Despite widespread use of MgSO4 in clinical practice, its effects on adult offspring are not well known nor have sex-specific differences in therapeutic response been explored. The objective of our study was to examine the long-term effects of perinatal neuroinflammation and the effectiveness of prenatal MgSO4/betamethasone treatments between males and females in a murine model via histologic and expression analyses. Our results demonstrate that male but not female offspring exposed to intrauterine inflammation demonstrated impaired performance in neurodevelopmental testing in early life assessed via negative geotaxis, while those exposed to injury plus treatment fared better. Histologic analysis of adult male brains identified a significant reduction in hippocampal neural density in the injured group compared to controls. Evaluation of key neural markers via qRT-PCR demonstrated more profound differences in gene expression in adult males exposed to injury and treatment compared to female offspring, which largely showed resistance to injury. Prenatal treatment with MgSO4/betamethasone confers long-term benefits beyond cerebral palsy prevention with sex-specific differences in response.Preterm infants are at significantly increased risk for lifelong neurodevelopmental disability with male offspring disproportionately affected. Corticosteroids (such as betamethasone) and magnesium sulphate (MgSO Damage to the central nervous system can occur through disruption of the delicate germinal matrix vasculature causing intraventricular hemorrhage (IVH) and as a result of hypoxia3. Long-term neurologic complications of prematurity include cerebral palsy4 and an increased frequency of attention-deficit/hyperactivity disorder5, among others.Infants born preterm are at increased risk of neurologic injury resulting in neurodevelopmental disability, a global term encompassing varying degrees of neurologic impairment6, with the hippocampus – a region of the brain within the limbic system – appearing particularly vulnerable8. Prematurity in the context of a very low birth weight may impact an individual’s intelligence9. Human and animal studies demonstrate that the type and severity of neurologic injury associated with preterm labor varies between genders, with increased susceptibility in males12. Despite mounting evidence of an interplay between gender and the degree of perinatal brain injury, the underlying molecular and cellular mechanisms remain poorly understood.Magnetic resonance imaging (MRI) studies suggest that children born preterm have decreased brain volumes compared to their term counterparts4) has emerged as a mitigating strategy. Multiple randomized controlled trials summarized in meta-analyses14 suggest that administration of MgSO4 can reduce some neurologic complications of prematurity, most notably cerebral palsy. The estimated number needed to treat is 1 in 4613. The molecular pathway(s) by which MgSO4 confers neuroprotection in some but not all patients is unclear. Potential mechanisms of action include promoting hemodynamic stability, prevention of neural excitatory injury, and through antioxidant and/or anti-inflammatory effects15. While none of the major randomized controlled trials evaluating the benefit of MgSO4 for neuroprotection stratified results based on gender, sex steroids do influence neurogenesis16. Within the developing hippocampus in particular the interplay between estrogens and endocannaboid pathways leads to greater suppression of GABAergic inhibition in females compared to males17. If MgSO4 works in part by preventing neural injury from excitatory neurotransmitters, it is conceivable that a differential response based on sex could exist.There is no definitive intervention to prevent or treat prematurity-related neurodevelopmental disability, though antepartum administration of magnesium sulphate . The primary purpose of corticosteroids in preterm labor is to promote fetal lung maturation18, though they also reduce other prematurity-related morbidities including IVH19. Steroids have known anti-inflammatory properties which may be beneficial in inflammation-induced preterm labor. In the largest U.S. study evaluating the benefit of MgSO4 for neuroprotection20, 97 percent of patients received a corticosteroid .In addition to MgSO21 given limitations on human research. Studies using a murine model have provided insight into the molecular mechanisms of brain injury related to pre-term birth24. These studies have demonstrated the effects of in utero neuroinflammation including reductions in hippocampal volume that are detectable in adolescence and analysed sex-specific differences with notation of a greater degree of injury in male offspring12. Whether MgSO4 and corticosteroids reduce these negative outcomes in experimental adult animals and if there is a sex-specific difference in their response has not been tested.The use of animal models is critical to understanding the effects of neuroinflammation on brain development4 and betamethasone will reduce the long-term impact of in utero neuroinflammation with male offspring benefitting to a greater degree than females. The objective of our study was to examine the long-term effects of perinatal neuroinflammation and the effectiveness of prenatal MgSO4 and betamethasone treatments between males and females in a murine model via histologic and expression analysis . Mice randomized to receive LPS followed by treatment with MgSO4 and betamethasone had an increased litter survival rate compared to LPS plus placebo; however, this difference was not significant (student’s t-test P > 0.05). There was no difference in the number of offspring per litter assessed on post-natal day 5 with the exception of PBS vs. PBS + MgSO4/betamethasone control groups (student’s t-test P < 0.05).Approximately 600 offspring were delivered between the four groups Fig. . All con4/betamethasone  and nine (P9) compared to PBS controls and those that prenatally received MgSOone Fig. . Stratifone Fig. . By post4/betamethasone treated males vs. females weighed more on P13, P21, and P40, whereas PBS controls were only significantly different on P50.Mouse weights compared between treatment groups or when stratified by sex were significantly higher in both males and females on P5, P9, P13, P21, and P40. Fig. . On P50 4/betamethasone and the PBS controls , the second most abundant protein in the brain and an essential component of myelination25, revealed reduced expression with LPS mediated injury in both males and females  was assayed to evaluate astrocytes. Results indicate a significant decline in Gfap in male brains between PBS controls and LPS exposed animals ; however, interrogation of targets linked to neurotransmission, glia, and stem cells generally revealed more profound injury in males with females largely unaffected. Similarly, histologic analysis of hippocampal neural density via NeuN staining identified a decline in male but not female offspring following injury  but not all genes . Largely resistant to neuroinflammation, female offspring appeared to derive little benefit from MgSO4/betamethasone therapy. Of note, females expressed greater levels of Grin2a which encodes the NMDA receptor 2 alpha subunit (NR2A) , may influence perinatal brain injury29. However, it is unclear from our study whether the differential response to therapy is due to sex-specific mechanisms related to glutamate signaling or other molecular factors.In adult offspring, both sexes demonstrated a decline in a key marker of myelination  Fig. . NR2A isChat between PBS controls  are used to reduce type I error, these approaches are more commonly employed for high-throughput gene analysis such as microarrays. The probability of false discovery is attributed in part to the sensitivity of such screening assays and the large number of genes analyzed. Our analysis relied on specific primers to examine a small number of pre-selected genes, thereby limiting the possibility of a type I error39. In particular, male infants born preterm are at increased risk for neurodevelopmental disability including cognitive deficits, language impairment, and cerebral palsy, among others41. The mechanism conferring increased susceptibility in preterm male offspring remains unknown though differences in intrauterine adaptations, rates of maturation, and fetal sex steroids have been proposed42. Further research into sex-specific responses to inflammation-induced neurologic injury may yield insight into specific vulnerabilities of the male fetus and allow more targeted interventions. Additionally, stratifying existing data from the major randomized controlled trials could determine if a sex-based difference in response to MgSO4 therapy exists