Access to a complete continuum of maternal and child health care has been recommended globally for better pregnancy outcomes. Hence this study determined the level  and predictors of successfully completing continuum of care (CoC) in Rwanda.We analyzed weighted secondary data from the 2019–2020 Rwanda Demographic and Health Survey (RDHS) that included 6,302 women aged 15 to 49 years who were selected using multistage stratified sampling. We analyzed complete continuum of care as a composite variable of three maternal care services: at least four ANC contacts, SBA, maternal and neonatal post-natal care. We used the SPSS version 25 complex samples package to conduct multivariable logistic regression.Of the 6,302 women, 2,131 (33.8%) (95% CI: 32.8–35.1) had complete continuum of care. The odds of having complete continuum of care were higher among women who had exposure to newspapers (adjusted odds ratio (AOR): 1.30, 95% CI: 1.11–1.52), those belonging to the eastern region (AOR): 1.24, 95% CI: 1.01–1.52), southern region (AOR): 1.26, 95% CI: 1.04–1.53), those with health insurance (AOR): 1.55, 95% CI: 1.30–1.85), those who had been visited by a field health worker , those with no big problems with distance to health facility (AOR): 1.25, 95% CI: 1.07–1.46), those who were married (AOR): 1.35, 95% CI: 1.11–1.64), those with tertiary level of education (AOR): 1.61, 95% CI: 1.05–2.49), those belonging to richer households (AOR): 1.33, 95% CI: 1.07–1.65) and those whose parity was less than 2 (AOR): 1.52, 95% CI: 1.18–1.95).We have identified modifiable factors , that can be targeted to improve utilization of the entire continuum of care. Promoting maternity services through mass media, strengthening the community health programmes, increasing access to health insurance and promoting girl child education to tertiary level may improve the level of utilization of maternity services.The online version contains supplementary material available at 10.1186/s12884-022-05109-9. Globally, almost all neonatal deaths 75%) occur in the first week of life . Majorit% occur iSustainable Development Goal (SDG) three targets to reduce maternal and neonatal mortality to less than 70 deaths per 100 000 livebirths and 12 per 1000 livebirths respectively .Although the 1994 genocide devastated Rwanda’s health system, efforts were put in place to ensure that the system is rebuilt which partly contributed to the country’s achievement of the Millennium-um Development Goals (MDG) 4 of reducing the under-five mortality rate by two-thirds and MDG 5 of reducing the maternal mortality ratio by three-quarters –16. HoweThe 2020 Rwanda Demographic and Health Survey (RDHS) reports variation in maternal care utilization with only 47% of the women accessing at least four ANC contacts compared to SBA of 94% . Given tAgent de Sante Maternelle (ASM) and these CHWs are responsible in delivering the first line of health services including maternal and newborn health services [Rwanda is a central-eastern African nation of about 12 million people , 25 whosservices , 28. Theservices , 29.The 2019-20 Rwanda Demographic Survey (RDHS) was used for this analysis employed a two-stage sample design with the first stage involving sample points (clusters) selection consisting of enumeration areas (EAs) [Eligibility for women to participate in the RDHS was being aged between 15 and 49 years and being either permanent residents of the selected households or visitors who stayed in the household the night before the survey . Out of Complete continuum of maternal and newborn healthcare was coded 1 and incomplete coded as 0 . CompletNineteen independent variables were categorized into women and household characteristics, and were chosen basing on previous studies –34 and aWe used the complex sample package of SPSS (version 25.0) statistical software which accounted for the multi-stage cluster study design . Individ± 6.74 years and 5.14 ± 1.86 members respectively.A total of 6,302 women were included in the analysis Table . Only 16Factors associated with CoC utilisation are shown in Table The current study reveals that only a third of Rwandan women received all elements of the continuum of care, and these women tended to be those from the Eastern region, those exposed to mass media (newspapers), those who had been visited by a field health worker, those who are from richer households, having tertiary level of education, married, low parity those with insurance and those with no big problems with distance to the nearest health facility.The prevalence of complete continuum of care in this study is lower than findings from Cambodia, Egypt and Zambia , 31, 36 Exposure to mass media (newspapers/magazines) showed a significant association with CoC utilization with the exposed women having higher odds of CoC. This finding is not surprising because several similar studies from other countries within in the region have reported this positive association between utilisation of mass media and the utilisation of MCH services , 42, 43.Women who had been visited by a field health worker had higher odds of CoC utilization. Rwanda’s health system has a strong community health based programme . Each viThese field based health workers are crucial in ensuring access to maternal care especially in areas with shortage of health facilities and large distances needed to be covered by women. The field health workers help equip mothers with knowledge on the dangers of using unskilled birth attendants and complications of pregnancies in addition to providing care, referring the complicated cases and also encouraging them to seek care within health facilities where possible . Further“geographical accessibility and spatial coverage modeling of the primary health care network in the Western Province” showed that when considering walking as the single mode of transportation, less than a third of the western province population is covered by the catchment area of the existing primary health facility network [Women in the eastern region had higher odds of CoC utilization. This could be partly attributed to the high focus of national and international non-governmental organizations (NGOs) in the eastern region after it experienced some of the worst effects of the genocide that negatively affected the health status , 15. The network . Fadelu  network . Hence tWomen who had no big problems with distance to the nearest health facility had higher odds of CoC utilization. Distance to health facilities has been found elsewhere in similar contexts to be an important determinant of maternal healthcare services utilization . Given tWomen with health insurance had higher odds of CoC utilization a finding that has been shown in other studies –63. RwanWomen belonging to richer households had more odds of having CoC compared to those from the poorest households a finding consistent with several SSA studies , 67, 68.Consistent with findings from Ethiopia, Ghana and Uganda, increased levels of education have been shown to be associated with higher odds of completing CoC , 71, 72.Married women had higher odds of having CoC compared to their non-married counterparts. Marital status’s positive association with utilization and outcomes of maternal healthcare has been documented in several countries , 75–78. Women with less parity had higher odds of having CoC compared to those whose parity was five and above. Increased utilization of maternal healthcare services among women with less parity has been shown in several other SSA countries –84. ThisOnly 3 out of 10 women were able to utilize the entire continuum of maternity care services. Regarding ANC utilization, less than half (47.2%) and only 0.3% of women were able to utilize at least four and eight ANC contacts respectively a finding showing that Rwanda is not on track towards achieving the recommended eight ANC contacts for each pregnant woman in the 2016 WHO ANC guidelines. Belonging to eastern and southern Rwanda, exposure to mass media, having been visited by a field health worker, having health insurance, having no big problems with distance to the nearest health facility, belonging to richer households, having tertiary level of education, being married and having low parity were positively associated with completion of maternal and newborn continuum of care. Thus, we recommend the different stakeholders engaged in maternal health care to focus on women from western region, those without insurance, from poor households, with low levels of education, high parity and unmarried to ensure utilization of complete continuum of maternal and newborn care. Promoting maternity services through mass media, strengthening the community health programmes, increasing access to health insurance, strengthening and promoting girl child education to post-secondary level, increased support to household economic development programmes may improve the level of utilization of maternity services. Utilization of mass media specially to sensitize the population on the benefits of continuum of care should be encouraged with great attention given for early initiation of ANC, increasing the number of ANC contacts, health facility and skilled birth attendance during childbirth and postnatal care. During ANC contacts in health facilities and in the community by CHWs unmarried and high parity women need to be given more time during care, awareness on the importance of having CoC and followed up actively.Below is the link to the electronic supplementary material.Supplementary Material 1


The purpose of this study is to evaluate the clinical effectiveness and determine the differences, if any, between the trifocal bone transport (TFT) technique and the bifocal bone transport (BFT) technique in the reconstruction of long segmental tibial bone defects caused by infection using a monolateral rail external fixator.A total of 53 consecutive patients with long segmental tibial bone defects caused by infection and treated by monolateral rail external fixator in our department were retrospectively collected and analyzed from the period January 2013 to April 2019, including 39 males and 14 females with an average age of 38.8 ± 12.4 years (range 19–65 years). Out of these, 32 patients were treated by the BFT technique, and the remaining 21 patients were managed by the TFT technique. The demographic data, operation duration (OD), docking time (DT), external fixation time (EFT), and external fixation index (EFI) were documented and analyzed. Difficulties that occur during the treatment were classified according to Paley. The clinical outcomes were evaluated by following the Association for the Study and Application of the Method of Ilizarov (ASAMI) criteria at the last clinical visit.p > 0.05). The mean defect size and OD in TFT  were larger than that in BFT  (p < 0.05). The mean DT, EFT, and EFI in TFT  were all less than those in BFT  (p < 0.05). Difficulties and complications were more prevalent in the BFT group than in the TFT group (p < 0.05).All patients achieved an infection-free union finally, and there was no significant difference between the two groups in terms of demographic data and both ASAMI bone and functional scores (Both the trifocal and BFT techniques achieve satisfactory clinical outcomes in the reconstruction of long segmental tibial bone defects caused by infection using a monolateral rail external fixator. The TFT technique can significantly decrease the DT, EFT, EFI, difficulties, and complications compared with the BFT technique. Long segmental tibial bone defects, a common clinical problem, can result from high-energy injury, the radical removal of contaminated bony fragments in an open fracture, a bone tumor resection, or a repeated debridement of infected non-union , 2. The The treatment of bone defects has been revolutionized by the bone transport technique as seen by its worldwide use. A lot of published data have declared that this distraction osteogenesis (DO) technique is an effective and practical method for the reconstruction of large bone defects due to the complete eradication of infection and its powerful effects –20. The Therefore, the purpose of this study is to evaluate the clinical effectiveness and determine the differences, if any, between the trifocal and the BFT techniques in the reconstruction of long segmental tibial bone defects caused by infection using a monolateral rail external fixator.A total of 62 consecutive patients with long segmental tibial bone defects caused by infection and managed by the bone transport technique with a monolateral rail external fixator  in our department