Individual and combined impacts of carbon dioxide enrichment, heatwaves, flow velocity variability, and fine sediment deposition on stream invertebrate communities.

Climate change and land-use change are widely altering freshwater ecosystem functioning and there is an urgent need to understand how these broad stressor categories may interact in future. While much research has focused on mean temperature increases, climate change also involves increasing variability of both water temperature and flow regimes and increasing concentrations of atmospheric CO2, all with potential to alter stream invertebrate communities. Deposited fine sediment is a pervasive land-use stressor with widespread impacts on stream invertebrates. Sedimentation may be managed at the catchment scale; thus, uncovering interactions with these three key climate stressors may assist mitigation of future threats. This is the first experiment to investigate the individual and combined effects of enriched CO2, heatwaves, flow velocity variability, and fine sediment on realistic stream invertebrate communities. Using 128 mesocosms simulating small stony-bottomed streams in a 7-week experiment, we manipulated dissolved CO2 (ambient; enriched), fine sediment (no sediment; 300 g dry sediment), temperature (ambient; two 7-day heatwaves), and flow velocity (constant; variable). All treatments changed community composition. CO2 enrichment reduced abundances of Orthocladiinae and Chironominae and increased Copepoda abundance. Variable flow velocity had only positive effects on invertebrate abundances (7 of 13 common taxa and total abundance), in contrast to previous experiments showing negative impacts of reduced velocity. CO2 was implicated in most stressor interactions found, with CO2 × sediment interactions being most common. Communities forming under enriched CO2 conditions in sediment-impacted mesocosms had ~20% fewer total invertebrates than those with either treatment alone. Copepoda abundances doubled in CO2-enriched mesocosms without sediment, whereas no CO2 effect occurred in mesocosms with sediment. Our findings provide new insights into potential future impacts of climate change and land use in running freshwaters, in particular highlighting the potential for elevated CO2 to interact with fine sediment deposition in unpredictable ways.

Platelet responses to agonists in a cohort of highly characterised platelet donors are consistent over time.

BACKGROUND AND OBJECTIVES: Platelet function shows significant inheritance that is at least partially genetically controlled. There is also evidence that the platelet response is stable over time, but there are few studies that have assessed consistency of platelet function over months and years. We aimed to measure platelet function in platelet donors over time in individuals selected from a cohort of 956 donors whose platelet function had been previously characterised. MATERIALS AND METHODS: Platelet function was assessed by flow cytometry, measuring fibrinogen binding and P-selectin expression after stimulation with either cross-linked collagen-related peptide or adenosine 5'-diphosphate. Eighty-nine donors from the Cambridge Platelet Function Cohort whose platelet responses were initially within the lower or upper decile of reactivity were retested between 4 months and five and a half years later. RESULTS: There was moderate-to-high correlation between the initial and repeat platelet function results for all assays (P ≤ 0·007, r2 0·2961-0·7625); furthermore, the range of results observed in the initial low and high responder groups remained significantly different at the time of the second test (P ≤ 0·0005). CONCLUSION: Platelet function remains consistent over time. This implies that this potential influence on quality of donated platelet concentrates will remain essentially constant for a given donor.

A multidisciplinary intervention programme for overweight and obese children in deprived areas.

Background: GPs can refer obese children living in deprived areas to multidisciplinary programmes for a weight loss intervention, though the effectiveness of these local initiatives targeted to this specific group is unknown. Objective: To evaluate the effectiveness of the Kids4Fit intervention in deprived areas on child's weight status. Methods: Design and setting: cohort study, including a waiting list control period. Subjects: children (N = 154) aged 6-12 years, who signed up for the Kids4Fit intervention programme, led by a dietitian, physiotherapist and child psychologist were included. Measurements of standardized body mass index (BMI-z) and waist circumference were taken at start of the waiting list period, at start and at the end of the intervention and after 52 weeks. Mixed model analyses (random effects models) were used, expressed in effect per week [ß with 95% confidence interval (CI)], compared to the waiting list expectancy over the 52-week study period. Results: Mixed model analyses showed a non-significant trend towards a lower BMI-z up to 52 weeks after start of Kids4Fit (ß: -0.0024; 95% CI: -0.0053; 0.0004), compared to the waiting list expectancy. A significantly lower waist circumference was found over time compared to the waiting list expectancy (ß: -0.0558; 95% CI: -0.0950; -0.0166). No differences were found in lifestyle and health-related quality of life. Conclusion: A local multidisciplinary intervention programme in deprived areas is effective in reducing waist circumference of obese children, compared to a waiting list expectancy, but no significant changes in lifestyle and quality of life were shown.