in humans.The increased morbidity and mortality of male offspring in the context of prematurity is well documented43. Thus, the impact of prenatal injury may be different in humans compared to mice. Furthermore, our study examines the benefits of MgSO4/betamethasone in brain injury resulting from endotoxin exposure not active bacterial infections. A comparison of MgSO4/betamethasone in models that rely on bacterial inoculation such as Group B Streptococcus44 and Escherichia coli46, would be informative in assessing sex-specific differences between pathogens commonly associated with preterm labor.The main limitation of this study is its reliance on an animal model. While we have selected one that uses an inflammation-based form of preterm labor, no model can completely reflect the complexities of human preterm labor. Rodents, including mice, are born at a more neurologically immature state than their human counterparts and brain development and growth continues into the postnatal period4/betamethasone but does not address if the effectiveness of these therapies varies with respect to dosing and timing of administration. It is conceivable that administering MgSO4 therapy prior to preterm labor may provide additional benefit, as indicated in trials for preenclampsia48. However, this agent is a “high risk” medication that currently requires hospitalization and a continuous intravenous infusion. Our results suggest that in the absence of neuroinflammation, MgSO4/betamethasone may negatively alter normal gene expression levels as evident in female controls with Gad1 and Mbp expression. Therefore, without biomarkers to predict preterm deliveries, the dosing and safety of these treatments would have to be carefully evaluated for prophylactic use.A second limitation is that our experimental design simulates the current clinical regimen for MgSOOur study has several strengths. We selected the model of preterm labor that is believed to most accurately reflect the most common form of preterm labor in humans rather than those that employ progesterone withdrawal or overt infection. Additionally, we carefully selected the timing and dosage of injury and therapeutic interventions based on prior published studies and worked to establish a phenotype of injury to correlate with histological and qRT-PCR findings.4/betamethasone partially ameliorated this damage but significant long-term benefit was only observed in male offspring. Our findings suggest that MgSO4/betamethasone confer long-term benefits beyond cerebral palsy prevention with sex-specific differences in response. Our study supports further research into sex-responses to perinatal neuroinflammation and development of precision medicine approaches that address the effectiveness of MgSO4/betamethasone treatments between male and female offspring.In a murine model, our research demonstrates that exposure to intrauterine inflammation produces a differential response based on sex, with male offspring more profoundly affected. Prenatal administration of MgSOet al. in 200351. CD-1 timed pregnant mice were shipped from the vendor  and arrived at our facility for a period of acclimation. On day 17.5 of a 19–21 day gestation, 88 dams were placed under general anesthesia and the bicorunate uterus was accessed by laparotomy . Animals involved in this study were maintained in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Research Council/Institute of Laboratory Animal Research (ILAR). We modified an existing model of inflammation-induced preterm labor and perinatal brain injury developed by Elovtiz omy Fig. .Escherichia coli  or an equivalent volume (100 μl) of vehicle (PBS)22. Thirty-four animals were assigned to each LPS group compared to 10 animals in the PBS control groups based on research indicating that 37 percent of dams exposed to this dose of LPS will deliver a surviving litter12.Animals were randomized to receive an intrauterine injection of 50 µg LPS derived from 4 (270 mg/kg loading dose followed by 27 mg/kg every 20 minutes for four hours injected subcutaneously). We selected this interval based on research demonstrating an increase in inflammatory serum cytokines that is measurable 30 minutes following LPS administration54. Dams remained in single cage housing and were closely monitored under the supervision of an attending veterinarian until delivery.Thirty minutes after surgery, dams were further assigned to receive a combination of betamethasone (0.1 mg subcutaneous injection) and MgSOSurviving litters were reared until postnatal day 60. Weaning took place on postnatal days 20–22. Once sex could reliably be determined, animal numbers were randomly reduced from up to 16 to six animals per litter with an equal number of male and female offspring. Euthanasia of pups was accomplished with a sodium pentobarbital-based compound , adolescents and adults underwent cervical dislocation under general anesthesia.55. For this test, the pup was placed head down on a screen angled 45 degrees and the time in seconds for the animal to convert to a heads up orientation was measured56. Animals acclimated to the testing room for at least 60 minutes before assessments. Testing was conducted at the same time each session and by the same core group of investigators. Animals were weighed prior to testing and individuals were tracked using tail markings. A subset of tests were filmed and audited by a separate investigator to ensure consistency in test performance and grading.Neurodevelopmental assessment in the form of negative geotaxis testing was performed on postnatal days 5, 9, and 13 to assess body righting mechanisms, strength, and coordination – deficiencies that may result from exposure to perinatal neuroinflammation57. NeuN staining was conducted following antigen retrieval with sodium citrate buffer pH 6.0 + 0.05% Tween 20 for 30 minutes at 95 °C and blocking with 2.5% horse serum for 30 minutes. Subsequently, the monoclonal rabbit anti-NeuN  was diluted at 1:2000 in PBS +1% BSA and applied overnight at 4 °C. The next day, the staining was visualized using the ImmPRESS anti-rabbit peroxidase kit and ImmPACT DAB substrate  in accordance with the manufacturer’s protocols. Hippocampal neural density was calculated from photographs of NeuN stainings in ImageJ  by analyzing the percentage of DAB (NeuN) staining within the hippocampal cross-sectional area58.On postnatal day 60, any remaining offspring were euthanized. Whole brains were collected for either histological or transcriptional analysis. Brains destined for histological analysis were fixed in neutral buffered formalin and embedded in paraffin. Fixed brains were subsequently sectioned 8 µM thick and stained for NeuN, a marker of mature neurons18s by the ΔCt method. All primers were generated by the Harvard PrimerBank, with the exception of 18s rRNA60. Primer sequences are listed in Supplemental Table For expression analysis, brains were flash frozen in liquid nitrogen and homogenized for total RNA isolation using the RNeasey Lipid Tissue Mini kit . First stand cDNA was generated using the GoScript Reverse Transcriptase System  for quantitative reverse-transcription PCR (qRT-PCR) with the FastStart SYBR Green master mix ). PCR reactions were run for 40 cycles using a Roche 480 II lightcycler. The relative expression of each target was calculated by normalizing to ribosomal Microtubule associated protein 2 (Map2) which encodes a cytoskeletal protein indicative of neurons61, was selected as a general neural marker. Choline acetyltransferase (Chat), expressed by cholinergic neurons, and Tyrosine hydroxylase (Th), essential for catecholaminergic processes63, were examined as markers of neurotransmission. Glutamate and myelination, two related processes that are connected with perinatal brain injury64 were also compared between groups using the targets Glutamate decarboxylase 67 kDa (Gad67) encoded by Gad165, Glutamate receptor ionotropic NMDA 2AGrin2a)66, and Meylin basic protein (Mbp)25. To assess astrocytes, the expression of glial fibrillary acidic protein (Gfap) was assayed67. Finally, neural progenitors and stem cells were evaluated by the expression of SRY-box 2 (Sox2) and Nestin68.Genes associated with neurons, neural transmission, glia, white matter, and stem cells were targeted for expression analysis. All genes tested as part of this study are reported as follows; t-test or one-way ANOVA. P values < 0.05 was deemed significant. In these statistical analyses, multiple comparisons were not corrected to circumvent type II error, though type I error - false discovery may arise. The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.For all experiments, statistical significance was determined by student’s Supplemental Table 1.


