by teachers’ confusion over the objectives of specific activities and inadequate planning to provide requisite materials. Despite these challenges, teachers largely reported using most of the HiFive tools and most were able to correctly identify the key messages of each activity.The overall rate of observed student handwashing in our sample was strikingly low. In control schools, students were observed washing their hands after using the toilet with at least water 14.8% of the time, and with soap only 2.5% of the time.P < 0.01), and 5.6 p.p. more likely to wash their hands with at least water (P = 0.03)  .Surveyed students dramatically overreported their handwashing behavior in both treatment and control schools. The difference between self-reported and observed rates of HWWS after toilet use was 74.4 p.p. in treatment schools and 73.7 p.p. in control schools; the small difference in overreporting between treatment and control schools was not statistically significant. It is likely that self-reported handwashing before eating suffers from similar rates of overreporting.P = 0.03). The script recall question may have been more successful in reducing self-reporting bias: in the control group, only 31.0% of students recounted washing their hands with soap when asked to describe the events preceding lunch. Students in HiFive schools were 6.4 p.p. more likely to recall washing their hands with soap (P = 0.04). The impact estimate from the list randomization method was statistically indistinguishable from zero.P = 0.01).Access to a functional handwashing facility near a toilet was high across all schools, especially relative to the rate of observed handwashing. In total, 90.4% of toilets in control schools had a functional handwashing facility nearby, compared with 95.4% of toilets in treatment schools (P < 0.01). Although the proportion of classrooms with a handwashing facility was similar across treatment groups, classrooms in treatment schools were 10.8 p.p. more likely to have a facility containing soap (P < 0.01).However, handwashing facilities in HiFive schools were more likely to be stocked with soap. Approximately 38.9% of handwashing facilities near toilets in control schools had soap, compared with 49.7% of facilities in treatment schools   and to reduce the incidence of illness (61.8%). However, students rarely responded using visceral language repeated in HiFive program tools such as “gross,” “yuck,” or “eww.” Reasons related to social motivators were also rarely mentioned: approximately 1% of respondents cited other students’ adverse reactions to unwashed hands as a motivator to HWWS (peer affiliation), and there was no difference between treatment and control groups ( = 0.91) .P = 0.16). Similarly, we do not find evidence that the HiFive program led to a notable difference in the proportion of students who strongly believe that other students should wash their hands.HiFive program tools were designed to strengthen the motivating force of peer affiliation by establishing classroom norms around handwashing. The goal was for students to perceive that their classmates wash their hands with water and soap and that students do not tolerate dirty, unwashed hands. We tested to see if the HiFive program entrenched these norms by asking students whether they believed that their peers wash their hands with soap at critical times  and whether they believed that students should wash their hands with soap at critical times (normative belief). In total, 25.6% of students in treatment schools reported that every student washes his/her hands with soap after using the toilet and before eating, compared with 22.4% of students in control schools, although the difference was not statistically significant . Other reasons cited by students included that they were in a hurry (24%), reasons related to lack of access to functioning handwashing stations (18%), and because students did not want to (2%).Three months after the conclusion of HiFive, we find that the program led to a modest gain in the prevalence of handwashing after toilet use. Results are suggestive of similar sized increases in the rate of handwashing before eating. Despite the positive gains, handwashing rates remained extremely low. Student-reported motivations to handwashing provide clues that HiFive did not successfully alter behavioral motivators explicitly targeted by the program. Reasons for handwashing related to peer affiliation and the specific language of disgust used by the HiFive program were rarely mentioned and were not more frequently cited by students in HiFive schools than in control schools. Survey evidence indicates that students’ expectations and normative beliefs around peer handwashing did not change. It is therefore unlikely that HiFive entrenched critical social norms required for peer affiliation to work as a social motivator.19The modest increase in HWWS observed in HiFive schools may be largely attributable to a greater opportunity for HWWS, rather than an increased desire to HWWS. In addition to increasing the presence of handwashing stations nearby toilets, the HiFive program led to sizable increases in the stocking of soap at handwashing facilities and the proportion of classrooms with a handwashing facility containing soap. However, it is unlikely that providing universal access to stocked handwashing facilities would dramatically improve the frequency of student handwashing. In over 90% of classrooms, students had the opportunity to wash hands with water after using the toilet, yet fewer than one in six did so. In more than half of classrooms, students had the opportunity to HWWS, yet the rate of HWWS remained in the single digits. Knowledge gaps did not seem to be the primary constraint. Student surveys indicate high levels of student knowledge on the health benefits of handwashing. This confirms findings from other studies that have shown that access and knowledge by themselves do not translate to sustained large improvements in student handwashing.33 and a handwashing intervention in Ethiopia that reduced parasitic reinfection by half involved weekly household visits over the course of 6 months.34 On the other hand, other handwashing interventions have been successful on an even shorter timeline and with fewer sessions than HiFive: the SuperAmma behavior change campaign in India, for instance, involved only 4 days of implementation over the course of a month and led to a 15 p.p. improvement in HWWS.20 The Povu Poa pilot study included a single day of behavioral change messaging and found a large impact on observed student handwashing.21 Thus, although a longer HiFive intervention period might have further entrenched social norms, we do not regard the 6-week implementation period to be the primary constraint to a greater impact.It is unclear why the HiFive program was unsuccessful in changing students’ motivation for handwashing. One possibility is that a 6-week campaign period may have been too short to lead to sustained behavior change, and students may have required refresher sessions over the school year to reaffirm key HiFive messages. In nonschool settings, successful handwashing promotion interventions are sometimes implemented over a longer time period: a handwashing intervention in Pakistan that halved diarrheal incidence among children involved weekly household visits over the course of a year,37Another possibility is that the relevance of social motivators may have been inadequate, given the context and student demographics. Despite formative research suggesting that disgust and affiliation were important motivational drivers among Filipino schoolchildren, we cannot rule out whether other social motivators or a combination of different approaches would have been more effective. For example, some researchers recommend an approach that targets multiple psychological determinants of handwashing at the same time, including motivators that trigger feelings of disgust and social pressure, as well as behavioral nudges that influence automatic or habitual responses.39 A proof-of-concept study of a nudge-based intervention targeting handwashing in Bangladeshi schools found that HWWS rates increased 14 p.p. when handwashing infrastructure was moved closer to toilet facilities, but 52 p.p. after a footpath from the toilet to the handwashing facility was painted on the ground.40 A follow-up randomized evaluation found a smaller, but sustained, impact of the paved painted footpath and found it to be as effective as a high-intensity hygiene education program.41 In Zambian public