Effect of multidisciplinary interventions on quality of life in obese children: a systematic review and meta-analysis.

PURPOSE: To assess the effect of multidisciplinary intervention (MI) programs for overweight and obese children on quality of life (QoL). METHODS: Medline, EMBASE, Web of Science and Cochrane databases were searched for relevant studies without date restrictions up to July 2014. Included were randomized controlled trials and controlled clinical trials evaluating an MI aimed to reduce weight in overweight children and reporting QoL. The risk of bias of the included studies was assessed using the Cochrane guidelines. Data were pooled for short- (up to 6 months) and long-term (12-18 months) effects using a random effects model. RESULTS: In total, 11 studies were included, studying a total of 997 children aged 3-18 years. No significant differences were found between MI and control interventions on short-term QoL outcomes [mean difference (MD) 1.73, 95 % confidence interval (CI) -0.26 to 3.73 on a 0-100 scale]. Long-term results showed a nonsignificant trend toward a higher QoL in children following an MI program compared with control interventions (MD 4.40 95 % CI -0.12 to 8.92). CONCLUSION: There is insufficient evidence that MI programs, aimed to reduce weight in overweight and obese children, improve QoL.

Resting-state connectivity deficits associated with impaired inhibitory control in non-treatment-seeking adolescents with psychotic symptoms.

OBJECTIVE: Psychotic symptoms are common in the population and index risk for a range of severe psychopathological outcomes. We wished to investigate functional connectivity in a community sample of adolescents who reported psychotic symptoms (the extended psychosis phenotype). METHOD: This study investigated intrinsic functional connectivity (iFC) during resting-state functional magnetic resonance imaging (fMRI; rs-fMRI). Following screening in schools, 11 non-treatment seeking, youth with psychotic symptoms (aged 11-13) and 14 community controls participated in the study. Seed regions of interest comprised brain regions previously shown to exhibit aberrant activation during inhibitory control in adolescents with psychotic symptoms. RESULTS: Relative to controls, adolescents with psychotic symptoms exhibited reduced iFC between regions supporting inhibitory control. Specifically, they showed weaker iFC between the right inferior frontal gyrus (IFG) and the cingulate, IFG and the striatum, anterior cingulate and claustrum, and precuneus and supramarginal gyrus. Conversely, the psychotic symptoms group exhibited stronger iFC between the superior frontal gyrus and claustrum and IFG and lingual gyrus. CONCLUSION: The present findings are the first to reveal aberrant functional connectivity in resting-state networks in a community sample of adolescents with psychotic symptoms and suggest that disruption in integration between distributed neural networks (particularly between prefrontal, cingulate and striatal brain regions) may be a key neurobiological feature of the extended psychosis phenotype.

Hypothalamic vasopressin neural densities are higher in male Mongolian gerbils after separation from a pair bond partner and may facilitate behavior to form a new bond.

Although pair bonding has been studied for several decades, only somewhat recently have researchers began studying the neural consequences of separation from a pair bond partner. Here we examined the impact of partner separation on the socially monogamous Mongolian gerbil. Using a within-subjects design, we assessed nonsocial, nonreproductive, and reproductive behavior in male gerbils pre- and post- either 4 weeks of cohabitation with or separation from a pair bond partner. We then conducted an immediate early gene study to examine the influence of partner separation on hypothalamic oxytocin and vasopressin neural responses to interactions with a novel, opposite-sex conspecific.

The ethical matrix as a method for involving people living with disease and the wider public (PPI) in near-term artificial intelligence research.