Sensory information undergoes substantial transformation along sensory pathways, usually encompassing sparsening of activity. In the olfactory bulb, though natural odorants evoke dense glomerular input maps, mitral and tufted (M/T) cells tuning is considered to be sparse because of highly odor-specific firing rate change. However, experiments used to draw this conclusion were either based on recordings performed in anesthetized preparations or used monomolecular odorants presented at arbitrary concentrations. In this study, we evaluated the lifetime and population sparseness evoked by natural odorants by capturing spike temporal patterning of neuronal assemblies instead of individual M/T tonic activity. Using functional imaging and tetrode recordings in awake mice, we show that natural odorants at their native concentrations are encoded by broad assemblies of M/T cells. While reducing odorant concentrations, we observed a reduced number of activated glomeruli representations and consequently a narrowing of M/T tuning curves. We conclude that natural odorants at their native concentrations recruit M/T cells with phasic rather than tonic activity. When encoding odorants in assemblies, M/T cells carry information about a vast number of odorants (lifetime sparseness). In addition, each natural odorant activates a broad M/T cell assembly (population sparseness). Odor representations undergo substantial changes across the consecutive layers of the olfactory network. For instance, evoked inputs elicited in olfactory sensory neurons (OSNs) undergo significant reshaping by the recurrent and lateral inhibition of interneurons in the olfactory bulb (OB)1249Recent works proposed that sparsening of M/T cell firing drives GABAergic-dependent pattern separation of odorant representations1011126Using a combination of optical imaging and tetrode recordings in awake head-restrained mice, we monitored the glomerular and M/T cells responses to a large set of natural odorants. As M/T cells encode odorant information with subtle phasic temporal changes of spiking915n = 29) at concentrations encountered when an animal is close to the odor source1117n = 81 M/T cells from 4 mice). As previously describedWe selected a large set of natural odorants n = 2 at conceAs most M/T cells did not exhibit tonic changes69blue curves in grey curves in black curves in To identify which neuron was informative about a particular stimulus, we developed an algorithm (based on a simple classifier) that tested whether specific neuronal ensembles could classify odorant identity. For each stimulus, we used our algorithm to distinguish the activity of the baseline from the odor epochs  and 3. WFor some odorants, both methods identified responding neurons. For example, most neurons  carried information about honey and kiwi, while onion and mouse urine were weakly or not encoded by the set of recorded cells . We thenTaken together, these results suggest that the spike temporal envelope of M/T ensembles evoked by odors is sufficient to carry information about a large fraction of odorants. Hence, the dense representation of natural odorants previously observed at the level of the glomerular layern = 8) activated a large number of glomeruli in all animals tested 6914192021n = 79 M/T cells from 4 mice) using the same 50-fold diluted natural odorants. Though M/T cell population activity still predicts natural odor identity at this concentration, the maximum performance was much lower than the one observed at high concentrations  emerged from low concentrations of natural odorants, probably mimicking observations reported in previous studies6In this study we evaluated how natural odorants are represented in the mouse OB and how concentration affects the density of the odor code. By using natural odorant concentrations activating dense glomerular patterns, we showed that M/T cell are more broadly tuned than previously reported, encoding for a large fraction of natural odorants tested . In contPrevious studies showed that M/T cells recorded in anesthetized animals are highly selective, which was considered to be a consequence of selective OSN activity and odor-specific sparse input maps679249677109Finally, all previous studies computed M/T cells tuning curves by focusing on single cell response profiles671516231527282930313267Two important points must be mentioned. First, when considering odorants that activate a large fraction of neurons, the responsive neurons are not all coactive at a given time; they indeed exhibit complex temporal discharges during the respiratory cycle. Since the odor code is distributed and dynamic in the population of M/T cells91113151711101235In conclusion, we found that M/T assemblies are encoding a broad panel of natural odorants at their natural concentrations, processing sensory information with subtle temporal changes of activity.All experiments were performed on 12 to 20 week-old male C57BL/6J mice (Charles River France). All experiments were in accordance with the Swiss Federal Act on Animal Protection and Swiss Animal Protection Ordinance. The experiments were approved by the University of Geneva and the state of Geneva ethics committees (authorization 1007/3387/2).The initial preparation procedure has been extensively presented elsewhere914All natural stimuli , Swiss chocolate, wine rosé, nutmeg, rosemary, mint, cinnamon, tobacco, cardamom, cloves, vinegar, garlic, onion, pineapple, basil, lemongrass, apple, pear, sesame oil, black pepper) except mouse food, urine and feces were purchased from local grocery stores. C57BL/6J male mouse urine was collected and stored at −80 °C until use. Mouse feces were collected every day from cages containing male mice. Natural odorants were prepared fresh each experimental day . Either four grams of dry solid (for mouse feces: 1g) or four milliliters (for mouse urine: 1 ml) of natural stimuli were placed in glass vials. They were thus at natural partial pressure.Through a custom made olfactometer, odorants were delivered during an animal’s expiration (the odor onset was the onset of the first inspiration after delivery) as described previously91417The procedure has been presented in detail elsewhere1438The procedures have been described extensively elsewhere96151625In awake mice, the duration of respiratory cycles (RC) is highly variable within and across trials. In order to consistently analyze the neural responses to odors across trials, the beginning of each cycle was temporally realigned to each other. All RC were artificially matched to the average breathing duration (405 ms ± 178 S.D.) over all trials: longer cycles were truncated and shorter ones were prolonged. Spike times were realigned using the same method. Importantly, relative action potential timings in spike trains were not affected by this method.n matrix (n respiratory cycles before and during odor presentation). For each odor, we further averaged all matrices computed for individual trials. The same process was applied for all the 29 odors and the averaged matrices were concatenated together . A principal component analysis (PCA) was computed with the concatenated matrix, which allowed representing each respiratory cycle (RC) as a vector in an 8 dimensional space, each dimension representing one of the respiratory bins. The 3 first components of the PCA transformation carried more than ~75% of the variance.The procedure has been presented in detail elsewheremean) and the standard deviation (σ) of the Euclidean distances between the CBRC centroid and each of the single CBRC. Then the distances (Ko) between each single ORC and the CBRC centroid were calculated. A cell was considered as responsive if:To define if neurons were responsive to odorants, we assessed whether the firing distribution in RC of the baseline and odor periods was significantly differentb) between TBRC and the CBRC centroid. Similarly, if:where λ = 1, 2, 3 … n, for at least one odor. However, this method may detect false-positive responses. In order to discard them, we computed the distances . The number λ of standard deviation σ was parametrically increased until only 5% of cells were considered as responsive using the CBRC template. We thus kept the value of λ extracted from the equation (2) and used it to satisfy the conditions in the equation (1). This led to a detection of responsive cells with a risk of false-positive inferior or equal to 5%.In this analysis, a cell had to respond significantly for at least 3 RCs to be considered as responsive. Similar results were observed by considering one or two significant RCs but the exact percentages of responsive cells were lower due to an increase of false positives in the baseline.We pooled all neurons recorded in different animals, assuming that they represent the same variability of neural responses as an equal number of cells in a single mouse. The activity of the neurons was organized in vectors, each dimension containing the average firing rate of a recorded cell computed over a certain time bin. Population vectors were built using 8  per breathing cycle time bins.To compute classification performance, one trial per stimulus was chosen to be a test set and removed from the remaining trials that were averaged and considered as the template responses9In order to identify which individual cells contain information about odorants, one has to determine if odor-evoked activity can be discriminated from baseline activity. To do that, single cell firing rates over a breathing cycle were binned in 8 time windows of equivalent durations (50 ms). Single cell activity was thus represented by series of 8 raw vectors. We then processed our classification algorithm as following. The two first odor-evoked activity vectors after odor onset were extracted and chosen as test vectors for a particular trial (To). An odor-evoked activity template was calculated by computing the centroid of remaining vectors (i.e. except To) over the first and second breathing cycle after odor onset. Similarly, the baseline activity template  was computed as the centroid of remaining vectors (i.e. except one vector of the baseline) for the 2 breathing cycles before the odor onset. The Euclidean distances between each To and the 2 centroids (Co and Cbt) were calculated. Trials were assigned to the closest template .st dimension to the corresponding 8-raw activity vector of another cell. This led to a series of 16-raw activity vectors made by the activity of 2 cells during successive breathing cycles. We recurrently varied the identity of the second cell in order to again obtain the minimal performance when using the classification algorithm with each possible 16-raws vector (every possible combination of 2 cells). This process was continued until obtaining a population vector constituted of a total of (n × 8) × 1 (where n = number of cells) concatenated bins, which gave rise finally to a maximum classification performance per odor  and computed a second baseline activity template (Cbc) corresponding to two other pre-odor breathing cycles (different from the precedent ones) before odor application. To define a threshold, two methods were then applied. For the first one, the baseline activity vector was computed using the same cell order obtained to build the vector for the precedent process, that is to say for reaching the maximal classification performance. The classification algorithm was applied to obtain a prediction curve .