schools, a simple toilet pass fashioned out of soap attached to a rope was a strong enough contextual cue to motivate student HWWS.42We consider nudge-based interventions to be a particularly promising avenue for promoting handwashing in primary schools. The most often cited reason students gave for not handwashing was because they forgot. Nudges could in theory trigger the automatic psychological responses that counter forgetfulness. In hospitals, universities, and public schools in low-income settings, nudges in public restrooms such as eyes near handwashing stations and arrows pointing from the toilet to the sink have been found to increase rates of HWWS.Finally, the HiFive program may have had a sound conceptual basis but was less effective at improving rates of HWWS because of limitations in the implementation design. Specifically, teachers reported that they felt insufficiently trained to deliver all of the activities in the HiFive curriculum. As a result, the HiFive program was overhauled ahead of the second year of implementation to address some of these implementation issues, including further integration of the program’s messaging through detailed lesson plans and more extensive teacher training through a train-the-trainers model. However, handwashing rates among students after implementation of the revamped HiFive program remained below 8%. Given the limited impact of the HiFive program and the effectiveness of behavioral cues in other settings, we are working with UNICEF and the DepEd to design, implement, and evaluate a nudge-based intervention in Filipino primary schools.We acknowledge several limitations to our evaluation and suggest topics for further research. First, because the HiFive program appears to have had a limited effect on students’ feelings of affiliation and disgust, we cannot disentangle problems in program delivery from problems with the underlying theory linking social motivators to handwashing behavior; either or both factors may have reduced the effectiveness of HiFive. At the same time, a revamped HiFive program with integrated detailed lesson plans and more extensive teacher training also failed to bring about large-scale behavior change, which makes us somewhat pessimistic about the effectiveness of triggering social motivators in isolation from other interventions. Future research should continue to look at the effect of social motivators, but particularly when complemented with other interventions such as behavioral nudges.Second, the fact that a majority of students were aware of the importance of handwashing likely led students to overreport handwashing rates in the student survey: students were more than 70 p.p. more likely to say that they washed their hands with soap after toilet use than were actually observed doing so, and it is likely that HWWS before eating was similarly overreported. It is possible that students in treatment schools were more likely to overreport handwashing than students in control schools because of the social desirability effects triggered by HiFive messaging about peer affiliation. For this reason, treatment effects based on self-reported handwashing may be overestimated, and we regard these results as less credible than results based on direct observation.Social desirability effects may have also led students to wash their hands more in the presence of enumerators than they would otherwise, inflating estimates of HWWS based on direct observation . Although we cannot rule out Hawthorne effects in our study, these effects would have been modest, given the extremely low rates of HWWS overall. We also took several steps to mitigate Hawthorne effects, including conducting direct observation before student surveys about handwashing and giving enumerators a script to explain their presence in the classroom to teachers and students that did not reference handwashing or sanitation. Although we consider it unlikely that large and differential Hawthorne effects biased our evaluation, future research could combine direct observation with other measures, such as measuring microbial contamination of hands or embedding sensors in soap dispensers.43Third, capturing student motivations for performing or failing to perform a specific behavior by asking children to be self-reflective about their actions is likely to be a poor proxy for true motives. Not only is asking these sorts of open-ended questions susceptible to social desirability bias but students may also conflate motives with what they are able to recall at that particular instance in time. As such, we consider self-reported motivations for handwashing from the student survey a relatively weaker form of evidence and encourage future studies to think creatively about ways to use student observation to measure emotions and motivators for handwashing.Fourth, 39% of classrooms in the study had a handwashing station within the toilet facility that could not be observed by enumerators (in addition to a handwashing station outside the toilet facility); the difference in the fraction of classrooms with a handwashing station within a toilet facility between treatment and control was not statistically significant. The presence of a handwashing station within a toilet facility could lead us to underestimate or overestimate the program effect if students in treatment schools use unobservable handwashing stations at higher or lower rates than students in control school, respectively. Although we find no differential treatment effect on handwashing for students in classrooms that have a handwashing station within a toilet facility versus students in classrooms that do not, we cannot rule out the possibility of either upward or downward bias on our full-sample estimates.18 Further research on interventions like HiFive that seek to entrench social norms to improve handwashing behavior may provide valuable evidence on how the effectiveness of these interventions changes over time.Fifth, we collected data on student handwashing at a single point in time, 3 months after the conclusion of the HiFive intervention. We may have failed to capture larger immediate program effects that tapered off or effects that materialized at a later date. Other studies of handwashing interventions collected outcome data at multiple points during and after the intervention, enabling a more thorough investigation of how treatment effects manifest over time.Finally, our study measured the effects of a handwashing intervention targeting social motivators in primary schools across two provinces in the Philippines. To be eligible for our study, schools required functioning handwashing stations and toilet facilities; 40% of schools in the region were not eligible because of a lack of critical handwashing infrastructure. In these schools and other regions, improving WASH infrastructure is a prerequisite to behavior change interventions that target handwashing practices. At the same time, fewer than half of handwashing facilities in our study schools had soap. Further research is needed to evaluate the effectiveness of handwashing interventions that target social motivators in educational settings that start with more and less extensive handwashing infrastructure.Supplemental materials


The intracellular chloride ion concentration ([Cl-]i) is higher in immature neurons than in mature neurons; therefore, γ-aminobutyric acid type A (GABAA) receptor activation in immature neurons does not cause chloride ion influx and subsequent hyperpolarization. In our previous work, we found that midazolam, benzodiazepine receptor agonist, causes less sedation in neonatal rats compared to adult rats and that NKCC1 blockade by bumetanide enhances the midazolam-induced sedation in neonatal, but not in adult, rats. These results suggest that GABA receptor activation requires the predominance of KCC2 over NKCC1 to exert sedative effects. In this study, we focused on CLP290, a novel KCC2-selective activator, and found that midazolam administration at 20 mg/kg after oral CLP290 intake significantly prolonged the righting reflex latency even in neonatal rats at postnatal day 7. By contrast, CLP290 alone did not exert sedative effects. Immunohistochemistry showed that midazolam combined with CLP290 decreased the number of phosphorylated cAMP response element-binding protein-positive cells in the cerebral cortex, suggesting that CLP290 reverted the inhibitory effect of midazolam. Moreover, the sedative effect of combined CLP290 and midazolam treatment was inhibited by the administration of the KCC2-selective inhibitor