were retrospectively collected and analyzed from the January 2013 to April 2019 period. The inclusion criteria were as follows: patients with tibial bone defects more than or equal to 6 cm treated by the trifocal and BFT techniques, age older than 18 years, with a 24-month minimum follow-up. We excluded patients with pathological fracture, age older than 65 years, vascular and nerve injury, bone tumor, poor compliance, smoking habit, any other illness that can affect bone healing (such as diabetes), and those managed by a circular external fixator.Staphylococcus aureus in 25 cases, Methicillin-resistant Staphylococcus aureus in 17, Pseudomonas aeruginosa in 7, and Escherichia coli in 4.After the application of exclusion criteria, 53 patients were rendered eligible for this study, including their 53 injured limbs (left limb in 22 and right limb in 31). There were 39 males and 14 females with an average age of 38.8 ± 12.4 years (range 19–65 years). Thirty-two patients were treated by the BFT, and the remaining 21 patients were managed by the TFT. The etiology of bone defects was chronic osteomyelitis in 36 patients and infected non-union in 17 patients. With regard to bone defect location, there were 8 cases in the proximal one-third of the tibial shaft, 29 cases in the middle, and 16 cases in the distal. The mean previous operation was 2.6 ± 1.0 times (range 1–5 times). A meticulous debridement of the affected tissues, the installation of a monolateral rail external fixator, and a percutaneous minimally invasive cortical osteotomy using the Gigli saw were performed in all the patients. The average bone defect size after radical debridement was 8.5 ± 1.9 cm (range 6–13 cm) measured intraoperatively. There were 11 injured limbs that suffered active infection with drainage and sinus. Samples of the infected tissues were cultured, and antibiotic susceptibility tests were conducted in each patient. The culturing results showed The demographic data, operation duration (OD), docking time (DT), external fixation time (EFT), and external fixation index (EFI) were collected and analyzed. All patients were followed up at a minimum of 2 years after frame removal, and none was lost. The clinical outcomes were evaluated by following the Association for the Study and Application of the Method of Ilizarov (ASAMI) criteria  at the lThe same team performed all surgical procedures. The patients were positioned supine on a radiolucent table under continuous general or regional anesthesia. The incision was carried down to the periosteum corresponding to the previous surgical incisions when possible, protecting the healthy skin or subcutaneous tissues. The infected and devitalized bone and soft tissues were radically resected after complete hardware removal, and the samples were sent to culture for a sensitive antibiotic examination. The bony ends were resected until cortical bleeding, called the paprika sign , which mAntibiotic-impregnated cement beads were applied for patients with severe infection. Meticulous hemostasis was performed before wound closure. The vacuum sealing drainage technique was used for those with soft tissue defects or those that could not be initially closed without tension. Subsequently, local tissue flap or direct suture without tension was performed to reconstruct the small soft tissue defects, whereas flap transfer or free skin grafting was used to cover the larger wound.Length and axis restoration of the injured extremity was achieved first when the monolateral external fixator was installed. Two or three Schanz screws fixed by the connecting rail were inserted into the proximal and distal bony ends, respectively. For the BFT, the other two screws were inserted into the planned transport bony fragment and fixated at the sliding block. Every screw needed to be on the same plane. According to the location of the bone defect, a percutaneous cortical osteotomy with minimal invasion was conducted using Gigli saw. The framework of the TFT was similar but with two intermediate transport fragments fixated by the corresponding sliding blocks, as well as two osteotomy sites at the appropriate site. Bone defects larger than 8 cm or those that exceeded 40% of the injured bone underwent a TFT procedure , 27. WheSensitive antibiotic therapy was performed until the infection had been resolved, depending on the clinical manifestations and laboratory indicators. The small soft tissue defects were treated by direct suture or local tissue flap. The larger soft tissue defects were managed by free skin grafting or flap transfer.All patients were encouraged to undergo the isometric muscle and joint range of motion exercise on the postoperative second day, and early full-weight-bearing was also encouraged. The foot was kept in a neutral position using a rigid shoe with an elastic band, preventing ankle equinus contracture. Regular pin site care was performed every day using medical alcohol or iodophors.After a 7-day latency period, bone transport started at a rate of 0.25 mm in the BFT group, 4 times per day. In the TFT group, the bony fragment near the bone defect was transported 0.5 mm 4 times per day, while the other bony fragment was transported at a rate of 0.25 mm 4 times per day (tandem transport). As for converging transport, each bony fragment on both sides of the bone defect was transported 0.25 mm 4 times per day. Notably, the transport speed was modified according to the patient’s tolerance level and the radiological quality of the regenerate. After docking, bone transport continued for 4 or 5 days to compress the docking site, and the regenerate was allowed to consolidate.During the bone transport phase, the patients were asked to pay regular clinical visits twice a month, while it was once a month during the consolidation period. The monolateral external fixator was dynamized and followed by removal when satisfactory consolidation (three uninterrupted cortices appeared at the distraction zone) and docking site complete union were achieved on the standard anteroposterior and lateral radiographs. After frame removal, a functional brace was used to prevent refracture at the docking site or bending in the distraction zone.t-tests were used to analyze the continuous variables, which were expressed as the mean, standard deviation, and range of the observations. The count variables were analyzed by using the χ2 or Fisher’s test, expressing as a number. A statistically significant difference was set at p < 0.05.Statistical analysis was performed with SPSS 22.0 . Independent-sample p > 0.05), except for the mean defect size (p < 0.05). The typical BFT is shown in There were no statistically significant differences in demographic data between the two groups p > 0.0, except p < 0.05). The DT in BFT was longer than that in TFT , as well as the EFT . All patients achieved an infection-free union finally. The average EFI in BFT (60.8 ± 1.9 days/cm) was larger than that in TFT (34.8 ± 2.1 days/cm) (p < 0.05). More details are given in The mean OD was 122.5 ± 11.2 min in the BFT group, while it was 161.9 ± 8.9 min in the TFT group (p > 0.05)  .p < 0.05). Complications were more prevalent in the BFT group (31 complications of 32 patients) than in the TFT group (11 complications of 21 patients) (p < 0.05). More details are given in Based on Paley, there were 43 problems, 20 obstacles, and 31 complications in the BFT group, and 17 problems, 8 obstacles, and 11 complications were observed in the TFT group. The mean difficulties in BFT (2.9/patient) were larger than those in TFT (1.7/patient) p < 0.0. ComplicFive patients in BFT and two patients in TFT suffered deep pin tract infection and were successfully managed by screw replacement and intravenous antibiotics. The axial deviation was observed in seven cases in BFT and three cases in TFT. These patients underwent apparatus modification, docking site revision, and malalignment correction with regional anesthesia under image intensifier control. Three patients in BFT and one in TFT suffered soft tissue incarceration. Surgical intervention was performed to resect the interposed soft tissues, freshen the bone ends, and reopen the medullary canal. Joint stiffness was observed in 16 patients (11 in BFT and 5 in TFT) and successfully treated by a surgical release. Four patients in BFT suffered delayed union, and the “accordion maneuver” technique contributed to satisfactory outcomes. One patient developed non-union in the BFT group and finally achieved bone union by autogenous iliac crest bone grafting. No neurovascular injury, psychological problems, and refracture were observed in the 53 patients.The ultimate way of treating bone defects is to reconstruct a fully functional extremity without any unacceptable deformities or limb length discrepancy . UndoubtAt present, the two common techniques for long bone defects are acute shortening followed by relengthening (AST) and bone transport using an external fixator. However, neurovascular injury, limb discrepancy, and blood circulation obstacles are problems worth considering when the AST is performed on patients with bone defects >5 cm , 20, 29.Several methods have been developed to reduce the EFI, reduce the potential complications, and achieve satisfactory clinical results. Gupta et al.  performeThe TFT technique was proposed to accelerate the defect closure and shorten the treatment duration. Considering the biological characteristic of incapable speed up lengthening in just one osteotomy site, the TFT was derived from the theoretical basis of distraction simultaneously using two osteotomy leads to effectively double lengthening speed. In this procedure, the regeneration time is theoretically decreased to 50%, and the total treating time is reduced accordingly . AdditioBorzunov  performeIn the present study, the clinical results of 53 patients with long segmental tibial bone defects treated by the bifocal or TFT technique were retrospectively analyzed and compared. The mean defect size was significantly increased in the TFT group . In contrast, the mean DT , the mean EFT , and the mean EFI  were significantly reduced. The results were comparable to those of the previous studies , 33.A previous study reported that hypoplastic bone formation was a common complication when bone defects exceed 5 cm or 40% of the injured bone when treated by BFT . The regAlthough additional surgical procedures of one transport sliding block and an osteotomy site in TFT may increase the complications corresponding to Schanz screws and the distraction zone, faster regeneration and early frame removal have counteracted this negative effect. In this study, the BFT group showed a statistically significant increase in the mean rate of difficulty (2.9 difficulties/patient in BFT and 1.7 difficulties/patient in TFT) and complication (1.0 complications/patient in BFT and 0.5 complications/patient in TFT), and this can be explained by the longer frame duration in these patients. The only shortcoming in the TFT group was the significantly increased operation time .Furthermore, compared with previous studies , 29, 33,The clinical results of the present study reveal that both BFT and TFT achieve satisfactory outcomes in the reconstruction of long segmental tibial bone defects caused by infection using a monolateral rail external fixator. Although there were no significant differences between BFT and TFT in the final bone and functional results, the EFI, difficulties, and complications were significantly decreased in the TFT group. Our experience suggests that the most vital step is radical debridement of the infectious tissues in the reconstruction of bone defects caused by infection. In addition, a comprehensive understanding of frames, prudent patient selection, appropriate pin insertion, meticulous care, early detection of complications, and proper intervention or psychological counseling all ensure satisfactory results.The present study may be limited by its retrospective nature with only a single-center small sample size, and, therefore, a conservative attitude should be adopted while making interpretations of our results. Multicentered trials with larger sample sizes, life quality assessments, and mental evaluations should be adopted in future investigations.Both the trifocal and the BFT techniques achieve satisfactory clinical outcomes in the reconstruction of long segmental tibial bone defects caused by infection using a monolateral rail external fixator. The TFT technique can significantly decrease the DT, EFT, EFI, difficulties, and complications compared with the BFT technique.