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Scientific Reports using confocal-microscopy based automated quantification of anti-synapsin labeled microglomeruli in the mushroom bodies of honeybee brains reports potentially incorrect numbers of microglomerular densities. Whereas several previous studies using visually supervised or automated counts from confocal images and analyses of serial 3D electron-microscopy data reported consistent numbers of synaptic complexes per volume, Peng and Yang revealed extremely low numbers differing by a factor of 18 or more from those obtained in visually supervised counts, and by a factor 22–180 from numbers in two other studies using automated counts. This extreme discrepancy is especially disturbing as close comparison of raw confocal images of anti-synapsin labeled whole-mount brain preparations are highly similar across these studies. We conclude that these discrepancies may reside in potential misapplication of confocal imaging followed by erroneous use of automated image analysis software. Consequently, the reported microglomerular densities during maturation and after manipulation by insecticides require validation by application of appropriate confocal imaging methods and analyses tools that rely on skilled observers. We suggest several improvements towards more reliable or standardized automated or semi-automated synapse counts in whole mount preparations of insect brains.A recent study by Peng and Yang in  Recent studies in the honeybee have shown that microglomerular synaptic complexes, termed microglomeruli (MG), in olfactory and visual input regions of the MB calyx express a high degree of structural synaptic plasticity during adult maturation11, in response to environmental factors during postembryonic development12, after sensory exposure13, and following stable long-term memory formation15. All of these studies used anti-synapsin labeling of relatively large presynaptic boutons of MG in the MB calyx, which, in some studies, was combined with f-actin phalloidin labeling of postsynaptic compartments or quantitative 3D serial electron microscopy analyses10. A recent review by Fahrbach and Van Nest16 provides a most comprehensive summary of these studies addressing possible links between brain plasticity and behavioral flexibility in social honeybees.The mushroom bodies (MBs) of the insect brain perform high-level sensory integration and are involved in learning and memory formation17 used anti-synapsin immunolabeling and volume quantification of MB-calyx MG in whole-mount brains of adult honeybees to analyze synaptic maturation in the MBs under normal conditions and after larval treatment with sublethal dosages of imidacloprid, a neonicotinoid pesticide. Changes in the densities of synapsin-positive boutons from confocal image series were quantified to ask whether exposure of honeybee larvae to this insecticide alters densities and total numbers of MB synaptic boutons in adult brains which, as a consequence, might affect pollination behavior. Whereas the goal of this study addresses a very interesting and highly topical issue, the confocal-imaging based automated counting method for synapsin-positive boutons in MG of the MBs contains serious flaws. Although the authors claim they are aware of the difficulties of automated quantifications compared with visually guided counts by a human expert blind to the experimental treatment18, the discrepancies in an order of magnitude compared with previous analyses are not supported by any visual analyses to provide a confirmation of results from automated counts. In contrast, sample confocal images  sections. The study by Groh et al.10, in general, greatly supports the reliability of visually guided MG counts obtained from fluorescently labeled synapsin-positive boutons in whole mount preparations. The obvious discrepancy of confocal imaging-based, automated volume counts by Peng and Yang is not supported by the confocal images they provided as the 1,000 µm3 tissue cube, even in a 2D view, clearly contains more than 0.8 to 2 synapsin-positive boutons . For an average number of 1.46 MG per 1000 µm3 in the total MB calyx volume, the representative images presented in Fig. 5c–e  could make up only a proportion of ~5% of the total MB calyx (as the average number is by a factor of approximately 20 lower than in the image shown). In consequence, 95% of the remaining MB calyx volume should be devoid of any MG if the average numbers reported by Peng and Yang were true. Earlier studies considered anatomical subcompartments in the MB calyx by sampling MG in selected volumes (regions of interest) and, for example, show that the MG distribution in the dense collar is very regular with densities around 65 MG/1000 µm3 and more heterogeneous with densities around 35 MG/1000 µm3 in the lip  lists a 40x and a 63x objective with working distances of only 0.1 mm - both objectives do not allow complete scans through the entire depth of the MB calyx . This discrepancy with earlier studies was also pointed out by Fahrbach and Van Nest16, but is largely ignored in the study by Peng and Yang17. In summary, we suggest that in the light of the serious methodological flaws outlined above the results by Peng and Yang on both maturation under normal conditions and after insecticide treatment should be viewed as preliminary and potentially flawed rather than conclusive.Peng and Yang17 with two other previous studies in the honeybee using automated MG counts based on confocal imaging9  of the MB calyx  and the results from visually guided counts (~25–36) in all previous studies including 3D serial EM quantification for the MB lip region15 , so far, have failed to produce reliable results compared with visually guided counts, even in image stacks at high optical resolution  image analysis algorithms. This represents a great future potential for enhancement of automated image analyses in large data sets after various treatments like manipulations by environmental, hormonal or genetic factors including the appropriate controls. To promote the development of such tools across laboratories working on the honeybee, we provide two high-resolution confocal image stacks in a central layer of the honeybee MBs (lip and collar) as supplementary data stacks  in the MB calyces of the honeybee and also various other species of bees and ants will continue to be highly promising candidates for quantitative manipulative studies on the adaptive flexibility of neuronal microcircuits and their role in behavioral plasticity in a social context.In the end, however, the best automated counting tool will still require some degree of visual confirmation by a human expert’s eyesApis mellifera carnica) were taken from the institutional apiary of Zoology II, University of Würzburg. All steps including materials for brain dissection, immunohistochemistry and anti-synapsin immunolabelling in whole mount brain preparations were similar to refs We provide two examples of high-resolution confocal image stacks from a central layer within the honeybee MBs (lip and collar) as 3D supplementary data stacks (supplementary data Supplementary dataset S1Supplementary dataset S2Supplementary Information S3


We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression  In order to facilitate normal axonal signal transduction  to and from the peripheral nervous system, the endoneurial microenvironment is strictly regulated6. Tight junction-forming perineurial myofibroblasts provide a critical interface between the endoneurial and epineurial interstitial fluid compartments that helps further regulate the endoneurial microenvironment; however, these cells are not in direct contact with circulating blood. Endoneurial microvessels are in direct contact with circulating blood, and are considered to form the blood-nerve barrier (BNB)7.Peripheral nerves are structurally divided into three compartments: the outermost epineurium that consists of longitudinal arrays of collagen fibers in which fenestrated macrovessels directly derived from the extrinsic peripheral nerve blood supply reside (known as vasa nervorum); the inner perineurium, which consists of concentric layers of specialized myofibroblasts with intercellular tight junctions, that surround the innermost endoneurium, consisting of a loose array of collagen fibers in which myelinated and unmyelinated axons and non-fenestrated tight junction-forming microvessels reside, with rare leukocytes and fibroblasts12. In situ, endoneurial microvessels share their basement membrane with pericytes that may act as specialized smooth muscle cells that regulate the microvasculature in response to differences in neural activity, blood pressure and volume13. Phenotypic and functional differences occur between micro- and macrovascular endothelial cells within the same tissue, between endothelial cells from different tissues and endothelial cells from the same tissues in different species, with molecular and functional adaptations occurring dependent on the cellular microenvironment23. Extrapolation of observations and functional mechanisms from other restrictive microvascular barriers, such as the blood-brain barrier, or epithelial cells, provides general knowledge on restrictive barrier systems that may not specifically apply to peripheral nerves.In mammals, the BNB is generally considered the second most restrictive mammalian microvascular barrier after the blood-brain barrier4. Such knowledge is paramount to understanding peripheral nerve angiogenesis, vascular differentiation and the role of the BNB in regulating the endoneurial microenvironment to facilitate normal axonal growth and myelination, as well as the development and maintenance of its specialized restrictive barrier and adaptations to physiological changes