INTRODUCTION: The rapid pace of research in the field of Artificial Intelligence in medicine has associated risks for near-term AI. Ethical considerations of the use of AI in medicine remain a subject of much debate. Concurrently, the Involvement of People living with disease and the Public (PPI) in research is becoming mandatory in the EU and UK. The goal of this research was to elucidate the important values for our relevant stakeholders: People with MS, Radiologists, neurologists, Registered Healthcare Practitioners and Computer Scientists concerning AI in radiology and synthesize these in an ethical matrix. METHODS: An ethical matrix workshop co-designed with a patient expert. The workshop yielded a survey which was disseminated to the professional societies of the relevant stakeholders. Quantitative data were analysed using the Pingouin 0.53 python package. Qualitative data were examined with word frequency analysis and analysed for themes with grounded theory with a patient expert. RESULTS: 184 participants were recruited, (54, 60, 17, 12, 41 respectively). There were significant (p < 0.00001) differences in age, gender and ethnicity between groups. Key themes emerging from our results were the importance fast and accurate results, explanations over model performance and the significance of maintaining personal connections and choice. These themes were used to construct the ethical matrix. CONCLUSION: The ethical matrix is a useful tool for PPI and stakeholder engagement with particular advantages for near-term AI in the pandemic era. IMPLICATIONS FOR PRACTICE: We have produced an ethical matrix that allows for the inclusion of stakeholder opinion in medical AI research design.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

Antiphospholipid antibodies in patients with purported 'chronic Lyme disease'.

BACKGROUND: Antiphospholipid antibody (aPL) positive patients and patients with purported chronic Lyme disease ('CLD') share many clinical features. After identifying significant aPL in sera of several index patients with 'CLD', we performed aPL tests on all patients referred in whom 'CLD' was suspected, diagnosed or treated. METHODS: All patients with suspected, diagnosed or treated 'CLD' and reportedly 'positive' Lyme assays were studied. aPL testing included anticardiolipin antibodies (aCL), anti-beta-2-glycoprotein-1 antibodies (anti-ß2GP1) and lupus anticoagulant (LAC). Patients were classified into four newly described categories of CLD and data was analyzed. RESULTS: One hundred and six patients were evaluated, of whom 82% had neurologic symptoms and 51% rheumatologic symptoms. Eighty-eight of 106 (83%) patients had positive Lyme serologies (enzyme-linked immunosorbent assay [ELISA] 62/106, 58.4%; western blot [WB] 64/106, 60%), while 18/106 (16.9%) were negative or equivocal. aPL was found in all 'CLD' categories. aCL and/or anti-ß2GP1 were positive in 85/106 (80%), with aCL present in 69/106 (65%) and anti-ß2GP1 present in 69/106 (65%). For all assays, IgM isotypes predominated: WB 55/64 (85%), aCL 63/69 (91%), anti-ß2GP1 52/69 (75%), aCL and/or anti-ß2GP1 74/85 (87%). Anti-ß2GP1 assays occurred in higher titer than aCL: 36/69 (52%) versus 63/69 (91%), p<0.001. Seventeen patients had aPL-related events. Only 12/106 (11.3%) had true post-Lyme syndromes (PLS), category IV, or late Lyme disease (LLD). Most patients had been treated for Lyme: 82/106 (79%). CONCLUSION: aPL occurs frequently in patients with 'CLD'. IgM anti-ß2GP1, IgM aCL and IgM WB were frequently found. Documented PLS or LLD was uncommon. The role of aPL in patients with 'CLD' needs further investigation.

Expanding ecological assessment by integrating microorganisms into routine freshwater biomonitoring.

Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.

Development of anterior cingulate functional connectivity from late childhood to early adulthood.

Human cerebral development is remarkably protracted. Although microstructural processes of neuronal maturation remain accessible only to morphometric post-mortem studies, neuroimaging tools permit the examination of macrostructural aspects of brain development. The analysis of resting-state functional connectivity (FC) offers novel possibilities for the investigation of cerebral development. Using seed-based FC methods, we examined the development of 5 functionally distinct cingulate-based intrinsic connectivity networks (ICNs) in children (n = 14, 10.6 +/- 1.5 years), adolescents (n = 12, 15.4 +/- 1.2) and young adults (n=14, 22.4 +/- 1.2). Children demonstrated a more diffuse pattern of correlation with voxels proximal to the seed region of interest (ROI) ("local FC"), whereas adults exhibited more focal patterns of FC, as well as a greater number of significantly correlated voxels at long distances from the seed ROI. Adolescents exhibited intermediate patterns of FC. Consistent with evidence for different maturational time courses, ICNs associated with social and emotional functions exhibited the greatest developmental effects. Our findings demonstrate the utility of FC for the study of developing functional organization. Moreover, given that ICNs are thought to have an anatomical basis in neuronal connectivity, measures of FC may provide a quantitative index of brain maturation in healthy subjects and those with neurodevelopmental disorders.