Physical fitness was compared to data collected on a cohort of students from the same institution and program, 30 years prior . Post30 had greater waist circumference  and lower estimated maximal aerobic fitness . Compared to Pre, male Post30 vertical jump  and female Post30 broad jump  were lower. A subsample (n = 65) of Post30 whose habitual activity was assessed, met the aerobic portion of Canadian physical activity guidelines (~400 min/week), but spent excessive amounts of time stationary (10.7 h/day). Current kinesiology students may not be immune to population decreases in physical fitness. Relative to previous group of students interested in movement, fitness was lower in our sample, potentially attributed to excessive stationary time. Regular assessment of physical fitness in kinesiology curriculums may be valuable to understand these declining trends in undergraduate students that mimic population declines in fitness.The physical fitness of Canadian adults has decreased over the past 30 years, while sedentary time has increased. However, it is unknown if university students studying human movement exhibit similar population declines. Physical fitness  and habitual activity  was measured in a cohort of kinesiology students (Post30; 2010–2016;  The health benefits of being physically active are well established , with adA large proportion of post-secondary students are excessively physically inactive ,8 and imPhysical activity is any bodily movement produced by skeletal muscle that results in energy expenditure while physical fitness is a set of attributes that are either health- or skill-related that can be measured with specific tests . PhysicaWe sought to characterize kinesiology students in a recent cohort (from 2010–2016) and compare them to students from the same school and similar program from 30 years prior (data collected in regular testing from 1984–1987). These comparative cohorts are from the same institution that has retained most aspects of what was previously a ‘physical education’ program into the, now named, ‘Bachelor of Kinesiology’ program. It was hypothesized that among the unique sample of activity-minded individuals, fitness levels of current kinesiology students would not be different than the physical education students 30 years prior. Due to the well-established impact of sex on physical fitness ,14, analn = 178), which were collected as part of the past degree’s mandatory fitness testing. Kinesiology students from 2010, 2015, and 2016  were recruited through a first-year Bachelor of Kinesiology compulsory course. Both cohorts of participants were collected from the same university in Atlantic Canada. This reference base is an appropriate cohort for comparison of students interested in the study of human movement, as the current Bachelor of Kinesiology program has preserved much of the practical and applied roots of the previous Physical Education program where many past and present students go on to teach physical education or pursue occupations in applied kinesiology and health professions.Participant testing and secondary use of previously collected data was approved by the institution’s Research Ethics Board (REB 15-61). Fitness testing results were obtained from the university’s Bachelor of Physical Education students of 1984–1987 , weight (kg), and waist girth (cm) using standard anthropometric protocol . BMI waschniques ,17,19. Tchniques . A 50-m 2max) was calculated based on the distance ran using the following equation: Predicted VO2max (mL/kg/min) = (Distance ran (m) × 0.0268) − 11.3 [Aerobic fitness was measured using the Cooper 12-min run on a 400-m track . This tr) − 11.3 .n = 65; 25 males) completed a Modified-Leisure Time Physical Activity Questionnaire (mLTPA-Q) [®, Steps Count Inc., Deep River, ON, USA) for one full week.A subsample of kinesiology students from 2015 and 2016 (mLTPA-Q)  and woremLTPA-Q)  physicalStudents instructed to wear the device a minimum of 10 h per day for one full week. PiezoRx monitors were selected due to their ability to measure steps, light-, (LPA), moderate- (MPA), and vigorous-intensity physical activity (VPA), total physical activity (TPA), and the number of 10-min bouts. Stationary time was calculated by subtracting TPA from wear time and represents the combination of sedentary time and standing time, which cannot be distinguished using waist-worn or wrist-worn accelerometers. The PiezoRx has been previously validated in controlled laboratory  and freed effects sizes were calculated between Pre and Post30 within each sex as (Pre − Post30) ÷ pooled standard deviation.Linear regression analysis was implemented within each sex to compare Pre and Post30. Pre/Post30 were inserted as the independent variable , and the physical fitness measure as the outcome variable. The unstandardized-β represented the mean difference between Pre and Post30, and statistical significance of this predictor variable indicated a group difference. Cohen’s p < 0.001). For males, waist circumference and sprint times were non-normal . For females, grip strength was non-normal (p < 0.001). While thresholds for skewness vary, median values with ranges are presented for outcome variables exceeding ±2 . Independent sample t-tests evaluated physical fitness differences in the sub-sample of 2015/2016 students who wore the PiezoRx® pedometers to the entire Post30 sample and confirmed that the sub-samples fitness was representative of the entire group . The sub-sample of participants with physical activity data were analyzed using regressions, with sex incorporated as the dichotomous predictor variable. Analysis was completed in SPSS Statistics . Statistical significance was defined as p < 0.05. All physical fitness and habitual activity variables were described using mean ± standard deviation.Normality was assessed via the Kolmogorov–Smirnov test within each sex. For both sexes, age, BMI, sit and reach, and the agility tests were non-normal  and males (n = 103) had mean ages of 19.4 ± 3.0  years and 19.4 ± 2.1  years, respectively. The Post30 cohort were primarily females (n = 219) with a mean age of 19.0 ± 2.0 years , while males (n = 129) had a mean age of 19.3 ± 2.0  years. Female Post30 weighed more than female at Pre . Both male and female Post30 had larger waist circumferences  compared to Pre of the same sex.Participant physical fitness characteristics are presented in p < 0.045). No differences between cohorts were observed for vertical jump power, sit and reach, Illinois agility test and 50 m sprint . The median and range for 50 m sprint for Pre and Post30 females was 8.2 (6.4–11.9) and 8.1 s (7.0–13.4), respectively. Post30 male’s grip strength was 12.3 kg lower compared to Pre (p < 0.001). Predicted VO2max was lower in Post30 males (63 vs. 55 mL/kg/min), and females (50 vs. 45 mL/kg/min) compared to the Pre groups . Most activity was of moderate-vigorous-intensity , with less time spent in LPA (24%). There were no significant sex differences for the number of resistance training sessions per week , or planned aerobic exercise sessions per week .Objectively measured PA of Post30 was not different between sexes, as shown in The purpose of this study was to characterize physical fitness levels of kinesiology students in comparison to an analogous cohort of students interested in the study of movement from 30 years prior that attended the same institution. We demonstrated that a recent cohort of kinesiology students are less aerobically physically fit and have a greater waist circumference than a similar cohort of students ~30 years ago. Despite both cohorts of students studying human movement and being educated on the multitude of benefits of physical fitness, students in our sample had increased adiposity and lower fitness, similar to the trend observed in the greater Canadian population .d, although without limitations [Waist circumference estimates abdominal obesity and has implications on obesity-related comorbidities, independent of BMI . Post30 itations , the difitations . Our stuitations . While t2max [Aerobic fitness is a strong predictor of morbidity and mortality in the population across a range of conditions ,34. Acco2max  our samp2max . For con2max . Even wi2max , whereas2max ,39. Whil2max  and in h2max . While t2max . Certain2max , whetherThis study adds to the current literature by evaluating physical fitness of a sample of university kinesiology students with a similar cohort of university students 25–30 years ago. Even among students who are knowledgeable on physical fitness, the Post30 kinesiology students in our study exhibited larger waist circumferences, poorer muscle power/strength, and worse predicted aerobic fitness. Our kinesiology students appear to follow the same declining health patterns of the public, highlighting the impact of modern lifestyles . The implementation of more frequent fitness testing in current curriculum may draw attention to what may be a growing problem. Regular fitness testing was common practice many years ago and may help to identify trends and promote healthier outcomes for current and future students. Students interested in movement may benefit most from the practical experience gained by having their fitness assessed and/or senior students being the fitness appraisers. The integration of this testing may prove a useful educational experience for this specific sample. As well, the amount of time spent sedentary and in vigorous activity is likely important in the degree of adiposity and aerobic fitness. Tracking physical activity and sleep levels  may be a learning opportunity for those students interested in movement to broaden their perspectives of the influence of physical activity on health to include sedentary behaviour, light physical activity, and vigorous physical activity  aside from structured exercise.The recent students are from the same academic program, that was previously a Physical Education program but is now a Bachelor of Kinesiology, that has maintained a similar focus over the years on a balance of content in both the science of movement as well as teaching and coaching and theory and practical and applied contexts (as opposed to a Bachelor of Science in Kinesiology that may focus purely on science aspects and cater to a different type of student). It is possible that differences may even be larger among science focused programs that focus less on professional or applied contexts whose curriculum more so emphasizes graduate or medical school preparation. As the specific composition of each program was not ascertained and is therefore a limitation of this observational analysis, the findings are still interesting and noteworthy.Our comparison of kinesiology students to similar students 30 years apart requires the assumption that students interested in either of these programs are interested in ‘movement’ to a similar amount. However, this cannot be assessed. Although objective activity was measured in current kinesiology students, there is no way to determine the activity level of past physical education students, so it is possible that the difference in fitness between the cohorts may be due to differences in the characteristics of the cohorts themselves. Many current kinesiology students are involved in varsity sports, recreationally competitive sports, and intramural sports, as was the assertion for physical education students of yesteryear, but without objective data on sport participation this is impossible to ascertain. The test–retest reliability may be influenced by different fitness appraisers between cohorts; however, this discrepancy is believed to be minimized by using senior exercise appraisal students supervised by trained exercise appraisers in both cohorts. Strong efforts were made to complete protocols according to published methods and standard protocols between the cohorts; however, no two testers were present for both testing cohorts 30 years apart.Despite having relatively high fitness levels, this sample of current students who are interested in the study of movement have more abdominal adiposity and are less aerobically fit than a fellow sample of students educated in human movements from the 1980s. Those educated on the importance of movement may not be immune to the trends of decreasing fitness experienced by the rest of the Canadian population, possibly due to spending too much time stationary.