in the systemic circulation. Furthermore, transport of water, ions and polar molecules, solutes, nutrients , macromolecules and drugs into and out of the endoneurium in physiologic and pathophysiologic states has direct relevance to peripheral nerve function in health and disease, including targeted drug delivery.There is limited knowledge on the molecular composition, and as a consequence, the mechanistic changes that occur at the human BNB during development, in healthy homeostatic states and in disease24. Animal model studies aimed at understanding peripheral nerve leukocyte trafficking in vivo should be guided by knowledge derived from human observational in situ data in order to increase biological relevance and translational potential.The BNB is a major interface of interaction between hematogenous leukocytes and peripheral nerve endoneurium, thus peripheral nerve immunosurveillance and adaptive or pathological alterations that facilitate leukocyte transmigration following injury, inflammation and in autoimmune neuropathies  requires identification of the relevant molecular componentsin vitro observational and functional studies relevant to understanding BNB angiogenesis, wound healing, response to extrinsic insult and metabolic derangements and alterations following physiological cytokine stimulus and normal/pathologic leukocyte trafficking33. However, current knowledge is incomplete as phenotypic changes may occur following primary endothelial cell isolation from endoneurial microvessels and culture compared to endoneurial endothelial cells in vivo, and immortalization may further alter the molecular composition and function of human BNB-forming endoneurial cells. Previous in situ peripheral nerve studies have identified several molecules expressed by the BNB using histochemical methods;39 however, such studies are limited by the quality of tissue preservation, availability and specificity of antibodies and other detection reagents, the clinical/scientific question being addressed, and possible ascertainment bias.Significant advances have been made following the recent isolation and successful development of primary and immortalized human endoneurial or peripheral nerve endothelial cell lines that have resulted in some in vitro with progressive culture and expressed by BNB-forming endoneurial microvessels in situ. We validated the human BNB transcriptome in situ by indirect immunohistochemistry of 31 expressed proteins performed on a histologically normal cryopreserved adult sural nerve from an individual not used for RNA-sequencing.In order to more completely elucidate the molecular composition of the normal human BNB, as required to provide an essential framework to understand endoneurial endothelial cell biology and the molecular determinants of physiologically relevant interactions between the systemic circulation and peripheral nerve endoneurium, we performed whole transcriptome shotgun sequencing or RNA-Sequencing on cultured early (P3)- and late (P8)-passage adult primary human endoneurial endothelial cells (pHEndECs) and laser-capture microdissected endoneurial microvessels from 4 cryopreserved normal adult human sural nerves to determine transcripts conserved 40 accurately identified endoneurial microvessels and epineurial macrovessels on Optimum Cutting Temperature (OCT)-preserved axial sural nerve cryostat sections  demonstrated normal axonal density Fig.  and normons Fig. . The minnce Fig. . Followince Fig.  and the nce Fig. . The minQuality (Q) Scores demonstrated a probability of base calling error for each base pair by the sequencer for each sample as <0.001 for all samples, negating the need to remove any low quality reads/bases from the datasets. cDNA from all 4 laser-capture microdissected endoneurial microvessel samples had >50% of sequenced reads align uniquely or to multiple loci of the human genome (range 50.62–85.46%) with average input read length 137 (range 123–145). P3 and P8 pHEndEC cDNA had >95% of sequenced reads align uniquely or to multiple loci of the human genome  with an input read length of 98 for both samples. For all samples, >20 million reads aligned to the human reference genome (range 22.3–40.6 million), with no chimeric reads detected.in vitro and in situ), we identified 18002 transcripts from a total of 60252 transcripts expressed by both P3 and P8 pHEndECs (the in vitro BNB [IVBNB]), with an 80.2% transcript overlap between these passages. 2122 transcripts were identified in the P3 pHEndECs only, associated by a total of 40 independent networks and subnetworks that predominantly reflected regulation of cell division and gene transcription (Supplementary Data file in situ BNB [ISBNB]).Using predefined criteria of Fragments Per Kilobase of transcript per Million (FPKM) >0.1  that are predominantly indicative of active cell division, but also indicate cytokine/chemokine binding and regulation of leukocyte trafficking networks not detected in situ  that include myelination, nervous system development, glial cell differentiation, neuronal action potential signaling and circulatory system processes. These networks implied significant exclusion of in situ contaminating Schwann cells, axons and pericytes from the deduced human BNB transcriptome , immunohistochemistry or western blot. We further validated the BNB transcriptome by demonstrating expression of 31 selected cell membrane, chemokine receptor, cytoskeletal, junctional complex and secreted proteins by endoneurial microvessels in situ by indirect fluorescent immunohistochemistry  and its tyrosine kinase component, RET , previously detected and biologically active in response to exogenous GDNF at the human BNB in vitro during restoration of restrictive barrier characteristics following diffuse injury27, were detected through this analysis. Other potentially relevant BNB transcripts that may have been lost with pHEndEC culture based on our detection criteria include ABCB4 , AQP7 , CDH19 , CELSR2 , CGN , GJC3 , SLC17A7 , SLC1A3 , SLC22A15 , SLC35F1 , SLC38A3 , and protocadherins, PCDHGC4  and PCDHGC5 .A total of 408 transcripts expressed There is a diverse repertoire of transcripts expressed by the deduced normal human BNB, supportive of an active role in homeostatic peripheral nerve function, and dispelling the notion that microvascular endothelial cells provide a relatively passive conduit for blood flow in normal healthy tissues. The repertoire of biological processes is depicted in Fig. in vitro or in situ43, provides a blueprint to determine which transporters are required for human endoneurial endothelial cell metabolism, as well as those responsible for maintaining endoneurial homeostasis by transporting essential ions, polar molecules, solutes, nutrients and macromolecules from circulating blood into the endoneurium for normal Schwann cell and axonal function, and removing metabolic waste products and xenobiotics from the endoneurium.The human BNB demonstrated 509 transporter transcripts that include members of the solute carrier transporter family (196), ATP-binding cassette transporter family (33), cation channels (76), anion channels (13), zinc transporters (14), solute carrier organic anion transporters (4) and aquaporins (3), as shown in Supplementary Data file 35. Tight junction transcripts include 6 members of the claudin family , occludin, zona occludens-1 and -2, junction adhesion molecules-1, -2 and -3, and endothelial cell adhesion molecule. Adherens junction transcripts include cadherins  and 36 protocadherins 44. Cell junction-associated or adapter transcripts include the catenin family , nectin cell adhesion molecule-1, -2 and -3, and 8 members of the membrane associated guanylate kinase family. Five gap junction channel transcripts  were also detected .The molecular composition of the intercellular junction complex provides insights to the components of a specialized restrictive microvascular barrier system in health, as required to determine alterations that may occur in disease states. We ascertained expression of 133 intercellular junctional complex transcripts, including 22 tight junction or junction-associated molecules, 45 adherens junction or junction-associated molecules, and 52 cell junction-associated or adaptor molecules by the human BNB  that serves to modulate chemokine gradients for CXCR4, implies an important autocrine effect at the human BNB, such as in vascular remodeling46. Interestingly, expression of CCL28 and one of its receptors, CCR10 by the human BNB similarly implies an autocrine role in normal endoneurial endothelial cell physiology 47, suggesting redundancy in an important microvascular biological process. CXCR5 48 is also expressed by the human BNB.We determined which chemokines and chemokine receptors may have homeostatic roles during immunosurveillance at the human BNB or actively participate in innate immune responses. These include CCL14 (induces enzyme release from monocytes), CCL2 (monocyte/macrophage and T-cell trafficking), CX3CL1 , CXCL16 , and CXCL3 (neutrophil trafficking)in vitro that were not observed in situ. Transcripts detected in vitro only include chemokines involved in neutrophil trafficking , T-helper 1 cell trafficking , T-helper 2 cell trafficking , T-helper 17 trafficking (CCL20), T-cell, and monocyte/macrophage trafficking as part of the innate and adaptive immune responses  and CCL16 implicated in dendritic cell maturation46. Atypical chemokine receptor CCRL2  is also expressed by the IVBNB. It is possible that these endothelial cells are activated by in vitro culture resulting in increased chemokine gene transcription or there is some chemokine system dysregulation in situ following nerve biopsy and cryopreservation precluding detection and inclusion as part of the normal human BNB. Nonetheless, these data support an active role of the human BNB in hematogenous leukocyte interactions in peripheral nerves, as previously implicated50.As  stated previously, chemokine binding and regulation of leukocyte trafficking networks were detected c fragment of IgG receptor and transporter) which binds to the monomeric region of IgG to facilitate its transcytosis to the apical or basolateral membrane of endothelial cells51 by the human BNB. However, PIGR (polymeric immunoglobulin receptor: binds dimeric IgA and pentameric IgM)51 was not expressed, suggesting that these large molecular weight antibody subclasses do not undergo human BNB transport in normal nerves or do so via undefined non-receptor-mediated transcytosis pathways. It remains to be determined whether adaptive or pathologic changes in FCGRT or PIGR expression