The resting brain: unconstrained yet reliable.

Recent years have witnessed an upsurge in the usage of resting-state functional magnetic resonance imaging (fMRI) to examine functional connectivity (fcMRI), both in normal and pathological populations. Despite this increasing popularity, concerns about the psychologically unconstrained nature of the "resting-state" remain. Across studies, the patterns of functional connectivity detected are remarkably consistent. However, the test-retest reliability for measures of resting state fcMRI measures has not been determined. Here, we quantify the test-retest reliability, using resting scans from 26 participants at 3 different time points. Specifically, we assessed intersession (>5 months apart), intrasession (<1 h apart), and multiscan (across all 3 scans) reliability and consistency for both region-of-interest and voxel-wise analyses. For both approaches, we observed modest to high reliability across connections, dependent upon 3 predictive factors: 1) correlation significance (significantly nonzero > nonsignificant), 2) correlation valence (positive > negative), and 3) network membership (default mode > task positive network). Short- and long-term measures of the consistency of global connectivity patterns were highly robust. Finally, hierarchical clustering solutions were highly reproducible, both across participants and sessions. Our findings provide a solid foundation for continued examination of resting state fcMRI in typical and atypical populations.

Brief telephone intervention increases testing for osteoporosis in patients treated in emergency departments for wrist fractures.

Previous studies show that identification and treatment of osteoporosis in patients with minimal trauma fractures treated as outpatients are poor. Our aim was to test two interventions designed to increase rates of identification and treatment. This prospective, action research study, using explicit medical record review and scripted telephone interview, was conducted at emergency departments (ED) of three hospitals from April 2007 to February 2008. Participants were patients aged over 50 years who were treated as outpatients with a minimal trauma wrist fracture. Data collected included demographic and fracture details, bone density testing and osteoporosis-related medication change. There were two interventions staff education in ED and fracture clinic and information provided to patients by telephone by a research nurse. These interventions were applied to all patients sequentially. The outcome measure of interest was the proportion of patients who underwent bone density testing (DEXA scans) in the follow-up period, analysed by intervention (clinic or phone). One hundred and seventeen patients were studied. Eighty-six per cent were female; median age 64 years. Ten per cent (12/117) of the ED/clinic intervention group had undergone testing at follow up. At follow up after the telephone intervention 55% (65/117) had undergone testing (P < 0.001, chi(2)). Patients undergoing testing were significantly more likely to have an osteoporosis-related medication change (relative risk 6.8, 95% CI 2.8-17.9). A brief telephone intervention and provision of information pack significantly improved testing rates for osteoporosis after minimal trauma wrist fracture. An ED/clinic-based intervention resulted in low rates of testing. Treatment of clinical osteoporosis remains suboptimal.

Regional variation in interhemispheric coordination of intrinsic hemodynamic fluctuations.

Electrophysiological studies have long demonstrated a high degree of correlated activity between the left and right hemispheres, however little is known about regional variation in this interhemispheric coordination. Whereas cognitive models and neuroanatomical evidence suggest differences in coordination across primary sensory-motor cortices versus higher-order association areas, these have not been characterized. Here, we used resting-state functional magnetic resonance imaging data acquired from 62 healthy volunteers to examine interregional correlation in spontaneous low-frequency hemodynamic fluctuations. Using a probabilistic atlas, we correlated probability-weighted time series from 112 regions comprising the entire cerebrum. We then examined regional variation in correlated activity between homotopic regions, contrasting primary sensory-motor cortices, unimodal association areas, and heteromodal association areas. Consistent with previous studies, robustly correlated spontaneous activity was noted between all homotopic regions, which was significantly higher than that between nonhomotopic (heterotopic and intrahemispheric) regions. We further demonstrated substantial regional variation in homotopic interhemispheric correlations that was highly consistent across subjects. Specifically, there was a gradient of interhemispheric correlation, with highest correlations across primary sensory-motor cortices (0.758, SD=0.152), significantly lower correlations across unimodal association areas (0.597, SD=0.230) and still lower correlations across heteromodal association areas (0.517, SD=0.226). These results demonstrate functional differences in interhemispheric coordination related to the brain's hierarchical subdivisions. Synchrony across primary cortices may reflect networks engaged in bilateral sensory integration and motor coordination, whereas lower coordination across heteromodal association areas is consistent with functional lateralization of these regions. This novel method of examining interhemispheric coordination may yield insights regarding diverse disease processes as well as healthy development.