VU0463271, suggesting indirectly that the sedation-promoting effect of CLP290 was mediated by KCC2 activation. To our knowledge, this study is the first report showing the sedation-promoting effect of CLP290 in neonates and providing behavioral and histological evidence that CLP290 reverted the sedative effect of GABAergic drugs through the activation of KCC2. Our data suggest that the clinical application of CLP290 may provide a breakthrough in terms of midazolam-resistant sedation.Immature neurons dominantly express the Na In the neonatal rat cerebral cortex, the NKCC1 expression level is usually highest around postnatal day (PND) 5 to 7; afterward, the KCC2 expression level gradually increases and becomes dominant i manipulation through the KCC2 pathway could minimize adverse effects [-]i in cultured injured neurons. Furthermore, in vivo experiments showed that systemic CLP290 administration in a rat model of neuropathic pain can improve the hyperalgesia threshold Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The study by Akiki Doi et al “CLP290 promotes the sedative effects of midazolam in neonatal rats in a KCC2-dependent manner: A laboratory rat study” provides evidence that activation of the KCC2 cotransporter isoform can facilitate midazolam—triggered sedation. This reviewer has a number of comments/questions to authors.Abstract. In the context of their results, what authors mean by saying: Contrastingly, CLP290 alone did not enhance the sedative effect of midazolam?Introduction. “GABAA receptor activation exhibits excitatory toxicity…” Membrane depolarization, usually leading to neuronal excitation, is a normal physiological event, not necessarily neurotoxic.Introduction. “there has been no study showing that these compounds inhibit GABAA receptor-induced excitation in immature neurons…” Present study also does not show this. What is shown is facilitation of benzodiazepines sedative effects. CLP290 alone does not do anything. It might change conformation of benzodiazepine binding site at GABAA receptor, directly or indirectly, in such a way that midazolam could change ion selectivity of the associated ion channel, or block it, for example, or whatever else. However, all these possibilities are too speculative. Omitting ungrounded speculations might benefit the present manuscript.Introduction. “p-CREB immunohistochemistry to determine the neural circuits underlying the sedative effect of CLP290…” pCREB is a transcriptional factor, which can be used as a nonspecific marker for protein synthesis, perhaps, neuronal activation, but not of neuronal excitation or excitability (here and throughout the manuscript). Of course, this immunohistochemistry may not “determine neuronal circuits". Please, correct these scientific/linguistic inconsistencies.Drugs. “…dissolved CLP290 in 10% 2-hydroxypropyl-β-cyclodextrin or suspended it in 0.5% carboxymethyl cellulose with a final concentration of 0.66% CLP290.” Why two different solvents  were used and in which experiments?Immunohistochemistry protocol. “At 75 min after midazolam or saline injection, the rats were deeply anesthetized…” Why this time interval has been chosen?Immunohistochemistry protocol. Indicate how many times the sections were washed between all steps .Immunohistochemistry protocol. “…(100 × 200 μm2) slices and thalamus (200 × 500 μm2)… This is not very clear and even confusing. Please, extend this description sufficiently for readers to understand what it means and to be able to replicate this sampling technique.Biochemistry. “Next, 1 mm brain slices were obtained using a brain slicer and the somatosensory cortex was extracted. Subsequently, the slices were incubated…” Please, explain how the somatosensory area was found and “extracted”. Not clear – only these extracted parts were than used or remaining slices.Statistics. Why authors opted for Mann-Whitney test instead of parametric ANOVA?Immunohistochemistry results and figure 2 legend. Statistics was done for “N = 9-12 slices/group”. This is not valid. The numbers obtained from all sections of a single animal should be averaged. N (number of rats) in each group should be given. I understand that sample sizes for some groups might be too small for statistical comparisons. Should be this the case, authors should consider the possibility of either omitting this part or removing the entire submission until the data are complete.Why “brain CLP290 levels were expected to have a slower increase than those of VU0463271”?Discussion. Authors compare the results obtained in the somatosensory cortex, thalamus and hippocampus, but I was not able to find data for the thalamus and hippocampus in the manuscript.Discussion. The hypothesis of distinct switching timing for cation-chloride cotransporters appears somewhat immature. It should be better explained and referenced. If not possible – omitted.There is an excessive use of phrases starting with “We…”. This makes the story a bit subjective. Although personal statements of interest and of hypotheses cannot be fully circumvented in scientific papers, the use of sentences written in the third person is preferential.Reviewer #2: In the current Ms, the authors want to investigate whether CLP290 promotes the sedative effects of midazolam in neonatal rats via an action on KCC2.This Ms is written in a clear structure and has some substantial interest. However, I have some concerns listed below.1) In P6, the authors mentioned that they used bentobarbital. Did the authors notice if the pentobarbital anesthetic effect was altered by the pharmacological drugs used in their study? Which dose of sodium pentobarbital has been used?2) To prove further their concept, the authors should perform some experiments showing the effect of midazolam on adult rat treated with VU0463271 or CLP290 during their behavioral experiments.3) In order to be able to state in the title of their Ms that CLP290 has an effect via an action on KCC2 I am expecting to see more convincing arguments or at least more direct evidences that KCC2 is involved.4) It would be good to provide supplemental figures in a more standard format.I have some few comments regarding the methods section:1) It would be interesting for the reader not familiar to the field to give a description (even brief) of the righting reflex test.2) Ln107: please could the authors explain why they disssolved CLP290 in 2 different vehicles? Which vehicle was used for which experiments?3) Ln161: Could the authors explain why they used isoflurane this time and not pentobarbital? I presume that the rats were sacrified before removing the brain, please could you give more details regarding this?4) Ln162-164: Please could you describe your dissection buffer and your ACSF?5) Ln163: Please give reference of the vibroslicer.The authors should check carefully the numerous typos or confusions present in the text, I have listed few of them below:1) Abstract ln29: Therefore, midazolam, a benzodiazepine agonist: I presume that you mean benzodiazepine receptor agonist.2) ln30 "This indicates that the balance between the NKCC1 and KCC2 is important for GABA agonist action". I don't think this conclusion was drawn from the midazolam experiments.3) Abstract, Ln37: « Contrastingly, CLP290 alone did not enhance the sedative effect of midazolam.” Not clear at all. What do you mean exactly? I guess you meant that CLP290 alone had no sedative effect?4) ln59: "higher intracellular [Cl-]I levels": I presume it should not be a capital I.5) Ln198 Strangely the legend of the figure 1 is at the end of the Methods section.6) ln255: please be consistent and use capital letters for CLP2907) ln311: “To our knowledge, this is the first study to report that CLP290 modulates GABAA receptor-mediated pharmacology”. I think this statement is not correct. CLP290 does not interfere with pharmacology at GABAA receptor but affect indirectly the effect of GABAA receptor activation.8) ln379 "“A possible resulting hypothesis is that higher midazolam doses might deeply attenuate neuronal activity in the thalamus deeply,”. Deeply is repeated twice.