occur at the human BNB with different disease states to modulate immunoglobulin transport between the blood circulation and the endoneurium.Of further clinical relevance is the transport of circulating immunoglobulins (pathogenic or therapeutic) across the tight junction-forming human BNB. We observed transcript expression of FCGRT  was used rather than the more recently released Reference 38 (hg38) as hg19 is currently the most widely accepted genome to align human data to, with most of the current human genome databases utilizing hg19 coordinates.27. Variations in pHEndEC shape and diameter with differences in cytoplasm-to-nucleus ratio in culture had been observed based on the proliferating state7. By selecting transcripts expressed by both P3 and P8 pHEndECs to define the IVBNB transcriptome, we sought to identify transcripts that persisted with continuous cell culture and eliminate induced transcript expression as a direct consequence of recent endothelial cell isolation from peripheral nerves that required ex vivo manual endoneurial bundle stripping, aggressive enzymatic digestion and density gradient centrifugation, as well as the effects of pHEndEC de-differentiation that may occur as a consequence of extended in vitro culture.pHEndECs isolated and purified from a single donor were used, and previous work demonstrated a ~95% purity at P3, using indirect fluorescent immunohistochemistry and flow cytometry, with loss of contaminating pericytes and fibroblasts with continued culture using our defined culture conditions that involved seeding these endothelial cells at 10,000–15,000 cells/cmBy selecting transcripts expressed in endoneurial microvessels from three of the four normal adult sural nerves, we sought to include conserved transcripts in unrelated individuals and reduce the effect of contaminating luminal or migrating leukocytes, pericytes that share a basement membrane with endothelial cells and myelinated axons that are commonly reside ~5–10 μm away from endoneurial microvessels as shown on glutaraldehyde-fixed, osmium tetroxide post-fixed, plastic-embedded sections Fig. . A high in vitro and in situ. The validity of approach is further evidenced by the inclusion of transcripts that were previously demonstrated at the BNB in vitro or in situ by PCR, immunohistochemistry or western blot, our indirect fluorescent immunohistochemistry validation of endoneurial microvessel protein expression in situ, evaluating transcripts with mean FPKM values between 0.8 and 750.5 in situ and 0.4 and 3012.4 in vitro using another histologically normal adult cryopreserved sural nerve biopsy, as well as uniformly expressed microvascular endothelial specific transcription factor orthologs (i.e. vascular identity factors) previously observed in mice, including cerebral microvascular endothelial cells42. We also identified a few additional transcripts, such as the previously validated GFRA1 and its receptor tyrosine kinase RET27, intercellular junctional complex molecules and transporters that were expressed by laser-capture microdissected endoneurial microvessels in situ that were lost with pHEndEC culture. It is important to recognize that some “classic” markers of pericytes, Schwann cells and fibroblasts are known to be expressed by vascular endothelial cells, emphasizing the importance of using biological networks and subnetworks deduced from PANTHER analysis to aid identify cell types based on known or expected biological function55.Taking into account our definition of the human BNB transcriptome, the probability that transcripts identified as the IVBNB (18002/60252) and ISBNB (15375/60252) occur in both independently derived transcriptomes is 0.076 [(18002/60252) x (15375/60252)], supporting our notion that the identified transcripts are highly conserved by normal adult endoneurial endothelial cells in vitro.This comprehensive list of transcripts and the associated networks provides the framework for biologically relevant studies on human BNB angiogenesis during peripheral nerve development and following injury, formation of restrictive intercellular junctions and adaptations under normal physiological and pathophysiological states, hydraulic conductivity  in response to changes in hydrostatic pressure and endoneurial interstitial volume and the transport kinetics of ions, polar molecules, solutes, nutrients, macromolecules (such as immunoglobulins) and drugs/ xenobiotics into and out of the peripheral nerve endoneurium in health and disease. Hypothesis-driven studies relevant to leukocyte-endothelial interactions unique to the human BNB during immunosurveillance would be guided by the established transcriptome profile of chemokines and other constitutively expressed cellular adhesion molecules. Furthermore, direct transcript database comparison of the human BNB transcriptome with restrictive and non-restrictive barrier-forming human microvascular endothelial cells, including the blood-brain barrier, would aid determine transcripts and specific biological networks potentially unique to the human BNB for functional assessment using pHEndECs Our overall strategy makes the assumption that any rare contaminating Schwann cells, pericytes, axons and endoneurial macrophages and fibroblasts present following pHEndEC isolation that may exist in early passage cultures would be lost with continuous culture in endothelial cell growth supporting conditions. Our data analysis showed 89.4% of transcripts expressed by P3 pHEndECs and 88.6% of transcripts expressed by P8 pHEndECs were expressed by both and included in the IVBNB transcript database, attesting to the fact that there was relatively little change in transcript expression between passages 3 and 8. This observation does not support preferential proliferation of contaminating cells such as pericytes with extended pHEndEC culture. Excluded transcripts were predominantly associated by networks involved in regulation of cell division and gene transcription (P3) and de-differentiation (P8).in situ, resulting in their inclusion as part of the human BNB transcriptome. In situ RNA degradation in OCT-cryopreserved sural nerves and subsequent degradation following endoneurial laser capture microdissection could have occurred, resulting in a failure to completely identify transcripts expressed by purified viable endoneurial endothelial cells in vitro. Another question is whether there is appreciable microvascular endothelial cell heterogeneity within the same or different parts of the peripheral nerve endoneurium. The sural nerve is one of the terminal branches of the sciatic nerve with its fascicles in direct continuation, so we do not necessarily expect differences in BNB molecular composition. We did not attempt to evaluate BNB diversity between normal adult nerves in this study; however deduction of the normal human adult BNB transcriptome provides a framework for future comparative studies. It is also quite possible that some of the expressed protein coding transcripts are not translated, or expressed in the presumed cellular compartments at the human BNB in vivo. The overall aim was to establish a comprehensive molecular signature of the normal human BNB, providing an essential blueprint or reference guide for future translationally relevant peripheral nerve biology studies in health and disease.We cannot exclude the possibility that cultured pHEndECs could have acquired some non-vascular transcripts due to early de-differentiation that overlap with these contaminating cells Targeting drug delivery across restrictive tight junction-forming microvascular barriers, such as the blood-brain barrier, and target site tissue retention at physiologically active concentrations have become essential considerations in therapeutic drug design strategies. This has implications for effective local and systemic drug administration targeting normal (e.g. anesthesia) and abnormal peripheral nerves  with limited off-target or unwanted side effects. Knowledge of the components and regulators of small molecule and macromolecular transport unique to the human BNB would aid medicinal chemists and pharmacologists develop drugs with high BNB influx permeability, utilizing the array of influx transporters, channels and receptor-mediated transcytosis components to aid drug design, with specific modifications made to modulate drug efflux in the opposite direction as a means of increasing peripheral nerve endoneurial bioavailability and efficacy.Detailed knowledge of the molecules relevant for BNB angiogenesis and intercellular junction complex formation could guide therapeutic strategies for peripheral nerve repair following traumatic injury, recognizing that rapid restoration of the normal endoneurial microenvironment may be an essential prerequisite for axonal regeneration and remyelination. Incorporation of specific essential vascular growth and differentiation factors in peripheral nerve grafts/conduits that facilitate BNB formation is an important consideration with translational potential to the clinic guided by the human BNB transcriptome.Similarly, this study also provides essential information on the possible determinants of leukocyte trafficking during normal immunosurveillance and the biological networks that may be implicated in peripheral nerve innate and adaptive immune responses. This knowledge could provide insights into understanding the mechanisms of human BNB response to injury, microbial entry from the bloodstream into peripheral nerves and response to viral infections. Our work should also guide studies designed to understand the interactions between the systemic immune compartment and peripheral nerves relevant to understanding the pathogenesis and targeted treatment of peripheral nerve-restricted autoimmune disorders such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy. It is hoped that the deduced human BNB transcriptome and its associated networks and subnetworks will stimulate hypothesis-driven mechanistic studies that would significantly increase our understanding of specialized restrictive barrier-forming human microvascular endothelium.OCT-cryopreserved sural nerve biopsies stored at −80 °C from 4 adults (2 men and 2 women) with histologically normal peripheral nerves defined as no visually detectable structural abnormalities on routine light microscopy with no known history of systemic or neurological disorders were obtained from the Shin J Oh Muscle and Nerve Histopathological Laboratory, University of Alabama at Birmingham. Informed consent for clinical and research purposes was obtained from every patient undergoing biopsy. The study