Interestingly, although small lakes (<1 km2) accounted for just 15% of the global lake area, they dominated the variability in total lake size in half of the global inland lake regions. The identified lake area increase over time led to higher lacustrine carbon emissions, mostly attributed to small lakes. Our findings illustrate the emerging roles of small lakes in regulating not only local inland water variability, but also the global trends of surface water extent and carbon emissions.Lakes are important natural resources and carbon gas emitters and are undergoing rapid changes worldwide in response to climate change and human activities. A detailed global characterization of lakes and their long-term dynamics does not exist, which is however crucial for evaluating the associated impacts on water availability and carbon emissions. Here, we map 3.4 million lakes on a global scale, including their explicit maximum extents and probability-weighted area changes over the past four decades. From the beginning period (1984–1999) to the end (2010–2019), the lake area increased across all six continents analyzed, with a net change of +46,278 km Lakes are essential components of the hydrological and biogeochemical cycles. Here, Pi et al develop a global lake dataset called GLAKES via high-resolution satellite images and deep learning to examine global lake changes over four decades. However, rapid lake changes have been identified worldwide in response to changing climate and escalating human activities10, threatening the ecosystem services provided by these lacustrine habitats. For example, lake desiccation has been observed in some populated regions due to unregulated water withdrawal, triggering water shortages, international conflicts, and other societal consequences12. Conversely, widespread glacier lake expansions have been detected due to climate warming-induced snowmelt and glacial melting14.Lakes play a major role in global hydrological and biogeochemical cycles15, and these similarities cause challenges when differentiating between lakes and rivers using satellite signals. A typical example is the recent map of changes in global surface water12, where the respective contributions of lakes and rivers to the total area or the detected changes remain uncertain. As lentic lakes and lotic river systems represent distinct hydrological and biochemical processes17, further efforts are warranted to quantify their distinct roles in these hydrological changes.A spatially explicit understanding of lake size changes is essential for evaluating the associated ecological, environmental, and societal impacts. In theory, determining lake dynamics using satellite images is straightforward, as remotely sensed imagery is available at high temporal frequencies with global coverage. However, terrestrial surface waters share similar optical features20, but these were largely generated using snapshots of historical imagery and did not consider seasonal and interannual fluctuations18. Previous studies on lake dynamics primarily focused on medium- to large-sized lakes with well-defined boundaries21. Small lakes (defined herein as lakes with an area <1 km2) are more variable than large lakes due to their high sensitivity to natural wet/dry transitions and human management activities22. Furthermore, small lakes have a disproportionally large contribution to global lacustrine systems in terms of their primary productivity23, biodiversity25, and carbon cycle26. However, these available global assessments of ecosystem parameters are usually based on uncertain estimates of the sizes and distribution of small lakes27, and few studies have examined how freshwater biogeochemical cycles are influenced by lake variation because changes in lake sizes over time have never been characterized globally. Here, we fill this existing knowledge gap and map more than 3.4 million lakes and reservoirs with surface area >0.03 km² (hereafter simply lakes unless otherwise specified) at the global scale. We used deep learning to identify lakes smaller than the minimum mapping unit for all global lake datasets that are publicly available (0.1 km²). We then examined changes in the global lakes over four decades and discussed the associated implications for lacustrine carbon emissions.Estimates of the global extent of lakes are available from several existing datasets2 (corresponding to ~33 Landsat image pixels), which greatly improves the minimum mapping unit and mitigates the issues of mis-accounted small lakes in previous lake datasets that are publicly accessible. Validation showed high accuracy levels across different size groups in our dataset , with 49.8% and 23.6% of the total number of lakes and total lake area located north of 60°N , medium (1–100 km2), and large (>100 km2); these size groups accounted for 94.39%, 5.56%, and 0.05% of the total number of lakes, respectively, and 15%, 26%, and 59% of the total lake area, respectively. Including lakes <0.1 km² in size (1.91 million lakes) resulted in a 30.2% larger bounded lake area in the small-lake group than that mapped by the recently published and widely used HydroLAKES dataset17  dataset and a deep-learning classification algorithm , 2000s: 2000–2009), and 2010s: 2010–2019) Fig. .Fig. 2La2. Lakes and reservoirs fed by glaciers or permafrost, representing 30% of the worldwide lake area, accounted for 48%  of the total increase  was attributed mostly (75%) to the expansion of reservoirs outside of glacial or permafrost regions  dominated the total areal increase from the 2000s to the 2010s, which was more than twice the amount of natural lake increases (3362 km2) in glacial or permafrost regions. Over half (53%) of the global grid cells exhibited inconsistent changes between the 1980–1990s to the 2000s and the 2000s to the 2010s. For example, the lake area has increased in southeastern Australia in the most recent decade, indicating the potential regional recovery of these lakes following the Millennium Drought in the 2000s31. Similar change patterns were also found in northwestern India, suggesting that this drought-prone region was likely to have suffered less from water stress in recent years32. In contrast, large lakes showed a net area decrease of 14,553 km2 from the 2000s to the 2010s, 50% of which was attributed to the human-induced shrinkage of the Aral Sea in Central Asia7.From the 2000s to the 2010s, although the total lake area increased, the net area gain represented only 16.3% of that identified from the 1980–1990s to the 2000s. The substantial expansion of reservoirs  over the past four decades, and 56% of the increase was attributed to reservoirs.Continuous lake expansions and shrinkages throughout the entire study period were found in 38% and 9% of the global inland lake regions , respectively, leading to an overall net area increase of 46,278 kmt-test, P < 0.05)  Fig. . Such diThe outsized role of small lakes can be further revealed by analyzing their contributions to lake size variability. We define variability as the proportion of absolute areal changes in small lakes within a given 1° × 1° grid cell (see Methods). The changes in small lakes showed dominant contributions (>50%) in approximately half of the examined inland regions  Fig. , and reg34. A common methodology used to estimate global emissions involves upscaling local measurements to the global scale by multiplying the mean lake-surface fluxes by the global area. Both CO2 and CH4 fluxes are reported to be influenced by lake size, and the data describing global lake size thus become crucial when upscaling carbon emissions26. With the detailed mapping of global lakes from GLAKES, we can update the total estimations of CO2 and CH4 emissions from global lakes.Although lakes cover just a small fraction of the Earth’s surface, they are important emitters of carbon gases2 and CH4 emissions from global lakes using our GLAKES dataset following the method proposed in a previous study26 (see Methods). We reached a global total estimate of 226 Tg C yr−1 for CO2 26. The difference could be partly due to the use of overestimated lake surface area by ref. 4 emission (1.4 Tg C yr−1) was also smaller than that previously estimated26. Nevertheless, our estimating approach was from ref. 40 , given their small share in the overall areal size of lakes (15%).We estimated the COCO2 Fig. , which i2 and CH4 emissions from global lakes throughout the study period (see Methods). Net increases in carbon emissions were found from the 1980–1990s to the 2010s, with +4.81 Tg C yr−1 for CO2 and +0.03 Tg C yr−1 for CH4, respectively . We have demonstrated an overall increase in inland lake areas worldwide over the past four decades. We corroborated previous findings regarding substantial lake expansions in glacial and permafrost regions14, whereas we further revealed that such climate warming-induced changes were not a major contributor to global lake dynamics throughout the entire period. Instead, we demonstrated that human-regulated reservoirs contributed to more than half of the overall areal increases, highlighting the dominant role of human alterations on global water dynamics41. Our results also showed a net loss trend in lake area within endorheic basins ; this result agrees with the findings obtained using GRACE-detected water storage measurements recorded between 2002 and 201610. However, our high-resolution mapping revealed the opposite trend when the desiccated Aral Sea was excluded; under this condition, we found continuously expanding small lakes in endorheic basins from the 1980–1990s to the 2010s , high spatial resolution (~30 m), long-term changes (four decades), spatiotemporal consistency , overall accuracy , and the delineation of small lakes  to gain a more comprehensive perception of the impacts of climate change and anthropogenic activities. Furthermore, our established dataset is essential for quantifying various aspects of aquatic systems  and identifying the potential transitions of ecosystem functions under climate change49. Our findings also underscore the urgent need for future management efforts focused on small lakes, given their crucial roles in regulating local hydrologic cycles and inland water-related carbon emissions.Our detailed mapping of the dynamics of 3.4 million lakes can potentially be used to better characterize regional-to-global hydrological budgets, as the changes in evaporation and water storage induced by lake size variability have often been ignored in past studies12 as the source data to determine the areal extents of lakes. The GSWO dataset provides global (from 60°S to 80°N) documentation of the location and frequency of water occurrences over nearly four decades (1984–2019) and was generated using 30-m-resolution Landsat images. Extensive validation of the GSWO dataset was conducted at the global scale, over the whole study period, and among all Landsat sensors involved. The results demonstrated the high accuracy of the surface water delineation in the GSWO datasets  and, consequently, the ability to afford comparable, continuous, and consistent mapping spatially, temporally and across sensors12. In addition, we used the GSW monthly water history (MWH) data collection to calculate the occurrence of water in different periods over the past four