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  15 Jan 2021Response to ReviewersResponse to PLoS One editor:1. Note also that statistical analysis was not performed in an appropriate manner. Particularly, repeated measures analysis of variance and two-way analysis of variance were not considered, respectively, in figures 1 and S1. This is only an example, but all your statistical approach requires a reappraisal. Thank you for your suggestion. As you mentioned, the ANOVA test is usually considered for parametric data. However, our behavioral data in this study did not have a normal distribution according to the test of normality and, therefore, the ANOVA should not be used. In this case, quantile regression is suitable because it can be used without an assumption of a specific distribution, and we performed the statistical analysis using quantile regression in the following results .2. Details on fixation are also missing from the manuscript. It is well known that pups require a different procedure when compared to adults to obtain an optimal brain preservation.Thank you for your comment. As you mentioned, the antigenicity in young brains decreases with overfixation. We have already optimized the fixation protocol for neonates, as shown in our previous paper [12], and this study used the same method. We added details of the fixation method to the methods section (Lines 152-156).http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols3. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: Thank you for your suggestion. We will try this procedure after this paper gets accepted.4. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (1) the total number of rats used in this study, (2) how often animals were checked on for welfare, and (3) whether decapitation was performed by trained personnel.Thank you for your comment. Following your instruction, we included the following information in our manuscript: (1) The total numbers of rats used were added to the methods section (Line 97); (2) all rats were checked for their well-being daily at the time of feeding (Line 101); and (3) we declared in the methods section that trained personnel performed the decapitation (Line 177).5. In your methods section, please provide the full and detailed criteria used to determine righting reflex latency times to ensure that other researchers can replicate and reproduce your experiments.Thank you for your comment. We added details of this method and criteria for determining the righting reflex latency to the methods section (Lines 115-119).Reviewer #1: The study by Akiki Doi et al “CLP290 promotes the sedative effects of midazolam in neonatal rats in a KCC2-dependent manner: A laboratory rat study” provides evidence that activation of the KCC2 cotransporter isoform can facilitate midazolam—triggered sedation. This reviewer has a number of comments/questions to authors.Abstract. In the context of their results, what authors mean by saying: Contrastingly, CLP290 alone did not enhance the sedative effect of midazolam?Thank you for your comment. We wanted to describe that “By contrast, CLP290 alone did not exert sedative effects ” and rephrased the corresponding sentence (Line 27).Introduction. “GABAA receptor activation exhibits excitatory toxicity…” Membrane depolarization, usually leading to neuronal excitation, is a normal physiological event, not necessarily neurotoxic.Thank you for your correction, and we fully agree with it. We rephrased the corresponding sentence (Line 61).Introduction. “there has been no study showing that these compounds inhibit GABAA receptor-induced excitation in immature neurons…” Present study also does not show this. What is shown is facilitation of benzodiazepines sedative effects. CLP290 alone does not do anything. It might change conformation of benzodiazepine binding site at GABAA receptor, directly or indirectly, in such a way that midazolam could change ion selectivity of the associated ion channel, or block it, for example, or whatever else. However, all these possibilities are too speculative. Omitting ungrounded speculations might benefit the present manuscript.Thank you for your comment with which we agree. In this study, we did not perform experiments directly showing that GABAA receptor activation induces neuronal excitation in neonates. Throughout our manuscript, we suggest that activation of KCC2 promotes the sedative effect of GABAA receptor activation in neonates. We rephrased the corresponding sentence (Lines 80-81).Introduction. “p-CREB immunohistochemistry to determine the neural circuits underlying the sedative effect of CLP290…” pCREB is a transcriptional factor, which can be used as a nonspecific marker for protein synthesis, perhaps, neuronal activation, but not of neuronal excitation or excitability (here and throughout the manuscript). Of course, this immunohistochemistry may not “determine neuronal circuits". Please, correct these scientific/linguistic inconsistencies.Thank you for your comment. We agree with the concerns expressed here. As you mentioned, we used p-CREB as a non-specific marker for neuronal activation, not excitation. That is to say, we performed p-CREB immunostaining to identify brain regions related to the promoting effect of CLP290 on midazolam-induced sedation, not to determine neuronal circuits. We rephrased the corresponding sentences (Lines 84-86).Drugs. “…dissolved CLP290 in 10% 2-hydroxypropyl-β-cyclodextrin or suspended it in 0.5% carboxymethyl cellulose with a final concentration of 0.66% CLP290.” Why two different solvents  were used and in which experiments?Thank you for this constructive question. In neonatal behavioral tests and immunostaining experiments, we used CLP290 kindly supplied by Dr. De Koninck. He indicated this drug had to be dissolved in hydroxypropyl-β-cyclodextrin (HPβCD). Two years ago, we were not able to contact him and, therefore, bought CLP290 from another company. CLP290 has very low solubility in water, and we could not find a way to dissolve it even in HPβCD. We consulted a chemist to find a way to dissolve it and finally purchased CLP290 from Sigma. Their drug can be dissolved in 5% carboxy methylcellulose, and we confirmed the same efficacy in behavioral experiments of this drug as that supplied by Dr. De Koninck. The experiments using VU0463271, adult behavioral tests, and western blot experiments were performed using CLP290 purchased from Sigma.Immunohistochemistry protocol. Immunohistochemistry protocol. “At 75 min after midazolam or saline injection, the rats were deeply anesthetized…” Why this time interval has been chosen?Thank you for your comment. Accordingly, we corrected this sentence. We have performed immunohistochemistry using the sample obtained from rats at 45 min after administration of midazolam or saline, as previously published [12]. (Line 149)Immunohistochemistry protocol. Indicate how many times the sections were washed between all steps .Thank you for your suggestion. We added the exact number of washing steps to the appropriate sentences in the methods section .Immunohistochemistry protocol. “…(100 × 200 μm2) slices and thalamus (200 × 500 μm2)… This is not very clear and even confusing. Please, extend this description sufficiently for readers to understand what it means and to be able to replicate this sampling technique.Thank you for this valuable suggestion. We added the detailed methodology of sampling for the immunohistochemistry staining to the methods section .Biochemistry. “Next, 1 mm brain slices were obtained using a brain slicer and the somatosensory cortex was extracted. Subsequently, the slices were incubated…” Please, explain how the somatosensory area was found and “extracted”. Not clear – only these extracted parts were than used or remaining slices.Thank you for your comment. We added the details regarding the method to identify the somatosensory region to the methods section (Lines 180-183).Statistics. Why authors opted for Mann-Whitney test instead of parametric ANOVA?Thank you for your comment. The righting reflex test is usually analyzed using a nonparametric test due to its non-normal distribution. Furthermore, our results obtained from biochemical experiments also showed non-normal distribution. Therefore, we performed median regression analysis in righting reflex experiment because median regression analysis does not require an assumption of a specific distribution and suitable for them, and used Mann-Whitney analysis in biochemical experiment .Immunohistochemistry results and figure 2 legend. Statistics was done for “N = 9-12 slices/group”. This is not valid. The numbers obtained from all sections of a single animal should be averaged. N (number of rats) in each group should be given. I understand that sample sizes for some groups might be too small for statistical comparisons. Should be this the case, authors should consider the possibility of either omitting this part or removing the entire submission until the data are complete.Thank you for your comment with which we fully agree. In the initial