was approved by the University of Alabama at Birmingham Institutional Review Board, with an exemption obtained to use archived pathological specimens for research (Protocol Number X140321012). All specimen handling and experiments were performed in accordance with relevant guidelines and regulations as stipulated by the University of Alabama at Birmingham. To reduce the impact of RNase activity, RNase-free reagents were utilized whenever available, and all materials and surfaces including tissue slides, cryotome blades and blade holder were cleaned with RNaseZap (Invitrogen) and 70% ethanol prior to sectioning.56.20 μm thick axial cryostat sections were transferred to polyethylene naphthalate (PEN) membrane glass slides  and stored in 50 mL centrifuge tubes at −80 °C for no longer than 10 days. In order to detect endoneurial microvessels, slides were fixed for 2 minutes in 95% ethanol, rinsed twice for 30 seconds in RNase-free phosphate buffered saline , and stained with 500 μL of 100 μg/mL FITC conjugated Ulex Europaeus Agglutinin (UEA)-1  in RF-PBS for 5 minutes. Slides were washed five times for 10 seconds each in RF-PBS and dehydrated in 70% ethanol for 30 seconds, 95% ethanol for 30 seconds, 100% ethanol for 1 minute and xylene (≥99% pure) for 5 minutes, adapting a protocol used for cerebral microvesselsUEA-1 FITC-positive endoneurial microvessels were imaged, annotated, and captured using the Arcturus XT system equipped with CapSure Macro laser-capture microdissection [LCM] Caps . Epineurial and perineurial macrovessels were excluded. Capture was performed using the following infrared (IR) settings: IR spot spacing = 80; IR spot diameter = 6; IR spot power = 80; IR spot duration = 50; IR spot/cut length = 500; tab length = 0; with ultraviolet (UV) cut length = 1500; and UV cutting speed = 1000. Additional xylene dehydration was required to reduce high background fluorescence on some slides prior to annotation and capture. Following capture, each cap was inspected for inappropriately captured tissue. Large extraneous areas were gently removed with tape, while smaller areas were ablated with the laser. Caps were placed in a 0.5 mL microcentrifuge tube with 50 μL RNA extraction buffer, inverted, and stored on dry ice or at −80 °C until RNA isolation. Unless otherwise stated, all reagents were from Thermo Fisher Scientific .Similarly, 20 μm thick axial cryostat sections were also transferred to polyphenylene sulfide (PPS) membrane slides  for LCM using a Leica Microsystems LMD6 scope and stored at −80 °C for no longer than 10 days. To identify endoneurial microvessels, slides were fixed for 2 minutes in 70% ethanol, and washed for 30 and 60 seconds in RF-PBS. Slides were stained 350 μL of 40 μg/mL UEA-1 FITC in RF-PBS for 10 minutes, washed for 30 and 60 seconds in RF-PBS, dehydrated for 2 minutes in 70% ethanol followed by 30 seconds in 100% ethanol, and air dried for 5 minutes. Endoneurial microvessels were microdissected using the Leica system using the “Move and Cut” mode over a 30 min period according to the manufacturer’s instructions, excluding epineurial and perineurial macrovessels. Laser alignment was tested prior to capture for each slide used. The LMD6 system UV (355 nm) laser uses a pulse frequency of 80 Hz, pulse length of < 4 ns and maximum pulse intensity of 70 μJ, with dissected material dropping into the collection tube cap by gravity. Sample humidity levels were controlled by limiting drying times and excessive static was alleviated by dipping slides in 70% alcohol. Following capture, 50 μL of Qiazol  was added to well of cap, the tube was closed and incubated inverted at room temperature for 5 minutes, and stored on dry ice or −80 °C until RNA isolation.RNA was isolated from endoneurial microvessels dissected with the Arcturus XT system using the Arcturus Pico Pure Isolation Kit (Thermo Fisher Scientific), according to the manufacturer’s directions. Caps were heated at 42 °C for 35 minutes (using Pico Pure extraction buffer (XB)) for 15 minutes with membrane removal using RNaseZap-treated forceps and placed in bottom of tube in XB for additional 20 minutes. Following RNA binding to the column, an on-column DNase digestion for 15 minutes using 80 μL DNase Mixture  was performed to remove contaminating genomic DNA. RNA was eluted using 20 μL RNase-free water supplied with the kit. RNA from endoneurial microvessels dissected with the Leica system was extracted using the miRNeasy kit , according to the manufacturer’s instructions. On-column DNase digestion was also performed to remove contaminating genomic DNA using the RNase-free DNase Set . Isopropanol-containing RNA was eluted using RNase-free water. Extracted RNA was stored at −80 °C prior to further use.7. pHEndECs were expanded on rat-tail collagen-coated Coring CellBIND® flasks or Petri dishes in freshly prepared growth medium consisting of RPMI-1640, 10% NuSerum, 10% fetal bovine serum, 1% vitamin solution, 1% non-essential amino acid solution, 1% sodium pyruvate, 1% penicillin-streptomycin, 2 mM L-glutamine, 10 mM HEPES buffer, and enriched with 50 μg/mL endothelial cell growth supplement, 1 ng/mL human recombinant basic fibroblast growth factor and 10 U/mL heparin, from the same vendors. pHEndECs were passaged at 80–90% confluence, and allowed to grow to complete confluence at early- (P3) and late- (P8) passages before total RNA was extracted 24 hours later using TRIzol reagent according to the manufacturer’s instructions, as previously published27. Extracted RNA was stored at −80 °C prior to further use.Primary human endoneurial endothelial cells (pHEndECs) were previously isolated and purified from the sciatic nerves of a decedent 38 year old woman with no known history of systemic or neurological disease via endoneurial stripping, and sequential enzymatic digestion and density centrifugation, and cultured as publishedRNA yield and quality were determined on an Agilent 2100 BioAnalyzer using the Pico Chip . RNA obtained from a minimum of 200 laser-capture microdissected endoneurial microvessels per adult nerve were pooled and concentrated on a Savant SpeedVac  or used as extracted from P3 and P8 pHEndECs. cDNA libraries were generated using the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian  and ribosomal cDNA was depleted prior to the final amplification step as per the manufacturer’s instructions. High Sensitivity DNA electropherogram traces were captured on the BioAnalyzer to confirm the quality of the RNA-Seq Libraries. The final libraries were quantified by quantitative PCR . Equal nanomolar amounts of libraries were mixed prior to loading on to the sequencer.http://www.pantherdb.org/)57.Next Generation Sequencing was performed by the UAB Heflin Center Genomics Core using the Illumina NextSeq. 500 (100 Gb/flowcell) Sequencing Platform setup to capture 75 base pairs (bp), paired end sequences  and 50 bp, paired end sequences (for pHEndECs), as per the manufacturer’s standard protocols (Illumina Inc. San Diego CA). Quality (Q) Scores  were determined for each base pair from each sample (in duplicate). Sequence alignment was performed using STAR program referenced to Genome Reference Consortium Human Reference 37 (hg19). Transcript annotation was performed using PANTHER . FPKMs were generated using Cufflinks and Cuffdiff software programsin vitro BNB (IVBNB) was defined as transcripts expressed by both P3 and P8 pHEndECs. The in situ BNB (ISBNB) was defined as transcripts expressed in at least 3 out of 4 adult sural nerve biopsy laser-capture microdissected endoneurial microvessel samples. The human BNB transcriptome was defined as transcripts expressed by both the IVBNB and ISBNB. These common transcripts should exclude pericyte, Schwann cell, fibroblast, leukocyte and axonal transcripts that may contaminate early passage pHEndECs or laser-capture microdissected endoneurial microvessels. To identify possible transcripts expressed by the human BNB in situ that may be lost with pHEndEC culture, transcripts expressed by the ISBNB and P3 pHEndECs were also identified. The average IVBNB and ISBNB FPKMs for each transcript was used for hierarchical clustering. Transcript overlaps between the P3 and P8 pHEndECs, and the IVBNB and ISBNB were generated using custom scripts. Cluster 3.0 and JAVA Treeview software packages were used for hierarchical clustering and heatmap generation respectively61.The 62. Lists of transporters, junctional complex components and chemokines/chemokine receptors expressed by the human BNB were generated by referencing PANTHER generated functional classification lists based on gene ID against the database of all identified human BNB transcripts and recently published references on these gene subfamilies to ensure completeness69.The human BNB transcriptome functional classification was performed using PANTHER. This program performs pathway enrichment analyses referenced to GO ontology and utilizes Fisher’s one-tailed test  with its specific multiple testing correction algorithm (g:SCS) to determine the probability that genes associated by a specific network/subnetwork occur by random chance. Lower p-values are generally indicative of higher odds that the assigned association is important and reveals relevant biological information. Statistical significance is set at p ≤ 0.05. Pie charts and bar histograms were generated to demonstrate BNB biological function, cellular component and protein class GO-terms distribution and quantityin situ by indirect fluorescent immunohistochemistry on 10 μm thick cryostat sections of a histologically normal cryopreserved sural nerve biopsy from a 44 year old woman using an array of 31 adhesion, cell membrane, chemokine receptor, cytoskeletal, junctional complex , secreted and transporter proteins with mean transcript FPKM values varying between 0.8 and 750.5 in situ and 0.4 and 3012.4 in vitro  or goat anti-rabbit IgG (H + L) Alexa Fluor® 594 conjugate  with UEA-1-FITC  in 2% NGS in 1X PBS for 1 hour at room temperature in the dark. Following a final wash, all sections were stained with 0.45 μM 4, 6-diamidino-2-phenylindole (DAPI) for 5 minutes to detect nuclei and mounted with ProLong® Gold antifade mounting medium . Digital photomicrographs were taken using an Eclipse Ci-S Upright epifluorescent microscope with a D5-Qi2 camera  and merged using the Nikon Elements software.Consecutive 10 μmthick axial cryostat sections were fixed in acetone at −20 °C for 5 minutes, washed with 1X Phosphate Buffered Saline (PBS) and air-dried at room temperature for 5 minutes prior to blocking with 10% normal goat serum (NGS) in 1X PBS for 30 minutes. Without washing, each slide was incubated with a single primary antibody diluted in 2% NGS in 1X PBS for 1 hour at room temperature. Following washing, slides were incubated with either goat anti-mouse IgG (H + L) Alexa Fluorhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107574). pHEndECs may be obtained through an MTA. Detailed isolation protocols have been previously published.The human blood-nerve barrier transcriptome data (including Supplementary Data file S3) have been deposited in National Center for Biotechnology Information's Gene Expression Omnibus and are accessible through GEO Series accession number GSE107574 (Supplementary informationDataset 1Dataset 2Dataset 3Dataset 4Dataset 5Dataset 6Dataset 7Dataset 8