decades. The smallest lake mapped in our study had a surface area of 0.03 km2, corresponding to approximately 33 Landsat pixels. The GSW dataset was developed by the European Commission’s Joint Research Centre and can be obtained from https://global-surface-water.appspot.com.We used the Global Surface Water Occurrence (GSWO) dataset50 and ice sheet mass balance inter-comparison exercise (IMBIE) Rignot data51 to determine the distributions of glacier-fed lakes. The RGI 6.0 data provide a global inventory of glacier polygons (see https://www.glims.org/RGI/rgi60_dl.html), and the IMBIE Rignot dataset includes an additional ice sheet polygon supplemental dataset representing Greenland (see http://imbie.org/imbie-2016/drainage-basins/). In addition, we used the permafrost distribution data provided by the National Snow and Ice Data Center of the National Aeronautics and Space Administration (NASA) to determine the locations of permafrost-fed lakes. This dataset provides the gridded permafrost distribution in percent area in the Northern Hemisphere52. The gridded permafrost data can be obtained through the NASA webpage (see https://neo.sci.gsfc.nasa.gov/view.php?datasetId=PermafrostNSIDC).We used the Randolph Glacier Inventory 6.0 (RGI 6.0) glacier data53 was used to identify endorheic lakes. HydroBASINS presents consistent and seamless watershed outlines at different scales (lev01–lev12) according to the Pfafstetter coding system, with a global spatial resolution of 15 arc-seconds. In this study, the lev12 watershed polygons were selected for analysis. In particular, HydroBASINS offers a variable called Endo to indicate whether each basin/subbasin is an endorheic basin. The HydroBASINS dataset was obtained from https://www.hydrosheds.org/page/hydrobasins.The HydroBASINS dataset54 was used to distinguish reservoirs from natural lakes. The GeoDAR dataset, taking advantage of multi-source dam/reservoir inventories, provides global documentation of reservoirs with detailed attribute tables and well-georeferenced spatial locations. The GeoDAR dataset is available at 10.6084/m9.figshare.13670527.The Georeferenced global Dam And Reservoir (GeoDAR) dataset56 to investigate the relationship between the human population and lake area changes. The GPW dataset provides gridded population density estimations based on proportional allocation models utilizing population information gathered across global administrative units. Due to the long time span of our study, two GPW data versions were combined to establish population censuses reflecting different time periods. The GPW version-3 data (resolution: 2.5 arc-minutes) were used for 1990 and 1995, while the GPW version-4 data (resolution: 30 arc-seconds) were applied for 2000, 2005, 2010, 2015, and 2020. Then, the GPW data reflecting different years were averaged to represent the long-term population density status throughout the study period. The GPW data are accessible through the Socioeconomic Data and Applications Center (https://sedac.ciesin.columbia.edu).We used the Gridded Population of the World (GPW) dataset60 and is proven to be a powerful and creative tool in detecting features of interest from satellite images63. A recent inspiring deep-learning application in remote sensing image processing was documented by Brandt et al.64, who detected tree crowns by combining the U-Net model with submeter high-resolution satellite images. The U-Net model used in Brandt et al.64 is a typical semantic segmentation technique that performs pixel-wise classification within an image for precise segmentation66. Compared to conventional classification tasks, U-Net yields not only the label category of a specific image but also its corresponding location. Upon the application of U-Net, Brandt et al.64 managed to map more than 1.8 billion non-forest tree crowns (>3 m2) in the West African Sahara and the Sahel, somewhat overturning the previous stereotype of tree scarcity in these dryland regions. Here, we modified the U-Net model developed by Brandt et al.64 and transferred its application to global lake mapping. We expect a well-trained U-Net model to perform well when classifying lakes and rivers using GSWO images, as lakes and rivers are already highly distinguishable in visual examinations . GLAKES also contains constructed impounded water bodies  that are closely related to human activities. Notably, some agricultural fields are also included in our dataset, although the proportion may not be large, and further segregation is under process  that are learned automatically through backpropagation. Except for convolution layers, there are various structures that are also essential in the modified U-Net architecture of Brandt et al.64, including activation function (enabling nonlinear classification), batch normalization (stabilizing and accelerating the training process), pooling (reducing data dimension and computation complexity), up-convolution  as well as concatenation (combing the higher-level feature map with a lower-level one to better learn representation). In addition, the U-Net model adopts the overlap-tile strategy, which makes it possible to perform a seamless segmentation for large images without losing information about the divided border regions66. This makes U-Net particularly applicable to our goal of pixel-wise lake classification through GSWO images on a global scale. The specific structure of U-Net model is presented in Supplementary Fig. Building upon a fully convolutional neural network16, the OpenStreetMap Water Layer (OSMWL) dataset72, and the HydroLAKES polygon dataset17. We used the GSWO map to mask land pixels and exclude low-confidence water pixels (some of which may be caused by the inherent classification errors of GSWO) with <5% occurrence . Notably, in floodplains, the occurrence threshold was set to 30% instead of 5% to capture the ‘core’ portions of lakes and segment these portions from the larger-scale floodwaters  each year). We used the OSMWL and GRWL masking products to exclude ocean and river pixels. Since the GRWL dataset was developed using a limited set of Landsat images and was thus inadequate for representing the full coverage of rivers , we visually confirmed that all river masks used to prepare our labels were correct. Moreover, we gave higher priority to lakes that had already been detected in the HydroLAKES database by ensuring that the lake pixels within the HydroLAKES polygons would not be affected by the overlying river/ocean mask. Finally, extensive visual examinations were performed from one sample region to another, where some lakes in hydrologically complex regions were given more attention, such as those in the large river basins and floodplain regions. Manual corrections were performed mainly on the following two situations: (1) river residuals resulting from the absent coverage of the corresponding river masks and (2) river-connected lakes that required further division from river channels. Of all sample polygons, Case 1 polygons frequently occurred, which could take up ~10% of the total lake samples and thus require careful inspection. On the contrary, the percentage of Case 2 polygons was minor (far less than 1%). Ultimately, we would like to ensure that all extracted lake boundaries  matched well with the water/land interfaces isolated on the GSWO maps. Notably, the lower size limit of the samples was set as 0.03 km2, according to our visual observations. The main objectives were to exclude small polygon residuals generated during this sample extraction procedure and to screen out small isolated agricultural fields.The first step in deploying a deep-learning model is to prepare reliable labels for the training, validation, and test sets. In this study, lake labels were generated through an automatic extraction method followed by manual modification by combining several existing datasets, including the GSWO dataset, the Global River Widths from Landsat (GRWL) database2–103 km2, with a median area of 5.91 × 102 km2 for the Normal Model and 5.69 × 102 km2 for the Floodplain Model. The major principles considered when selecting samples included the selection of samples from different regions globally, the coverage of all typical hydrological conditions, and ensuring balance in the numbers of the five major region types.We delineated a variety of globally distributed sample regions with varying sizes to create lake labels  were randomly generated within each sample region, and the lake patterns within the patches were extracted and interpreted by the model. In addition, we applied the same local normalization method from Brandt et al.64. First, we enlarged the patch sizes of the input images  from 256 × 256 pixels  to 512 × 512 pixels  to enhance the capability of the model to capture the characteristics of large lakes. Additionally, the distance-weighted map between gaps used in the initial model for the segmentation of partially overlapped objects66 was not considered in our loss function since we did not face this issue when using GSWO maps as the input images. In terms of the selection of hyperparameters, we kept the same type of loss function and optimizer as those used in the original model but made substantial changes to some other hyperparameters, such as batch size, epoch numbers, and iteration numbers . MIoU is a widely used image segmentation performance indicator that fully considers true positives and false negatives73. The IoU of each class was calculated as the area of overlap divided by the area of union between the labels and predictions of that class. Then, the IoU from different classes was averaged to estimate MIoU.According to our own research objectives, we made some essential modifications to the U-net model proposed by Brandt et al.After the selection for the final Normal and Floodplain Model, predictions were obtained for each 512 × 512-pixel patch in each GSWO map, resulting in a raw global lake classification map. Then, several postprocessing procedures were conducted to further enhance the classification accuracy. We first overlaid the OSMWL and GRWL masking products to exclude ocean and river pixels, similar to the procedure followed to prepare the labels. This step was necessary because our model performed well when distinguishing lakes from small rivers but exhibited some limitations with regard to oceans and large rivers, mainly induced by the relatively small patch size  used for training. For coastal lakes, we further excluded lake polygons that directly intersected with the 10 m buffer of the global ocean boundaries defined by the OSMWL dataset; next, more detailed manual corrections were performed on near-coastal regions. For rivers, we minimized the residuals caused by the incomplete coverage of the GRWL and OSMWL river masks using the area ratio of the polygons representing the same lake before and after the application of the river masks . Within each buffer, the area of seasonally flooded non-lake and non-river waters was calculated by summing the area of all GSWO pixels with occurrence <75% (to exclude the permanent and near-permanent water), except for those already being defined as rivers (by GRWL mask) and lakes (by rasterized HydroLAKES polygons). Finally, buffer zones where the ratio of their containing flooded area to the corresponding buffer area exceeded the flooding threshold were flagged as “floodplain”. In this study, the flooding threshold was set as 0.1 through trial and error.Except for these postprocessing procedures, the lakes from the Normal Model and the Floodplain Model were combined to generate our final version of global lake polygons (referred to as the GLAKES dataset). Specifically, given that both the Normal Model and Floodplain Model yielded lake predictions at global coverage, we applied predefined river buffer zones to determine whether the extracted lake polygons from the Normal Model or Floodplain Model should be used for our final GLAKES dataset. Specifically, for buffer zones flagged as “floodplain”, lake polygons (within these buffer zones) extracted from the Floodplain Model were selected as a part of GLAKES dataset, while the corresponding outputs from the Normal Model were discarded. In contrast, for all remaining areas , lake polygons from the Normal Model were included in our final dataset. The basic principle for the determination of the exact flag  for each buffer zone is to measure the extent of seasonally flooded non-lake and non-river waters within the buffer zone. A 1 km buffer was applied to each vectorized polygon of global rivers documented in the GRWL Mask V01.01 product  to estimate the numbers and area of lakes in different size groups  for lake samples should probably be responsible for such kind of issue  were relatively high for small lakes  compared to those obtained for medium and large lakes. It is noteworthy that the setting of the predefined cutting threshold . The dataset is organized into three levels, focusing on large water body polygons, smaller water body shorelines, and the rasterized extents of potential wetlands. Based on the Shuttle Radar Topography Mission Water Body Data74 (for most lakes between 56°S and 60°N), CanVec75 (for the majority of North American lakes), as well as other lake datasets (see HydroLAKES documentation), the most widely used global lake database HydroLAKES17 was developed, along with intensive automated and manual corrections. The HydroLAKES dataset contains ~1.42 million individual lake polygons with surface area >0.1 km2. Downing et al.19 focused on the scaling relationships and size distributions of lakes; these factors were tested in different regions and integrated into a global Pareto distribution model to extrapolate the global lake extent. By using the GeoCoverTM program circa 2000 based on Landsat 7 images in combination with a corresponding water-extraction algorithm, Verpoorter et al.20 developed the GLObal WAter BOdies database (GLOWABO), which contains 117 million lakes with the area of the smallest lake polygons set at 0.002 km2. In this study, we made a more detailed spatial comparison using the latest version of the global lake dataset  at both the 1° × 1° grid scale  with the corresponding values reported in several previously established global lake datasets2) in our dataset resulted in a lake area of 1.05 × 105 km2 (30.2%) larger than that indicated by the small-lake group in the HydroLAKES dataset17. Moreover, we found a substantial number of missing lakes in eastern Canada and Scandinavia in our dataset compared to HydroLAKES, as well as lake overestimations with varying degrees in other regions, such as Siberia and major river floodplains . This is significant for the long-term monitoring of the lake surface water area dynamics. As a comparison, the Global Lake area, Climate, and Population (GLCP) dataset provides annual time series lake surface area records from 1995 to 2015 for all HydroLAKES polygons76. Nevertheless, GLCP faces the same limitations as HydroLAKES in terms of the lake size limit and spatial consistency. Besides, since the HydroLAKES polygons did not represent the maximum water extent, a fixed buffer zone around lakes was generated in GLCP for area estimation, which might result in fallaciously inclusion of water coverage that did not belong to the target lakes or missed detection of water area due to the insufficient coverage of the buffer outlines. On the other hand, large discrepancies were found between the estimates obtained using GLAKES and several other previously reported estimates20  constrained by the lake boundary defined in our GLAKES dataset and 12 by normalizing the number of water presence (Nw) incidences against the number of valid observations (Nvo) within a period. In practice, both Nw and Nvo can be derived using the GSW MWH data collection. In the MWH dataset, each pixel was assigned to one of the three values ; for each pixel within a given time period, we estimated Nw as the number of images corresponding to a value of 2 and set Nvo as the number of images corresponding to pixel values >0.We estimated the water probability-weighted area, compared the water probability-weighted area among different periods , and investigated the significance of small lakes on global lake dynamics. The probability-weighted lake area |∆Asmall | ) to the total areal changes  identified within a grid cell ). Notably, grid cells with insufficient satellite coverage in each period  were excluded from the cross-period comparison   all lakes and (2) only small lakes  classified to estimate the total gas emissions for each size class and subsequently calculated the total emissions for global lakes. We repeated this step by using the upper/lower bounds of the flux values to estimate the ranges of global lacustrine CO2 and CH4 emissions. We also estimated the carbon gases emitted from global lakes in different periods (1980–1990s–2010s) as well as the corresponding emission changes over time. Similar to the procedures described above, we combined the size-dependent CO2/CH4 fluxes with the probability-weighted lake area to estimate global lake carbon emissions for each period.Following the method outlined by Holgerson and Raymond4). Please see Supplementary Note Several uncertainties and limitations should be acknowledged in this study, both during the process of lake mapping along with related change analysis of lake area and carbon emissions. In lake mapping, these could be further categorized into the following major sources: lake definition, auxiliary datasets, U-Net model, and postprocessing. The temporal change of probability-weighted lake area among different time periods, otherwise, may be influenced by seasonal lake dynamics. As for the estimation of global carbon emissions as well as their long-term changes, the accuracy of our results was closely related to the representativeness of the average emission rates used for global upscaling, the impacts of lake dynamics at shorter timescales, and the quantification of emissions through different pathways (for CHSupplementary InformationPeer Review File


Fetal growth and birthweight depend on placental function, as this organ transports substrates to the developing fetus and it acts as a source of endocrine factors, including steroids and prolactins that are required for fetal development and pregnancy maintenance. To advance our knowledge on the aetiology of fetal growth disorders, the vast majority of the research has been focused on studying the transport function of the placenta, leaving practically unexplored the contribution of placental hormones in the regulation of fetal growth. Here, using mice and natural variability in fetal growth within the litter, we compared fetuses that fell on or below the 10th percentile (classified as SGA) with those that had adequate weight for their gestational age (AGA). In particular, we compared placental endocrine metabolism and hormone production, as well as fetal brain weight and expression of developmental, growth and metabolic genes between SGA and AGA fetuses. We found that compared to AGA fetuses, SGA fetuses had lower placental efficiency and reduced capacity for placental production of hormones . Brain weight was reduced in SGA fetuses, although this was proportional to the reduction in overall fetal size. The expression of glucose transporter 3 (Slc2a3) was reduced despite the abundance of AKT, FOXO and ERK proteins were similar. Developmental (Sv2b and Gabrg1) and microglia genes (Ier3), as well as the pregnancy-specific glycoprotein receptor (Cd9) were lower in the brain of SGA versus AGA fetuses. In this mouse model of SGA, our results therefore demonstrate that placental endocrine dysfunction is associated with changes in fetal growth and fetal brain development.Conditions such as small for gestational age (SGA), which is defined as birthweight less than 10 CompareFetal growth is regulated, in part, by the placenta which is a fetal organ responsible for the transport of gases and nutrients between the mother and the fetus. Aberrant placental function related to failure of substrate transport or deficits in vascular growth have been linked to fetal growth abnormalities, including fetal growth restriction, as well as stillbirth –14. HoweIn this scenario, animal models in which fetal and postnatal outcomes can be monitored are essential. Much of the investigations focused on fetoplacental development have been conducted in rodents, rabbits, sheep and pigs –36. HoweNumerous animal models of pregnancy complications have been created, including those that mimic environmental and maternal conditions , 29, 36.22.1ad libitum with a standard chow diet . Females were time-mated with C57BL6/J males and the day a copulatory plug was detected was designated as gestational day (GD) 1 (term occurs ∼GD20). On GD16, pregnant females were killed by cervical dislocation, the uterus removed and fetuses cleaned from feto-placental membranes. Each fetus (and its corresponding placenta), was identified based on its position in the uterus, dried on tissue paper, weighed and immediately decapitated. Fetal brains were removed, weighed and immersed in liquid nitrogen for rapid freezing. The placental Jz was dissected from maternal decidua and placental labyrinth zone, and subsequently snap-frozen in liquid nitrogen. All frozen fetal and placental samples were stored at −80°C until tissues were powdered in individual pieces of foil using a hammer and dry ice for RNA/protein extraction . Only viable fetuses were used in the study and litter sizes ranged from 6 to 11 pups.All animal work was performed under the UK Animals (Scientific Procedures) Act 1986. Experiments were conducted with a total of 11 C57BL6/J wild-type female mice (4 months old) housed at the University of Cambridge Animal Facility under a 12/12 dark-light system and fed 2.2Actb and Gapdh), while placental genes were normalized using Actb and Ywhaz as reference genes. These housekeeper genes remained stably expressed between the groups. Analysis was performed using the 2-ΔΔCt method  was extracted using the RNeasy Plus Mini Kit (Qiagen) and the quantity of RNA obtained was determined using a NanoDrop spectrophotometer (NanoDrop Technologies) as previously described . RNA wast method .2.3Total protein was extracted from fetal brain and placental Jz (5 samples per group) using RIPA buffer  supplemented with mini EDTA-free protease inhibitor cocktail mix (Roche), 1mM β-glycerophosphate  and 1mM sodium orthovanadate . Lysates were centrifuged at 2,500 rpm for 15 minutes at 4°C and protein concentration determined using