Functional connectivity of the human amygdala using resting state fMRI.

The amygdala is composed of structurally and functionally distinct nuclei that contribute to the processing of emotion through interactions with other subcortical and cortical structures. While these circuits have been studied extensively in animals, human neuroimaging investigations of amygdala-based networks have typically considered the amygdala as a single structure, which likely masks contributions of individual amygdala subdivisions. The present study uses resting state functional magnetic resonance imaging (fMRI) to test whether distinct functional connectivity patterns, like those observed in animal studies, can be detected across three amygdala subdivisions: laterobasal, centromedial, and superficial. In a sample of 65 healthy adults, voxelwise regression analyses demonstrated positively-predicted ventral and negatively-predicted dorsal networks associated with the total amygdala, consistent with previous animal and human studies. Investigation of individual amygdala subdivisions revealed distinct differences in connectivity patterns within the amygdala and throughout the brain. Spontaneous activity in the laterobasal subdivision predicted activity in temporal and frontal regions, while activity in the centromedial nuclei predicted activity primarily in striatum. Activity in the superficial subdivision positively predicted activity throughout the limbic lobe. These findings suggest that resting state fMRI can be used to investigate human amygdala networks at a greater level of detail than previously appreciated, allowing for the further advancement of translational models.

Functional connectivity of default mode network components: correlation, anticorrelation, and causality.

The default mode network (DMN), based in ventromedial prefrontal cortex (vmPFC) and posterior cingulate cortex (PCC), exhibits higher metabolic activity at rest than during performance of externally oriented cognitive tasks. Recent studies have suggested that competitive relationships between the DMN and various task-positive networks involved in task performance are intrinsically represented in the brain in the form of strong negative correlations (anticorrelations) between spontaneous fluctuations in these networks. Most neuroimaging studies characterize the DMN as a homogenous network, thus few have examined the differential contributions of DMN components to such competitive relationships. Here, we examined functional differentiation within the DMN, with an emphasis on understanding competitive relationships between this and other networks. We used a seed correlation approach on resting-state data to assess differences in functional connectivity between these two regions and their anticorrelated networks. While the positively correlated networks for the vmPFC and PCC seeds largely overlapped, the anticorrelated networks for each showed striking differences. Activity in vmPFC negatively predicted activity in parietal visual spatial and temporal attention networks, whereas activity in PCC negatively predicted activity in prefrontal-based motor control circuits. Granger causality analyses suggest that vmPFC and PCC exert greater influence on their anticorrelated networks than the other way around, suggesting that these two default mode nodes may directly modulate activity in task-positive networks. Thus, the two major nodes comprising the DMN are differentiated with respect to the specific brain systems with which they interact, suggesting greater heterogeneity within this network than is commonly appreciated.

Sarcoplasmic reticulum and T tubules in differentiating rat skeletal muscle.

An electron microscope study has been made of the distribution of membrane couplings between the sarcoplasmic reticulum (SR) and either the plasmalemma or the T tubules in fetal and neonatal rat intercostal muscle. Within primitive muscle cells at 12 days of gestation, the SR forms both simple and specialized membrane junctions with the plasmalemma; caveolae are very few, and T tubules are not detected. Undifferentiated cells neighbor muscle cells. Occasionally these cells contain subsurface couplings between the endoplasmic reticulum and plasmalemmae. Possible relationships between these couplings and the peripheral couplings of muscle cells are discussed. By 15-18 days of gestation, caveolae and beaded T tubules, comparable to those of cultured muscle, develop; T tubules lie along-side myofibrils and are rarely transverse. SR couples both to T tubules and to plasmalemmae during this period. T tubules with lineal profiles appear after further development and their orientation transverse to A-I junctions becomes increasingly evident. Membrane couplings between SR and T tubules also increase in number, whereas the incidence of peripheral coupling declines rapidly Evidence suggests that peripheral couplings are swept into myotubes as caveolae proliferate and T tubules form. SR thus appears to initially couple with the plasmalemma and then to await T tubular growth. This contrasts with the developmental pattern described in cultured chick muscle in which peripheral couplings are not reported and T tubules with diads and triads occur at very primitive stages of muscle differentiation.

The histogenesis of rat intercostal muscle.

