The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% D TEM range: 79.5–134.3) compared to ZetaView (% D TEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.

The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

Efficient neuronal conductance requires that axons are insulated by glial cells. For this, glial membranes need to wrap around axons. Invertebrates show a relatively simple extension of glial membranes around the axons, resembling Remak fibers formed by Schwann cells in the mammalian peripheral nervous system. To unravel the molecular pathways underlying differentiation of glial cells that provide axonal wrapping, we are using the genetically amenable Drosophila model. At the end of larval life, the wrapping glia differentiates into very large cells, spanning more than 1 mm of axonal length. The extension around axonal membranes is not influenced by the caliber of the axon or its modality. Using cell typespecific gene knockdown we show that the extension of glial membranes around the axons is regulated by an autocrine activation of the EGF receptor through the neuregulin homolog Vein. This resembles the molecular mechanism employed during cell-autonomous reactivation of glial differentiation after injury in mammals. We further demonstrate that Vein, produced by the wrapping glia, also regulates the formation of septate junctions in the abutting subperineurial glia. Moreover, the wrapping glia indirectly controls the proliferation of the perineurial glia. Thus, the wrapping glia appears center stage to orchestrate the development of the different glial cell layers in a peripheral nerve.

Graphical Abstract Highlights d Septate junction (SJ) proteins are stable for days d SJ strands are prefigured during embryogenesis and are unfolded during larval stages d The G-protein coupled receptor Moody suppresses interdigitating cell-cell protrusions d Moody promotes formation of continuous SJ strands SUMMARYThe blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. Here, we used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. We show that septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish.