Neurexin and neuroligins jointly regulate synaptic degeneration at the Drosophila neuromuscular junction based on TEM studies.

The Drosophila larval neuromuscular junction (NMJ) is a well-known model system and is often used to study synapse development. Here, we show synaptic degeneration at NMJ boutons, primarily based on transmission electron microscopy (TEM) studies. When degeneration starts, the subsynaptic reticulum (SSR) swells, retracts and folds inward, and the residual SSR then degenerates into a disordered, thin or linear membrane. The axon terminal begins to degenerate from the central region, and the T-bar detaches from the presynaptic membrane with clustered synaptic vesicles to accelerate large-scale degeneration. There are two degeneration modes for clear synaptic vesicles. In the first mode, synaptic vesicles without actin filaments degenerate on the membrane with ultrafine spots and collapse and disperse to form an irregular profile with dark ultrafine particles. In the second mode, clear synaptic vesicles with actin filaments degenerate into dense synaptic vesicles, form irregular dark clumps without a membrane, and collapse and disperse to form an irregular profile with dark ultrafine particles. Last, all residual membranes in NMJ boutons degenerate into a linear shape, and all the residual elements in axon terminals degenerate and eventually form a cluster of dark ultrafine particles. Swelling and retraction of the SSR occurs prior to degradation of the axon terminal, which degenerates faster and with more intensity than the SSR. NMJ bouton degeneration occurs under normal physiological conditions but is accelerated in Drosophila neurexin (dnrx) dnrx273, Drosophila neuroligin (dnlg) dnlg1 and dnlg4 mutants and dnrx83;dnlg3 and dnlg2;dnlg3 double mutants, which suggests that both neurexin and neuroligins play a vital role in preventing synaptic degeneration.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae.

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy.

The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. Here, we reported an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that was about total 80 microns thickness, and 30-40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain.

Comparative Morphology of the Lungs and Skin of two Anura, Pelophylax nigromaculatus and Bufo gargarizans.

The lungs and skin are important respiratory organs in Anura, but the pulmonary structure of amphibians remains unclear due to the lack of a suitable procedure. This study improved the procedure used for fixing lungs tissues and used light microscopy, transmission electron microscopy and scanning electron microscopy to reveal the differences in the lung and skin morphologies between Pelophylax nigromaculatus (P. nigromaculatus) and Bufo gargarizans (B. gargarizans). In P. nigromaculatus and B. gargarizans, the cystic lungs comprise a continuous outer pulmonary wall on which primary, secondary, and tertiary septa attach, and a number of regular lattices form from raised capillaries and the pulmonary epithelium on the surfaces of the pulmonary wall and septa. Each lattice in P. nigromaculatus consists of several elliptical sheets and flat bottom, and the septa are distributed with denser sheets and have a larger stretching range than the pulmonary wall. The lattice in B. gargarizans consists of thick folds and an uneven bottom with several thin folds, and the septa have more developed thick and thin folds than the pulmonary wall. However, the density of the pulmonary microvilli, the area of a single capillary, the thicknesses of the blood-air barrier, pulmonary wall and septum, and the lung/body weight percentage obtained for B. gargarizans were higher than those found for P. nigromaculatus. In P. nigromaculatus, the dorsal skin has dense capillaries and a ring surface structure with mucus layer on the stratum corneum, and the ventral skin is slightly keratinized. In B. gargarizans, the stratum corneum in both the dorsal and ventral skins is completely keratinized. A fine ultrastructure analysis of P. nigromaculatus and B. gargarizans revealed that the pulmonary septa are more developed than the pulmonary walls, which means that the septa have a stronger respiratory function. The more developed lungs are helpful for the adaptation of B. gargarizans to drought environments, whereas P. nigromaculatus has to rely on more vigorous skin respiration to adapt to a humid environment.

Neurexin and Neuroligins Maintain the Balance of Ghost and Satellite Boutons at the Drosophila Neuromuscular Junction.

Neurexins and neuroligins are common synaptic adhesion molecules that are associated with autism and interact with each other in the synaptic cleft. The Drosophila neuromuscular junction (NMJ) bouton is a well-known model system in neuroscience, and ghost and satellite boutons, respectively, indicate the poor development and overgrowth of the NMJ boutons. However, the Drosophila neurexin (DNrx) and Drosophila neuroligins (DNlgs) are mainly observed in type Ib boutons, indicating the ultrastructural and developmental phenotypes of the Drosophila NMJ. Here, we identified the ultrastructural and developmental features of ghost and satellite boutons by utilizing dneurexin (dnrx) and dneuroligins (dnlgs) fly mutants and other associated fly strains. Ghost boutons contain synaptic vesicles with multiple diameters but very rarely contain T-bar structures and swollen or thin subsynaptic reticulum (SSR) membranes. The muscle cell membrane is invaginated at different sites, stretches to the ghost bouton from different directions, forms several layers that enwrap the ghost bouton, and then branches into the complex SSR. Satellite boutons share a common SSR membrane and present either a typical profile in which a main bouton is encircled by small boutons or two atypical profiles in which the small boutons are grouped together or distributed in beads without a main bouton. Electron and confocal microscopy data showed that dnrx, dnlg1, dnlg2, dnlg3, and dnlg4 mutations led to ghost boutons; the overexpression of dnrx, dnlg1, dnlg2, dnlg3, and dnlg4 led to satellite boutons; and the dnlg2;dnlg3 double mutation also led to satellite boutons. These results suggested that DNrx and DNlgs jointly maintain the development and function of NMJ boutons by regulating the balance of ghost and satellite boutons in Drosophila.

Different morphologic formation patterns of dark patches in the black-spotted frog (Pelophylax nigromaculata) and the Asiatic toad (Bufo gargarizans).

The black-spotted frog (Pelophylax nigromaculata) and Asiatic toad (Bufo gargarizans), two relatively distantly related species, live in different habitats with different adaptive dark patches. To explain the formation of dark patches, the distribution patterns of melanin granules were examined with light microscopy and transmission electron microscopy. Melanin granules were produced and gathered into the "cap" structures on top of the nuclei in most epidermal cells. The "cap" structures may play a role in forming the dorsal dark patches coupled with three-layer melanophores, which can give rise to three layers of interconnected melanin networks in the dorsal dermis in P. nigromaculata. Epidermal melanocytes are rare and do not have a definitive role in forming dorsal dark patches in either P. nigromaculata or B. gargarizans. In B. gargarizans, the dermal melanophores only give rise to a single-layered melanin network, which hardly results in dark patches in the dorsal skin. However, the dermal melanophores migrate twice and form into pseudostratified networks, leading to dark patch formation in the ventral skin in B. gargarizans. The melanin granules precisely coregulate dark patches in the dermis and/or epidermis in P. nigromaculata and B. gargarizans. The dark patch formation depends on melanin granules in the epidermis or/and dermis in P. nigromaculata and B. gargarizans.