manuscript, we analyzed the immunohistochemical data by indicating with n the number of slices, which is not a valid approach. In the revised manuscript, we performed additional experiments to add animals to each group and analyzed the data such that n indicates now the number of animals, and analyzed using the Tukey-Kramer test .Why “brain CLP290 levels were expected to have a slower increase than those of VU0463271”?Thank you for your comment. We mentioned that VU0463271 was administered intraperitoneally, whereas CLP290 was administered orally. Therefore, it is expected that compared to VU0463271, the concentration of CLP290 in the brain, as well as in the blood, increases slower. We added a sentence to explain this (Lines 309-310).Discussion. Authors compare the results obtained in the somatosensory cortex, thalamus and hippocampus, but I was not able to find data for the thalamus and hippocampus in the manuscript. Thank you for your comment. In this study, we focused on the thalamocortical pathway known to be related to sedation. Therefore, the description regarding the hippocampus was unnecessary and has been deleted. We added representative images and a graph showing the number of p-CREB-positive neurons in the thalamus to S1 Fig and also revised the corresponding figure legend (Lines 612-618).Discussion. The hypothesis of distinct switching timing for cation-chloride cotransporters appears somewhat immature. It should be better explained and referenced. If not possible – omitted.Thank you for your comment. In our manuscript, we added some references suggesting the following lines of evidence (Lines 400-414): 1. the expression of KCC2 in mice is still lowere at postnatal day (PND) 7 in the cerebral cortex [27] ,whereas in that in the thalamus increases from embryonic day 14  and become almost comparable with adults at PND0 [7]; 2. In neonatal neurons around PND7-10, GABA receptor activation induces neuronal activation in the cerebral cortex, whereas GABA receptor activation induces neuronal inhibition in the thalamus, similar to that in adults [6]. These reports are consistent with our p-CREB experiments showing that administration of midazolam with activation of KCC2 by CLP290 reduced neuronal activation in the cortex but no change was observed in the thalamus even though with CLP290 administration . Our hypothesis is that the regional differences in p-CREB expression in response to CLP290 administration may be related to differences in KCC2 expression levels and the GABA equilibrium potential at PND7. To strengthen this hypothesis, further research is needed in the future.There is an excessive use of phrases starting with “We…”. This makes the story a bit subjective. Although personal statements of interest and of hypotheses cannot be fully circumvented in scientific papers, the use of sentences written in the third person is preferential.Thank you for your suggestion. We rephrased these sentences accordingly.Reviewer #2: In the current Ms, the authors want to investigate whether CLP290 promotes the sedative effects of midazolam in neonatal rats via an action on KCC2.This Ms is written in a clear structure and has some substantial interest. However, I have some concerns listed below.1) In P6, the authors mentioned that they used pentobarbital. Did the authors notice if the pentobarbital anesthetic effect was altered by the pharmacological drugs used in their study? Which dose of sodium pentobarbital has been used?Neonatal rats were intraperitoneally administered 342 mg/kg of pentobarbital  (Line 151), and with this dose, all rats fell asleep within 2 min. Since an excess dose of 10 times the dose required for sedation in adult was administered, the synergistic effect with CLP290 and midazolam could not be directly evaluated. 2) To prove further their concept, the authors should perform some experiments showing the effect of midazolam on adult rat treated with VU0463271 or CLP290 during their behavioral experiments.Additional experiments were conducted to investigate whether CLP290 enhances the sedative effect of midazolam in adult rats, as well as in neonatal rats (Lines 120-133). As shown in Fig 1, a dose-response curve for midazolam was created for both neonatal and adult rats, showing that the 20% effective dose (ED20), equivalent to 20 mg/kg of midazolam in neonates, was 8.6 mg/kg in adult rats (Lines 239-241). Thus, 2 h after oral CLP290 administration, midazolam 8.6 mg/kg was intraperitoneally administered to adult animals, followed by the righting reflex test. In contrast to neonatal rats, CLP290 at 100 mg/kg exerted little influence on the sedative midazolam effect at ED20 . The CLP290 effect was not statistically significant and, therefore, we did not perform subsequent experiments using VU0463271 in adult animals. 3) In order to be able to state in the title of their Ms that CLP290 has an effect via an action on KCC2 I am expecting to see more convincing arguments or at least more direct evidence that KCC2 is involved.Thank you for your comment. To date, CLP290 has been used as a KCC2 activator in the following disease models: spinal cord injury [15], neuropathic pain , ischemic seizures [10], nicotine-induced ethanol overdose [17], and streptozotocin-induced diabetes [22]. Furthermore, VU0463271, a specific KCC2 inhibitor, is available . Our study also showed that antagonization of KCC2 using VU0463271 attenuated the promoting effect of CLP290 on midazolam-induced sedation, suggesting that the effect of CLP290 is directly or indirectly mediated by KCC2 (Lines 417-427).4) It would be good to provide supplemental figures in a more standard format.Thank you for your comment. According to your suggestion, and we have revised the supplemental figure. I have some few comments regarding the methods section:1) It would be interesting for the reader not familiar to the field to give a description (even brief) of the righting reflex test.Thank you for your suggestion. We added details of this method and criteria for determining the righting reflex latency to the methods section (Lines 115-119).2) Ln107: please could the authors explain why they disssolved CLP290 in 2 different vehicles? Which vehicle was used for which experiments?Thank you for this constructive question. In neonatal behavioral tests and immunostaining experiments, we used CLP290 kindly supplied by Dr. De Koninck. He indicated this drug had to be dissolved in hydroxypropyl-β-cyclodextrin (HPβCD). Two years ago, we were not able to contact him and, therefore, bought CLP290 from another company. CLP290 has very low solubility in water, and we could not find a way to dissolve it even in HPβCD. We consulted a chemist to find a way to dissolve it and finally purchased CLP290 from Sigma. Their drug can be dissolved in 5% carboxy methylcellulose, and we confirmed the same efficacy in behavioral experiments of this drug as that supplied by Dr. De Koninck. The experiments using VU0463271, adult behavioral tests, and western blot experiments were performed using CLP290 purchased from Sigma.3) Ln161: Could the authors explain why they used isoflurane this time and not pentobarbital? I presume that the rats were sacrificed before removing the brain, please could you give more details regarding this?Thank you for your comment. As you mentioned, the rats were sacrificed before removing the brains. In this biochemical experiment, some animals were processed simultaneously for brain sample collection, and we considered that anesthesia using a desiccator filled with isoflurane was easier for achieving this purpose. (Lines 176-177)4) Ln162-164: Please could you describe your dissection buffer and your ACSF?Thank you for your comment. The compositions of the dissection buffer (Lines 178-180) and ACSF (Lines 184-185) were added to the manuscript.5) Ln163: Please give reference of the vibroslicer.Thank you for your comment. We added this information to the revised manuscript (Line 181).The authors should check carefully the numerous typos or confusions present in the text, I have listed few of them below:1) Abstract ln29: Therefore, midazolam, a benzodiazepine agonist: I presume that you mean benzodiazepine receptor agonist.Thank you for your comment. We corrected this sentence (Line 21).2) ln30 "This indicates that the balance between the NKCC1 and KCC2 is important for GABA agonist action". I don't think this conclusion was drawn from the midazolam experiments.Thank you for your comment, and we agree with you. We revised the part of the abstract related to this sentence (Lines 23-25).3) Abstract, Ln37: « Contrastingly, CLP290 alone did not enhance the sedative effect of midazolam.” Not clear at all. What do you mean exactly? I guess you meant that CLP290 alone had no sedative effect?Thank you for your comment. We rephrased this sentence as follows: “By contrast, CLP290 alone did not exert sedative effects” (Line 27).4) ln59: "higher intracellular [Cl-]I levels": I presume it should not be a capital I.Thank you for your correction. We corrected this typo (Line 52).5) Ln198 Strangely the legend of the figure 1 is at the end of the Methods section.Thank you for your comment. We changed the position of this figure legend in the revised manuscript. 