Quantum dots are known to confine electrons within their structure. Whenever they periodically aggregate into arrays and cooperative interactions arise, novel quantum properties suitable for technological applications show up. Control over the potential barriers existing between neighboring quantum dots is therefore essential to alter their mutual crosstalk. Here we show that precise engineering of the barrier width can be experimentally achieved on surfaces by a single atom substitution in a haloaromatic compound, which in turn tunes the confinement properties through the degree of quantum dot intercoupling. We achieved this by generating self-assembled molecular nanoporous networks that confine the two-dimensional electron gas present at the surface. Indeed, these extended arrays form up on bulk surface and thin silver films alike, maintaining their overall interdot coupling. These findings pave the way to reach full control over two-dimensional electron gases by means of self-assembled molecular networks. Arrays of quantum dots can exhibit a variety of quantum properties, being sensitive to their spacing. Here, the authors fine tune interdot coupling using hexagonal molecular networks in which the dots are separated by single or double haloaromatic compounds, structurally identical but for a single atom. They aggregate to form QD solids whose final properties are based upon their cooperative interaction, suitable for many technological applications5. Ideal QD solids demand truly monodisperse building blocks to prevent undesirable anomalies6, but real ones exhibit significant structural variations. Digital structural fidelity is achieved on surfaces through atom-by-atom7 and molecular manipulation2 or by self-assembled molecular nanoporous networks10. Control of the potential barriers between neighboring QDs is essential to alter the crosstalk (interaction) between the existing units and engineer two-dimensional electron gases (2DEG)15.Quantum dots (QDs) are analogous to artificial atoms as they confine electrons with discrete energy levels16 that induce confinement of scattered surface 2DEG electrons inside its nanocavities15. Such regular nanostructures can be conceived as QD arrays17, where the surface adsorbed molecules (and adatoms) change the local surface potential landscape20. The confining barriers are characterized by a certain width and amplitude that affect the neighboring QDs' coupling degree17. Electronic engineering of 2DEGs can be achieved by tuning the molecular building blocks, thereby altering the QD dimensions, the barrier amplitude and/or the barrier width20. Moreover, changing the molecular compounds may also modify their interaction with the substrate12, consequently altering the QD electron confinement strength. Whenever long-range regular structures are achieved, the surface 2DEG is modulated thereby generating new electronic bands whose dispersion relates to its interpore coupling strength and array dimensions13. This long-range periodicity is desired for implementation into devices, if bottom-up fabrication methods are used. However, the fine control in terms of the interpore distance (barrier width), while maintaining the potential barrier and pore dimensions is still elusive. In an ideal case where only the barrier width is disrupted, two paths can be envisioned to widen the interpore walls: the first, changing the length/width ratio of the molecule  and, the second, laterally stacking different number of constituents .These self-assembled two-dimensional (2D) nanoporous networks are periodic extensions of quantum corralsIn this work, we target this second path by means of two haloaromatic compounds that differ just in a single atom in their structure. Two different hexagonal molecular networks on a Ag(111) substrate are self-assembled that show single-molecular and double-molecular separation between their identical pores. The communication between neighboring QDs is investigated following the 2DEG modification through a combination of scanning tunneling microscopy/spectroscopy (STM/STS), angle resolved photoemission spectroscopy (ARPES) and extended model calculations.21 and furan (Br-DNF). Intermolecular electrostatic attraction between the positive cap (σ hole) and the electron-rich regions  are ress22 Fig.  show ext23. The pores of the Br-DNT network are separated by a single molecule  with a CO functionalized tipule Fig. , whereasule Fig. . Note thule Fig. . The conI/dV) spectra acquired at the center of the pores of SW and DW networks and referenced to the clean Ag(111) substrate. Clear energy shifts of the pristine surface state onset (−65 meV) are induced by the QD confinement, peaking at 72 meV for the SW network and 45 meV for the DW network. These values are unexpectedly inverted since a stronger confinement (higher peak energy) is anticipated for the wider barrier (DW). It could be argued that it originates from a difference in the molecule-substrate interaction affecting the potential amplitude20. However, a larger interaction is expected for Br-DNF due to the extra oxygen electronegativity and the measured full width at half maximum of the confined state for the DW network (32 meV) is narrower than that for the SW network (45 meV). Moreover, the conductance maps at the peak energies show high conductance regions mainly located within the pores  become undetectable. In order to push both confined STS peaks into the occupied region, we grew them onto a 3 monolayer (ML) Ag thin film on Au(111), which shifts the Ag(111) surface state by −100 meV while preserving its 2DEG character . The network band periodicities relate to their interpore distances and match our DFT calculations and STM data  for the molecules  and constant non-zero potential (Veff) for the molecular positions  that scatter the electrons. The m∗ and Veff parameters are determined by an iterative fitting using both STS and ARPES data 19. The fundamental energy is established by the bonding state and the overall bandwidth  is limited by the anti-bonding ones. The reduced bandwidth of the DW network compared to the SW . Consequently, the peak lineshapes are generally asymmetric with maxima displaced towards the top of the band . These organic nanoporous networks are generated on bulk and thin Ag films alike by substitution of a single atom in the precursor molecule, reminiscent of a butterfly effect. The extended and periodic nature of these arrays provides access to their distinct band structures, which are directly compared with their local density of states and merged through calculations. Such complementary experimental and theoretical synergy provides complete fundamental insight into the nature of QD intercoupling processes. Even though substrate contributions cannot be discarded, our findings clearly suggest that the reduction of the QD coupling (from SW to DW) is associated with a flattening of the band dispersion and increase of the effective mass. This work not only complements the toolbox for tuning surface electronic properties, which started in the 90’s with the quantum corrals31, operating at 4.8 K in UHV. A clean Ag(111) surface was in-situ prepared by repeated cycles of standard sputtering and annealing. The W tip of a tuning fork sensor was ex-situ sharpened by focused ion beam milling technique and was then in-situ covered with Ag atoms by contacting to the sample surface.All experiments were performed with Omicron STM/AFM with a qPlus configuration21 and 3,9-dibromodinaphthofuran (Br-DNF) α (hν = 21.2 eV) source. The channel plate slit lies along the rotation axis of the manipulator. All the presented data were recorded approximately at 130 K.Our home laboratory angle resolved photoemission (ARPES) setup consists of a display type hemispherical analyzer (Phoibos150) with an energy/angle resolution of 40 meV/0.1° and a monochromatized source He I32. Afterwards, the sample was heated up to ~450 K to improve the surface quality. Br-DNT and Br-DNF were deposited on the substrate at ~130 K. After each preparation step, we controlled the sample quality by measuring its electronic structure.A clean Au(111) surface was in-situ prepared by repeated cycles of standard sputtering and annealing. To form Ag films of controlled monolayer thickness on Au(111), a wedge-like mask was positioned in front of the substrate and was moved slowly during the Ag deposition at 150 KThe combined Plane Wave Expansion (PWE) and Electron Boundary Element Method (EBEM) have been developed by García de Abajo and represents a scalar variant of the electromagnetic PWE/BEM extensively used for solving Maxwell’s equations and optical response for arbitrary shapes. It is based on Green’s functions for finite geometries and electron plane wave expansion for periodic systems. For the band structure calculations, the particle-in-a-box model is extended to infinite 2D systems by defining an elementary cell and using periodic boundary conditions. Within the PWE code, solutions of the Schrödinger equation are represented as a linear combination of plane waves and a satisfactory convergence was achieved with a basis set consisting of ~100 waves.33. A wavelet basis set was used to expand the wavefuntion of the valence electrons while the core electrons were removed using norm-conserving HGH pseudopotentials34. Calculating the electrostatic potential (and the electric field) for a surface system is uniquely precise by the Poisson solver of this DFT code that allows to apply periodic boundary conditions along two in-plane directions while keeping free boundary conditions out of the plane direction35.The calculations were carried out within the local density approximation of density functional theory (DFT) as implemented in the BigDFT codeThe data that support the findings of this study are available from the corresponding authors on request.transparent peer review FileSupplementary info