Intercostal muscle from fetal and newborn rats was examined with the electron microscope. At 16 days' gestation, the developing muscle was composed of primary generations of myotubes, many of which were clustered together in groups. Within these groups, the membranes of neighboring myotubes were interconnected by specialized junctions, including tight junctions. Morphologically undifferentiated cells surrounded the muscle groups, frequently extended pseudopodia along the interspace between adjacent myotubes, and appeared to separate neighboring myotubes from one another. At 18 and 20 days' gestation, the muscle was also composed of groups of cells but the structure of the groups differed from that of the groups observed at 16 days. Single, well differentiated myotubes containing much central glycogen and peripheral myofibrils dominated each group. These large cells were interpreted as primary myotubes. Small, less differentiated muscle cells and undifferentiated cells clustered around their walls. Each cluster was ensheated by a basal lamina. The small cells were interpreted as primordia of new generations of muscle cells which differentiated by appositional growth along the walls of the large primary myotubes. All generations of rat intercostal muscle cells matured to myofibers between 20 days' gestation and birth. Coincidentally, large and small myofibers diverged from each other, leading to disintegration of the groups of muscle cells. Undifferentiated cells frequently occurred in the interspaces between neighboring muscle cells at the time of separation. Myofibers arising at different stages of muscle histogenesis intermingled in a checkerboard fashion as a result of this asynchronous mode of development. The possibility of fusion between neighboring muscle cells in this developing system is discussed.

The fine structure of motor endplate morphogenesis.

The fine structure of the developing neuromuscular junction of rat intercostal muscle has been studied from 16 days in utero to 10 days postpartum. At 16 days, neuromuscular relations consist of close membrane apposition between clusters of axons and groups of myotubes. Focal electron-opaque membrane specializations more intimately connect axon and myotube membranes to each other. What relation these focal contacts bear to future motor endplates is undetermined. The presence of a group of axons lying within a depression in a myotube wall and local thickening of myotube membranes with some overlying basal lamina indicates primitive motor endplate differentiation. At 18 days, large myotubes surrounded by new generations of small muscle cells occur in groups. Clusters of terminal axon sprouts mutually innervate large myotubes and adjacent small muscle cells within the groups. Nerve is separated from muscle plasma membranes by synaptic gaps partially filled by basal lamina. The plasma membranes of large myotubes, where innervated, simulate postsynaptic membranes. At birth, intercostal muscle is composed of separate myofibers. Soleplate nuclei arise coincident with the peripheral migration of myofiber nuclei. A possible source of soleplate nuclei from lateral fusion of small cells' neighboring areas of innervation is suspected but not proven. Adjacent large and small myofibers are mutually innervated by terminal axon networks contained within single Schwann cells. Primary and secondary synaptic clefts are rudimentary. By 10 days, some differentiating motor endplates simulate endplates of mature muscle. Processes of Schwann cells cover primary synaptic clefts. Axon sprouts lie within the primary clefts and are separated from each other. Specific neural control over individual myofibers may occur after neural processes are segregated in this manner.

Development of muscle fiber specialization in the rat hindlimb.

The appearance of fast and slow fiber types in the distal hindlimb of the rat was investigated using affinity-purified antibodies specific to adult fast and slow myosins, two-dimensional electrophoresis of myosin light chains, and electron microscope examination of developing muscle cells. As others have noted, muscle histogenesis is not synchronous; rather, a series of muscle fiber generations occurs, each generation forming along the walls of the previous generation. At the onset of myotube formation on the 15th d of gestation, the antimyosin antibodies do not distinguish among fibers. All fibers react strongly with antibody to fast myosin but not with antibody to slow myosin. The initiation of fiber type differentiation can be detected in the 17-d fetus by a gradual increase in the binding of antibody to slow myosin in the primary, but not the secondary, generation myotubes. Moreover, neuromuscular contacts at this crucial time are infrequent, primitive, and restricted predominantly, but not exclusively, to the primary generation cells, the same cells which begin to bind large amounts of antislow myosin at this time. With maturation, the primary generation cells decrease their binding of antifast myosin and become type I fibers. Secondary generation cells are initially all primitive type II fibers. In future fast muscles the secondary generation cells remain type II, while in future slow muscles most of the secondary generation cells eventually change to type I over a prolonged postnatal period. We conclude that the temporal sequence of muscle development is fundamentally important in determining the genetic expression of individual muscle cells.