6) ln255: please be consistent and use capital letters for CLP290Thank you for your correction. We ensured that CLP290 is spelled consistently throughout the entire manuscript. 7) ln311: “To our knowledge, this is the first study to report that CLP290 modulates GABAA receptor-mediated pharmacology”. I think this statement is not correct. CLP290 does not interfere with pharmacology at GABAA receptor but affect indirectly the effect of GABAA receptor activation.Thank you for very much your comment. The proposed mechanism underlying the observed CLP290 effect is to change the [Cl-]i by activating KCC2, followed by indirect modification of the action induced by GABA receptor activation. We changed the manuscript as follows: “ However, as far as we know, the effect of CLP290 in naive neonatal rats has not been described yet, and this is the first report demonstrating that CLP290 enhances the sedative effect of midazolam in neonatal rats.” (Lines 371-373).8) ln379 “A possible resulting hypothesis is that higher midazolam doses might deeply attenuate neuronal activity in the thalamus deeply,”. Deeply is repeated twice.Thank you for your correction. Accordingly, we corrected this sentence (Lines 461-463).Besides response to the comments from reviewers, we changed following sentences to improve readability;1. Lines 18-23　　before “therefore, their intracellular chloride ion ([Cl]-i) concentration is higher than that in mature neurons; further, hyperpolarization does not occur due to γ-aminobutyric acid (GABA) receptor activation. Therefore, midazolam, a benzodiazepine agonist, does not exert a sufficient sedative effect in neonates.”　　after “The intracellular chloride ion concentration ([Cl-]i) is higher in immature neurons than in mature neurons; therefore, γ-aminobutyric acid type A (GABAA) receptor activation in immature neurons does not cause chloride ion influx and subsequent hyperpolarization. In our previous work, we found that midazolam, benzodiazepine receptor agonist, causes less sedation in neonatal rats compared to adult rats and that NKCC1 blockade by bumetanide enhances the midazolam-induced sedation in neonatal, but not in adult, rats.”2. Lines 33-37　　before “To our knowledge, this is the first study on the effects of CLP290 in neonates. It provides behavioral and histological evidence that CLP290 improves the sedative effect of GABAergic drugs through KCC2 activation. We expect that the clinical application of CLP290 could provide a breakthrough in midazolam-induced resistance and adverse effects.”after “To our knowledge, this study is the first report showing the sedation-promoting effect of CLP290 in neonates and providing behavioral and histological evidence that CLP290 reverted the sedative effect of GABAergic drugs through the activation of KCC2. Our data suggest that the clinical application of CLP290 may provide a breakthrough in terms of midazolam-resistant sedation.”3. Lines 54-56before “The switch in the functional role of the GABAA receptor from excitatory to inhibitory occurs between PND 8 to 14, which differs depending on the brain region [4-6].”after “The switch in the functional role of the GABAA receptor from excitatory to inhibitory GABA signaling occurs between PND8 and 14, which correlates with the switch in dominant expression from NKCC1 to KCC2 [6-8].”4. Lines 82-90　　before “We hypothesized that KCC2 activation promotes GABAA receptor-induced neuronal inhibition and sedative effects of midazolam in neonatal rats. To test this hypothesis, we conducted loss of righting reflex behavioral tests to examine the supportive effect of CLP290 on midazolam-induced sedation in neonatal animals. Moreover, we conducted p-CREB immunohistochemistry to determine the neural circuits underlying the sedative effect of CLP290. Further, we measured the cell surface expression of KCC2 to investigate the biochemical processes underlying the CLP290 effects in vitro.”　　after “We hypothesized that activation of KCC2 promotes the sedative effect of GABAA receptor activation in immature rodents. To test this hypothesis, we assessed the righting reflex behavior of neonatal and adult animals treated with midazolam and CLP290. Next, we conducted phosphorylated cyclic adenosine monophosphate-response element-binding protein (p-CREB) immunohistochemistry to determine the brain region related to the CLP290 effect on midazolam-induced sedation. Moreover, to elucidate the underlying mechanism, we investigated whether the sedation-promoting effect of CLP290 in neonates can be antagonized by the KCC2-selective antagonist VU0463271. Finally, we measured the cell surface expression of KCC2 to elucidate the biochemical processes underlying the in vivo effect of CLP290.”5. Lines 134-143　　before “3. Neonatal rats were orally administrated with CLP290 at 100 mg/kg or an equal vehicle volume. Subsequently, we intraperitoneally injected VU0463271 or an equal vehicle volume after 20 and 50 min at 20 ml/kg and 10 ml/kg, respectively. Additionally, we intraperitoneally injected midazolam at 20 mg/kg 2 h after CLP290 or vehicle administration.”　　after “3. To assess the involvement of KCC2 in the promotion effect of CLP290 on midazolam-induced sedation in neonatal rats, rats were pretreated with CLP290 followed by VU0463271 prior to the midazolam administration. That is, neonatal rats were randomly assigned to two groups and orally pretreated with CLP290 (100 mg/kg) or an equal volume of vehicle 2 h prior to the midazolam administration. Subsequently, each group was subdivided into two groups, and VU0463271 or an equal volume of vehicle was injected twice intraperitoneally at 0.02 ml/g and 0.01 ml/g 20 min and 50 min, respectively, after the first pretreatment. All rats received midazolam at 20 mg/kg intraperitoneally 70 min after the second administration of VU0463271 or vehicle. The righting reflex latency of all rats was measured 30 min after administration of midazolam (n = 6).”6. Lines 145-149　　before “We conducted the immunohistochemistry experiments with the purpose of assessing the neuronal excitability using the combination of midazolam and CLP290 . We orally administered neonate and adult rats with CLP290 at 100 mg/kg or an equal vehicle volume 2 h prior to intraperitoneal midazolam injection at 20 mg/kg or an equal saline volume.”　　after “This immunohistochemistry experiment was conducted to assess the neuronal activity following the separate or combined administration of midazolam and CLP290. Neonatal rats were randomly assigned to two groups and orally treated with CLP290 (100 mg/kg) or an equal volume of vehicle. Subsequently, 2 h after CLP290 or vehicle administration, each group was subdivided into two groups to receive midazolam at 20 mg/kg or an equal volume of saline intraperitoneally.”7. Lines 241-246　　before “To examine whether KCC2 activation promoted the sedative effect of midazolam, we administered CLP290 to PND 7 rats. We found that CLP290 administration significantly prolonged the righting reflex latency compared with vehicle administration at 15 min  and 30 min  after midazolam injection. Moreover, the administration of CLP290 alone did not have an effect on the righting reflex latency . This indicates that KCC2 activation through oral CLP290 administration promoted the sedative effect of midazolam in the neonates while a similar midazolam dose without CLP290 pretreatment had no sedative effect in the neonates unlike the adults.　　after “In PND7 rats, showing resistance to midazolam-induced sedation, CLP290 pre-administration significantly prolonged the righting reflex latency compared to vehicle at 30 min after midazolam injection . Moreover, the administration of CLP290 alone had no effect on the righting reflex latency . This suggests that prior activation of KCC2 by CLP290 might promote the sedative effect of midazolam in neonates whereas CLP290 has no sedative effect by itself.”8. Lines 279-280　　before “To examine whether the CLP290-induced sedative effect resulted from an inhibition of neuronal excitation, we conducted p-CREB immunohistochemistry to determine the number of excitatory neurons in awake-related brain regions.”　　