the Pierce BCA protein assay kit . Samples were mixed with SDS gel loading buffer  and protein denaturation was performed at 90°C for 5 minutes. After gel electrophoresis, proteins were transferred from the gel onto 0.45 µm nitrocellulose membranes . Membranes were then blocked either with 5% fetal bovine serum  or semi-skimmed milk (Marvel) for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies described in 2.4th percentile ≤321 mg; AGA fetuses: 321-409 mg and LGA fetuses: 90th percentile≥410 mg). ROUT test was used to identify outlier values, which were then excluded for statistical purposes. Normality and homogeneity of variance of variables for western blotting and qPCR data were performed using the Shapiro-Wilks test in GraphPad Prism. For comparisons between two groups, the Student’s t-test or Mann-Whitney test were applied according to the normality of the variable. For data with more than two groups, one-way ANOVA coupled with Bonferroni post hoc test was employed. The relation between fetal intrauterine position as well as litter size with the presence of SGA, AGA and LGA was analysed by Chi square test using a contingency table in GraphPad Prism software. Pearson r correlations were performed with GraphPad Prism software and presented in a double gradient colour mapping graph that was subsequently modified with Adobe Illustrator to facilitate visualization. qPCR and western blotting data are expressed as individual data points and reported as mean ± SEM. P-values <0.05 were considered statistically significant.No sample size calculation was conducted prior to undertaking this study. For the purpose of this paper, the fetus, and not the mother, was designated as the experimental unit. GraphPad Prism software (version 9) was used to determine statistical differences between groups. The distribution of fetal weights (graphed as a histogram) was constructed with a non-linear regression (Gaussian distribution) and percentiles calculated with the descriptive statistical analysis tool of GraphPad Prism software. Then, data in Excel were organized according to fetal weight and separated in three experimental groups based on the fetal weight threshold (SGA fetuses: 1033.1th percentile) and they were obtained from 5 different litters. On average, fetal weight was significantly lower by 19% in SGA fetuses when compared to AGA fetuses (th percentile) were obtained from 3 different litters and these were on average, 12% heavier than the AGA group. All litters had at least 3 AGA fetuses. Analysis of placental weight did not show significant differences between the SGA, AGA and LGA fetuses , pregnancy specific glycoprotein genes  and prolactins  in the Jz of SGA and AGA fetuses  did not change between SGA and AGA fetuses; however, the mRNA levels Slc2a3 were significantly reduced by 30% in SGA compared to AGA fetuses  . We found that Sv2b and Gabrg1 were reduced by 45% and 12% in SGA compared to the AGA group, respectively   and founogenesis  , prolactins (Prl3a1) and PSGs (Psg21) were compromised in fetuses classified as SGA. Moreover, SGA fetuses have a proportional reduction in brain weight, low expression of glucose transporter 3 (Slc2a3), axogenesis (Sv2b and Gabrg1) and microglia (Ier3) genes along with reduced expression of the PSG receptor (Cd9) in the fetal brain of SGA fetuses compared to AGA. Finally, we found a significant link between the placenta and fetal brain development, as shown by the reduced brain-placental weight ratio in SGA fetuses. Together, our data reveal that placental hormones could be additional regulators of fetal growth with implications for fetal brain development. Furthermore, mis-communication of placental hormones with the developing fetal brain may have immediate and long-lasting implications for neurocognitive outcomes.In this study we have shown that the defects in placental endocrine function are associated with reduced fetal growth and brain development in a mouse model of SGA. In particular, we found that the placental capacity to respond metabolically (FOXO1) and produce sex steroids (Prlr mRNA levels detectable as early as on GD8 (hGH/CSH) cluster in the SGA placenta  and impaired axogenesis markers (as inferred by the low expression levels of Sv2b and Gabrg1) in SGA fetuses. Of note, we did not find any changes in PI3K-AKT-mTOR signalling, which is critical for biological processes like nutrient uptake, cell growth and migration   receptor was reduced. Prior work has found that Sv2b is regulated by the maternal gut microbiota during mouse fetal brain development   and its s (TLR3) . In miceammation . Therefoelopment . This geelopment , 94. Othelopment . Hence, nn cells . Given tSlc2a1 and Slc2a3 , we observed that placental efficiency is affected by both, the intrauterine fetal position and the number of fetuses per litter. Previous work performed in rats have identified greater blood flow at the cervical and ovarian ends compared to middle of the uterus, which may partly explain our findings  and brain development. Moreover, animal studies deploying in vivo approaches to specifically delete genes in mouse placenta endocrine cells would be highly useful for decoding the communication between placental hormones and fetal organogenesis  Act 1986 and underwent review by the University of Cambridge AnimalWelfare and Ethical Review Body.JL-T performed research. JL-T and AS-P wrote the paper and designed the study. All authors contributed to the article and approved the submitted version.


Oligodendrocyte precursor cells (OPCs) are a population of glia cells that tile across the brain and retain the ability to proliferate. They can become myelinating oligodendrocytes or remain in the precursor state. An unresolved question has been what other functions OPCs might carry out. Using high-throughput transmission electron microscopy and dense 3D reconstructions, we were able to visualize evidence of ingestion and breakdown of axons by OPCs. Analysis of the abundant tertiary lysosomes or phagolysosomes (PLs) in OPC branches confirmed the presence of 40-nm vesicular structures within their chambers. Furthermore, analysis of PLs in microglia demonstrated that their numbers were significantly lower and rarely contained vesicles. This study provides evidence of engulfment and pruning by OPCs. Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development. Oligodendrocyte precursor cells (OPCs) emerge from several germinal zones in late prenatal development following the sequential generation of neurons and astrocytes, migrate into the expanding cortex, and then proliferate to establish a grid-like distribution, with individual cells occupying distinct territories. Genetic fate tracing and time lapse imaging in vivo have demonstrated that these progenitors play a critical role in generating oligodendrocytes and thus myelin throughout the central nervous system (CNS) . HoweverOur knowledge about the structure and function of OPCs has been severely limited by incomplete ultrastructural information, because their fine, highly branched processes are difficult to unambiguously recognize in electron microscopy (EM) studies without complete reconstructions  to conne5Past EM ultrastructural studies defined several common morphological characteristics of OPCs, such as their oblong nucleus containing low heterochromatin and presence of centrioles in their cytoplasm , consistSI Appendix, Fig. S1 A–H), which contained numerous filopodia on their tips. 3D renderings of adjacent OPCs revealed that these cells, like astrocytes and microglia , and contacted synapses , and were remarkably variable in both size and shape, ranging from elongated or bean shaped to smooth or irregular with a rough surface . OPCs were readily distinguished from other glial cell types by these ultrastructural features, as well as having: 1) larger nuclei that were elongated and contained less heterochromatin than those of microglia . Primary cilia were found on all OPCs in both datasets and this feature helped distinguish them from premyelinating and mature oligodendrocytes  picroglia L and M; icroglia M; and 5)drocytes . The sizSI Appendix, Fig. S3A) frequently contacted axons . Moreover, the cytoplasm of these processes often contained numerous membrane-bound organelles, most prominently phagosomes, lysosomes , suggesting that OPCs at this age engage in phagocytosis.Previous in vivo imaging experiments revealed the dynamic nature of OPC processes, which exhibit continuous branch remodeling as they migrate through gray matter, making transient interactions with various cellular constituents . Analysiysosomes B, and diMovie S2) that were readily distinguishable from the larger multichambered PLs measuring 500–750 nm in diameter , revealing that PLs were widely distributed in all 18 OPCs that were analyzed in both datasets and particularly enriched at near the tips of processes. Quantitative analysis from reconstructed OPCs revealed that 38 ± 14% of the PLs contained vesicles at P36 , indicating that this is a widespread phenomenon. Since the vesicles found inside the PLs were of the same size as the ones found in synaptic boutons, we asked if OPCs were engulfing portions of nearby axons and associated synapses.The process of phagocytosis begins with the recognition of a target, followed by phagosome formation (engulfment), late phagosome maturation, and finally fusion of the phagosome with a lysosome, forming the highly acidic phagolysosome . If OPCsdiameter B. Seldomdiameter , 26. Altdiameter D. These diameter D and E. SI Appendix, Figs. S1 A–I and S3) suggests the possibility of presynaptic terminals or portions of axons containing terminals are engulfed by these cells. Analysis of the 3D reconstructions revealed that indeed, small cytoplasmic protrusions and small branches of axons were often surrounded by OPC processes . The complex anatomy of the branches and the need to annotate and quantify the ingestions made this analysis particularly challenging. To simplify the analysis, ten individual main branches from ten distinct OPCs in the P36 dataset were surveyed for ingested materials . First, we categorized the ingestions as phagosomes (PS) or (PLs), and found they were present in every OPC in variable amounts (range 5–27 PS and 6–50 PL) . In these types of ingestions, there was no evidence of injury or destruction to the axon, only local contact with OPC cytoplasmic processes that encased the collateral or terminal region of the axon but did not fully encapsulate it. Two types of axon engulfment were observed: either small bulb like axonal buds ranging from 300–500 nm3 or small branches ranging from 1–4 μm  surrounded by an OPC process, in which the engulfed material remained connected to the parent axon .The appearance of clusters of 40 nm clear vesicles inside a subset of PLs in every OPC (6–50 PL) G. Next, SI Appendix, Fig. S7). Since the volume did not contain entire neurons, we were unable to identify the cell type of axonal origin and only whether they were excitatory (E) or inhibitory (I), based on anatomical features of the pre- and postsynaptic membranes, specifically their electron density. Because the engulfed axons usually formed multiple synapses, we used the nature of the postsynaptic structure as further confirmation of our classifications, since