after “To identify brain regions involved in the CLP290 effect on midazolam-induced sedation, p-CREB immunohistochemistry was conducted .” 9. Lines 282-292before “Contrastingly, midazolam administration to PND 7 rats pretreated with CLP290 significantly decreased the number of p-CREB positive cells compared with vehicle administration, which was consistent with the righting reflex test results . However, there was no significant among-group difference in the number of p-CREB positive cells in the thalamus . These findings indicated that the administration of 20 mg/kg midazolam alone had an insignificant effect on neuronal status; however, CLP290 pretreatment promoted the inhibitory effect of midazolam in the somatosensory cortex in neonatal rats.”　　after “By contrast, midazolam administration to neonatal rats pretreated with CLP290 significantly decreased the number of p-CREB-positive cells compared with vehicle administration . Moreover, the administration of CLP290 alone had no effect on the number of p-CREB-positive cells . These findings are inversely correlated to the level of sedation as assessed by the loss of the righting reflex . However, the numbers of p-CREB-positive cells in the thalamus were not significantly different among groups . These findings showed that a single dose of 20 mg/kg midazolam had little effect on neuronal activity in the cerebral cortex and thalamus of PND7 rats. Midazolam administration after pretreatment with CLP290 exerted inhibitory effects on neuronal activity exclusively in the somatosensory cortex of neonatal rats, which is consistent with the behavioral results in the righting reflex test shown in Fig 2.”10. Lines 317-323　　before “As aforementioned, the righting reflex latency after midazolam injection with CLP290 pretreatment was significantly longer than that without CLP290; further, VU0463271 co-administration significantly attenuated the effect of CLP290 on the righting reflex . These results indicated that the CLP290 effect of promoting the sedative effect of midazolam in the neonatal rats was largely KCC2-dependent.”　　after “There was a significant interaction between the effect of CLP290 and VU0463271 (p = 0.011). The main effect of CLP290 was significant (p < 0.001), whereas that of VU0463271 was not significant (p = 0.785). A post-hoc median regression analysis was performed as follows: First, the righting reflex latency after midazolam injection with CLP290 pretreatment was significantly longer than that without CLP290 administration . Furthermore, VU0463271 coadministration significantly attenuated the effect of CLP290 on the righting reflex . 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A systematic study of the genomic architecture of CSF biomarkers in Parkinson’s disease has not yet been conducted. Here, genome-wide association studies of CSF biomarker levels in a cohort of individuals with Parkinson’s disease and controls (N = 1960) were performed. PD cases exhibited significantly lower CSF biomarker levels compared to controls. A SNP, proxy for APOE ε4, was associated with CSF amyloid beta1–42 levels . No genome-wide loci associated with CSF alpha-synuclein, total tau, or phosphorylated tau181 levels were identified in PD cohorts. Polygenic risk score constructed using the latest Parkinson’s disease risk meta-analysis were associated with Parkinson’s disease status (p = 0.035) and the genomic architecture of CSF amyloid beta1–42 . Individuals with higher polygenic risk scores for PD risk presented with lower CSF amyloid beta1–42 levels (p = 7.3 × 10−04). Two-sample Mendelian Randomization revealed that CSF amyloid beta1–42 plays a role in Parkinson’s disease (p = 1.4 × 10−05) and age at onset (p = 7.6 × 10−06), an effect mainly mediated by variants in the APOE locus. In a subset of PD samples, the APOE ε4 allele was associated with significantly lower levels of CSF amyloid beta1–42 (p = 3.8 × 10−06), higher mean cortical binding potentials (p = 5.8 × 10−08), and higher Braak amyloid beta score (p = 4.4 × 10−04). Together these results from high-throughput and hypothesis-free approaches converge on a genetic link between Parkinson’s disease, CSF amyloid beta1–42, and APOE.Alpha-synuclein is the main protein component of Lewy bodies, the pathological hallmark of Parkinson’s disease. However, genetic modifiers of cerebrospinal fluid (CSF) alpha-synuclein levels remain unknown. The use of CSF levels of amyloid betaThe online version of this article (10.1186/s40478-020-01072-8) contains supplementary material, which is available to authorized users. SNCA gene, which encodes α-Syn, correlates with cerebrospinal fluid (CSF) α-Syn levels and a more severe PD phenotype -Pittsburgh Compound B (PIB) acquisition and analysis were performed according to published methods . Brieflyfunction , 50. MeaThe neuropathological analysis was done at WUSTL, as previously reported . Briefly−06) were excluded from downstream analyses. The X chromosome SNPs were used to determine sex based on heterozygosity rates. Samples in which the genetically inferred sex was discordant with the reported sex were removed. Whole-genome sequence data from the PPMI cohort was merged with imputed genotyped data; only variants present in both files were included in further analyses. Pairwise genome-wide estimates of proportion identity-by-descent tested the presence of unexpected duplicates and cryptically related samples (Pihat > 0.50). Unexpected duplicates were removed; the sample with a higher genotyping rate in the merged file was kept for those cryptically related samples. Finally, principal components were calculated using HapMap as an anchor. Only samples with European descent, an overall call rate higher than 95%, and variants with minor allele frequency (MAF) greater than 5% were included in the analyses.All cohorts, except PPMI, were genotyped using the Global Screening Array (GSA) Illumina platform. Genotyping quality control and imputation were performed using SHAPEIT  and IMPUSOMAscan).The three-stage single variant analysis was performed due to differences in time and platform for biomarker quantification. PLINK1.9 , 56 was APOE locus. GCTA estimates the phenotypic variance explained by genetic variants for a complex trait by fitting the effect of these SNPs as random effects in a linear mixed model.The genome-wide complex traits analysis (GCTA) software  was usedSOMAScan platform) [181 were not available in different tissues.The levels of α-Syn were measured in CSF, plasma, and brain  using an aptamer-based approach (latform) . After slatform) . A multilatform)  was appl181 were available for the PPMI cohort. A simplified PRS (detailed below) was used to test if the genetic architecture of PD was predictive of biomarker level progression. The PD PRS using sentinel SNPs from the META-PD [2 > 0.90 was used. The weight of each variant was calculated using the binary logarithm transformation of the reported Odd ratios. The final PRS is the sum of the weighted values for the alternate allele of all the sentinel SNPs.PRS is constructed by summing all trait-associated alleles in a target sample (META-PD and CSF biomarkers separately), weighted by the effect size of each allele in a base using different p-value thresholds. SNPs in linkage disequilibrium (LD) are grouped together to avoid extra weight into a single marker. The optimal threshold is considered the one that explains the maximum variance in the target sample. The association was tested using the default parameters and nine p-value cutoffs. The PRSice2 software  was used META-PD  was mode META-PD , 42, 68.2 statistic. I2 statistic, instead of F statistic, is a better indicator of instrument strength for the two-sample summary data approach [MR requires that the genetic instruments are associated with the modifiable exposure of interest (GWAS of CSF biomarkers), and any association between the instruments and the outcome (PD risk) is mediated by the exposure . A two-sapproach . The R papproach  . Finally, the summary statistics of the GWAS for α-Syn CSF levels generated in the current study were used. There was no overlap between CSF biomarker datasets and PD risk datasets. Instrumental variables for each GWAS were obtained by clumping each GWAS summary statistics based on the LD structure of the exposure (CSF biomarker levels) and a significance threshold of 1.0x10−5 using PLINK1.9 [The latest and largest meta-analysis for PD genetic risk was used to perform the MR analyses . Summarylso used . Deming lso used . None ofPLINK1.9 . InstrumPD = − 0.05, p = 2.10 × 10−04; Aβ42: betaPD = − 0.34, p = 4.38 × 10−05; t-tau: betaPD = − 0.23, p = 4.58 × 10−03; and p-tau181: betaPD = − 0.25, p = 2.46 × 10−03—Fig. 181 in the PPMI cohort (N = 785). These results suggest that CSF dementia biomarkers are associated with PD status.A generalized linear model (CSF biomarker levels ~ Age + Sex + Status) including PD cases (N = 700) and controls N = 189) from two independent datasets  were identified in these analyses , PD cases and controls (N = 889), AD cases only (N = 386), AD cases and controls (N = 575) and controls only (N = 189) were also performed. None of these analyses revealed any genome-wide significant locus, suggesting that these sample sizes might be underpowered to uncover the genetic modifiers of CSF α-Syn.181, individual cohort analyses revealed three genome-wide significant loci , brain (N = 380), and CSF (N = 835) using the 9, brain APOE ε4, rs769449, was associated with CSF levels of Aβ42 in the WUSTL , and ADNI cohorts . This association did not pass the genome-wide multiple test correction threshold in the PPMI cohort  and was not significant in the Spanish cohort  and a locus in the HLA region  remained significant in the meta-analysis  but not the HLA region . In the combined analysis of all cohorts (N = 1960), the APOE locus accounted for 36.2% of the CSF Aβ42 levels variance (p = 2.35 × 10−03). Overall, these results revealed a strong and highly significant association between APOE locus and lower CSF Aβ42 levels in PD cohorts.A proxy SNP for 181 levels  with a correlation coefficient (R2) of 2.29%. In PD cases and controls only, the correlation remained significant (p = 4.78 × 10−08), with an R2 of 2.36%. In PD patients with both GWAS and CSF biomarker data, the CSF levels of each biomarker were analyzed by quartiles of the PRS calculated from META-PD risk. A significant difference (p = 7.30 × 10−04) was found among the top and the bottom quartiles; higher PRS values exhibit lower levels of CSF Aβ42 , and a significant causal effect for CSF Aβ42 on PD  , but not in Chang et al., 2017  and the main driver of the causal effect of CSF Aβ42 on PD. Other SNPs contribute in a smaller proportion to the causal effect . No difference in the APOE ε4 frequency was found between cases (0.14%) and controls (0.11%). However, the CSF Aβ42 levels were significantly different between controls (p = 3.00 × 10−02) and cases (p = 3.80 × 10−06) when stratifying by the presence of APOE ε4 allele  and number of APOE ε4 alleles  . A linear regression model indicated that CSF Aβ42 and APOE ε4, explain 48% of the variance of MCBP. APOE ε4 is also significantly associated with MCBP  in analysis with 200 participants that included sex and age as covariates. APOE ε4 and age at onset explain 20% of the MCBP variance in this larger cohort. The presence of APOE ε4 did not affect the MCBP in controls (p = 0.19). However, PD patients carrying APOE ε4 exhibit significantly (p = 5.80 × 10−08) higher levels of MCBP than non-carriers  revealed that MCBP increased with age-at-onset  Fig. a, but deAPOE genotype were available from 92 PD cases. Individuals carrying an APOE ε4 allele had significantly (p = 4.40 × 10−04) higher Braak Aβ stage  and diffuse plaques , but not with neuritic plaques . The best multiple linear regression model for the Braak Aβ stage, which included age at onset and APOE ε4, explained 42% of the variance of the Braak Aβ stage. Altogether, these results suggest that APOE ε4 drives the Aβ deposition in PD participants.Neuropathological data and age Fig. c. APOE ε181 levels were significantly lower in PD cases compared with controls, as we previously reported with a smaller sample size [181. A SNP proxy for APOE ε4 was genome-wide associated with CSF Aβ42 levels. The PRS calculated using META-PD was associated with PD status and correlated with the genomic architecture of CSF Aβ42; in fact, individuals with higher PRS scores exhibit lower CSF Aβ42 levels. Two-sample MR analysis revealed that CSF Aβ42 probably plays a role in PD and PD age-at-onset, an effect mainly mediated by variants in the APOE locus. Using a subset of participants from the WUSTL cohort with additional clinical and neuropathological data, we found that the APOE ε4 allele was associated with lower levels of CSF Aβ42, higher cortical binding of PiB PET and higher Braak Aβ score.CSF α-Syn, Aβ42, t-tau, and p-tauple size . GWAS we−3). No correlation was found between the genetic architecture of PD with cross-sectional or longitudinal CSF α-Syn levels, consistent with what we have previously reported [This is the first comprehensive analysis of CSF α-Syn and AD biomarkers using GWAS, PRS, and MR in PD. We found lower levels of CSF α-Syn in PD cases compared to controls in a cross-sectional analysis but no significant differences in the longitudinal study (PPMI). CSF α-Syn, as measured with ELISA-based assays, is not a clinically useful diagnostic marker for PD, and utility as an outcome measure for clinical trials or progression is still controversial , 53. CSFreported . Using MAPOE locus, CSF Aβ levels, and PD. MR is used to test if the genetic variation associated with a trait has a causal relationship with a health outcome [APOE locus with CSF Aβ) should not be associated with the outcome (PD risk). The third MR assumption is the exclusion restriction, which means that SNPs do not affect PD risk except through CSF Aβ levels. The two sample MR used here requires two additional criteria: both cohorts must have similar genetic background but no overlap with each other. Here, samples used for the MR analysis, summary statistics from Deming, et al. [APOE locus. Thus, we inferred that the lifetime effect of the APOE locus is causal in relation to PD.MR analyses suggest that Aβ42 could play a causal role in PD. Our MR results consistently identified a causal correlation between the  outcome . MR is n outcome . Here, i outcome . A secon, et al.  and Nall, et al. , met botAPOE locus and CSF Aβ42 levels were GWAS significant in the meta-analysis. The association of the APOE locus with CSF Aβ42 levels has been previously reported in AD [APOE locus is the most significant locus associated with sporadic LBD, [The ed in AD  but not ed in AD . The APOdic LBD, , 60, anddic LBD, , but notdic LBD, . Here, wAPOE locus. Here, we also provided further evidence by showing that PD patients carrying the APOE ε4 allele presented with lower levels of CSF Aβ42 (p = 3.8 × 10−06), higher MCBP (p = 5.80 × 10−08) and higher Braak Aβ scores (p = 4.40 × 10−04). These results support the synergistic relationship between α-Syn and Aβ pathology in AD, PD and LBD brains [APOE ε4 drives the production of Aβ, the accumulation of Aβ fibrils in AD patients [APOE in human synucleinopathies is probably more complex. In LBD patients, the APOE ε4 effect on α-Syn pathology could be dependent on concurrent Aβ and/or tau pathology [APOE ε4 also promotes α-Syn pathology independently [APOE ε4 increased the α-Syn phosphorylation, worsened motor impairment, and increased neuroinflammation and neurodegeneration in different mouse models [The results from unbiased analyses like GWAS, PRS and MR demonstrated a link between PD genetic risk with CSF Aβ42 levels and the D brains , and thepatients , exacerbpatients  and affepatients . Howeverathology , howeverendently , 65 and endently . We recee models .This is the largest sample size used for discovering CSF α-Syn genetic modifiers to date and yet no GWAS significant locus was found. It is possible that the complexity of α-Syn genetic architecture makes the current sample size insufficiently powered to detect signals with a smaller effect. Here, we found lower levels of CSF α-Syn in PD patients, which aligns with previous reports. However, neither PRS nor MR analysis revealed evidence of the causal link of CSF α-Syn with PD risk. In fact, it has been reported that α-Syn aggregation is neither necessary nor sufficient for neurodegeneration or clinical parkinsonism , 32. TheAPOE locus. These findings were further validated by strong significant associations of APOE ε4 with Aβ deposition in cortical regions of living and postmortem PD patients.PD is a heterogeneous disorder with different identifiable clinical-pathological subtypes based on symptom severity and predominance . It is cAdditional file 1. Supplemental Figures.Additional file 2. Supplemental Tables.