excitatory axons make most of their synapses with spines while inhibitory axons make most of their synapses with somata and dendritic shafts.OPCs form synapses with both excitatory and inhibitory neurons in the developing cortex , 3 and aSI Appendix, Figs. S5 A–H and S6) with 6.3% undetermined. Approximately 7.7% of the identifiable ingested axons were inhibitory, suggesting that OPCs were mainly removing portions of excitatory axons in this dataset. Although interneurons comprise 10–20% of all neurons in the neocortex, the association of OPCs with interneurons may be lower because the P36 dataset was restricted to layer 2/3 where the interneuron density is lower. Overall, the numbers of all types of ingestions were variable among the ten OPC branches, possibly indicative of the phagocytic capability of the individual cells. The association of OPCs with axon fragments and small axon branches was observed in all OPCs from both P36 and P54 datasets, indicating that axon engulfment is a conserved function of OPCs during this period. Such associations may represent the predecessor to large-scale axonal pruning and suggest that small segments of axon collaterals are targeted for removal by OPCs.This analysis showed the predominant type of axons within the ten isolated branches of OPCs were excitatory (85.9 %) or inhibitory (7.7%) . Although some OPCs are larger than microglia at this age, the density of PLs was still significantly higher in OPCs when normalized to cell volume ; suggesting that OPCs are more actively engaged in neuronal process engulfment than microglia at this age.The presence of abundant PLs in OPCs was unexpected, as these cells have mostly been seen as precursors to oligodendrocytes. Moreover, most structural pruning of neurons is thought to be mediated primarily by microglia , 30. To l volume B (PL denMovie S4). Occasionally, we observed dendritic spines encompassed within the OPC branches, but this was also rare – in the P36 dataset, five excitatory synapses and six dendritic spines were found in the ten OPC branches. Like the axonal collateral branches, these encapsulations were connected to their parent axon or dendrites and did not appear to be damaged or unhealthy. The scarcity of 40-nm vesicles inside microglia PLs and their heterogeneity in both size and contents compared to those of OPCs suggest that these two glial cell types recognize and engulf distinct cellular elements.Microglia have been shown to engulf diverse materials, including cell corpses and myelin fragments , 32. AltSI Appendix, Fig. S8 A and B), indicating a high investment in cellular turnover in the developing cortex.To further support the finding of the prevalence of these organelles in OPC branches, OPCs isolated from the visual cortex of 1-m-old mice were immunolabeled for lysosome-associated membrane protein 2 (LAMP-2), which is associated with both lysosomes and PLs , and NG23; P54 OPCs, 0.085 ± 0.03 PLs/µm3; P = 0.01, Student’s t test, and n = 9, 11, respectively). Vesicles were also apparent in all P54 OPCs that were examined . Together, these results indicate there is a temporal shift in OPC behavior over time and that OPCs may contribute to an early phase of structural remodeling of neurons during the final phase of the critical period in the visual cortex.During the critical period of ocular dominance plasticity in the mouse visual cortex from P21 to P35, microglia engulf and eliminate synapses , 35 and SI Appendix, Fig. S9 A–D). Analysis of their somata and processes revealed that premyelinating oligodendrocytes had lower densities of PLs than OPCs  . In fully mature oligodendrocytes, PLs were extremely rare , suggesting that engulfment of neuronal processes declines rapidly as OPCs undergo differentiation.OPCs undergo dramatic changes in gene expression and morphology as they differentiate into myelin forming oligodendrocytes . To detepdgfra and olig2 and premyelinating/myelinating oligodendrocytes were identified by expression of opalin .The 3D EM reconstructions indicate that OPC processes are filled with numerous PLs that contain the neuronal material, suggesting that they assist in remodeling nascent neuronal circuits through engulfment of axons and occasionally synapses. Phagocytosis requires a complex array of proteins to recognize and engulf distinct cargos, the components for which are highly conserved among different species. To further support our EM findings of the presence of PLs in OPCs, we asked whether OPCs express genes that would enable phagocytosis. For this, we analyzed existing single-nucleus RNA-seq and DNA-methylation datasets derived from the mouse motor cortex (P56)  for bothf opalin A–C. Exprf opalin A and B, f opalin  and exprMertk, Ptprj, and Lrp1. Mertk (Mer tyrosine kinase) is a member of the TAM (Tyro3-Axl-Mer) family of receptors that signal engulfment, which astrocytes use to refine connectivity in the adult mouse hippocampus by phagocytosing excitatory synapses   , engage d Sirpa) , 51. Pard Sirpa) , 53. It d Sirpa)  and myeld Sirpa) . For exad Sirpa) , 56. OPCd Sirpa) , able tod Sirpa) . While td Sirpa) . Neverthd Sirpa) . They shd Sirpa) . Emergind Sirpa) –61 and pd Sirpa) . Althougd Sirpa) , 63, thed Sirpa) , 65.Mertk, Megf10)   and part Megf10) . The nerThe robust dynamics and broad distribution of OPCs place them in an ideal position to engage in the modification of neural circuits. Remarkably, OPC processes contained a higher density of PLs than surrounding microglia, raising the possibility that they are responsible for a significant amount of fine scale structural modifications at this stage of maturation of the cortex. However, it is also possible the engulfment and digestion of axonal elements by OPCs proceed more slowly than in microglia, leading to higher phagolysosome accumulation in their branches. It is conceivable that distinct types and temporal mechanisms of phagosome formation, maturation, and progression of lysosomal to phagolysosome formation in the two types of glia may exist but this is not known. This may be reflected in their genomic differences as OPCs express fewer phagocytic and lysosomal receptors. Previous studies documented that OPCs are a diverse and constantly changing population, with continuous cell turnover as a result of differentiation and cell death . Could iAlthough the presence of PLs within OPC processes declined with age, the persistence of OPCs throughout the CNS, which retain a similar morphology and dynamics, raises the possibility that they may retain the capacity to modify circuit maturation and clear cellular debris induced by injury or normal aging, consistent with conclusions made in other histological studies , 70. Som71Together, these findings raise new questions about how distinct glial cell types coordinate their phagocytic behavior to shape brain connectivity and contribute to plasticity, homeostasis, and disease processes. The interactions between different glial types and how they might coordinate large- and small-scale pruning activity, is fodder for investigation. Future studies selectively manipulating the ability of OPCs to phagocytose and degrade cellular material at different developmental stages may reveal the complex role of these progenitors in early refinement of neural circuits and lead to a deeper understanding of how the behavior of distinct glial cell types is coordinated to promote the maturation of the nervous system.Detailed description of methods is available in the supplementary information and also in refs. https://www.iarpa.gov/index.php/research-programs/microns) to study the functional connectivity of neurons; however, they also provided rich and unprecedented details of the fine morphology of glial cell types. Here, we use these datasets to evaluate the population of OPCs and microglia in the 3D environment of the mouse brain visual cortex. Dataset 1 was taken from a 200-µm-thick sample containing layer 2/3 of the mouse visual cortex (P36) and measured approximately 250 µm × 140 µm × 90 µm  in the mouse primary visual cortex. These data sets were collected as part of the IARPA Machine Intelligence from Cortical Network (MICrONS) consortium project , 2) tTA driver: B6;CBA- Tg(Camk2a-tTA)1Mmay/J (Jax: 003010<https://www.jax.org/strain/003010>), 3) GCaMP6f Reporter: Ai93(JAX 024103) (Allen Institute) (Mouse P 36 was a triple-heterozygote for the following three genes: 1) Cre driver: Slc17a7-IRES2_Cre (Jax: 023527<stitute) . Mouse PSI Appendix, Methods for more details regarding perfusion and sample handling.The histology protocol used here is based on the work of refs. The tissue block was trimmed to contain the neurophysiology recording site which is the region of interest (ROI) then sectioned to 40-nm ultrathin sections that were manually collected onto slot grids coated with a 50-nm-thick LUXFilm support or automatically onto a grid tape . The imaSI Appendix, Methods.Aligning the individual image tiles and sections into a coherent three-dimensional volume and segmenting the cellular morphology dataset was performed as previously described within , 16, 82.Using the multicut tool in Neuroglancer, individual branches of ten OPCs were removed at their trunk near the soma. Using the annotation tool in Neuroglancer, phagosomes, PLs, and axon ingestions were tagged along the lengths of fine branchlets. Axon ingestions were still attached to their parent axon, so that the axon could be followed along its length in both 3D and ultrastructural modalities. This made it possible to determine the class of axon, excitatory or inhibitory by examining the ultrastructure of individual boutons. By EM, excitatory axons have a large, electron-dense postsynaptic membrane while inhibitory axon boutons lack this feature, having instead thinner symmetric densities. If there was uncertainty, those were marked as unsure. Since the engulfed axons usually formed multiple synapses, we used the characteristics of the postsynaptic structure as further confirmation of our classification, since excitatory axons make most of their synapses with spines while inhibitory axons make most of their synapses with somata and dendritic shafts. These criteria were also confirmed using the axons of excitatory and inhibitory cells whose soma is present in the volume .Appendix 01 (PDF)Click here for additional data file.Movie S1https://vimeo.com/747877539Satellite Glia. All types of glia can be in the satellite position, in close apposition to neuronal cell somas. This movie shows both an OPC and a microglial cell together on the soma of a pyramidal neuron with their glial branches intertwined. Movie S2F) back to its soma. Green ball shows the position in the branch in the insert. Many cellular organelles are present in the branch. https://vimeo.com/747877039Following an OPC branch through the neuropil. This movie follows the branch of an OPC  is engulfed within the cytoplasm of the OPC (pink). The axon tip remains attached the parent axon and that allows its identification as inhibitory. See SI Fig 5 Movie S4G-L. https://vimeo.com/747877237Spine synapse engulfed by OPC. The dendritic spine in blue makes an excitatory synapse with the axon in gray. Both are still attached to their parent cell. There were few examples of whole synapses being ingested in this dataset. See Fig. 4