Ultrastructural study of the in vitro interaction between Biomphalaria glabrata hemocytes and Schistosoma mansoni miracidia

Biomphalaria glabrata and Schistosoma mansoni relationship was studied by light microscopy (LM) and freeze-fracture replica technique (FFR). We observed very thin cytoplasmic extensions of hemocytes in the LM, which then surround immobilize the miracidia. FFR images showed that the contact site between hemocytes cytoplasmic extensions and the external tegumentary coat involved only superficial layers of miracidia. Numerous vacuoles and filopodia were observed in the hemocyte cytoplasm, the latter binding with those from neighboring cells. In spite of the close interfilopodia contact, no cellular junctions were seen at these sites nor between filopodia-miracidia contact areas. The observed migration of hemocytes and their disposition in layers surrounding the miracidia in vitro correspond to previous studies.     

The Fine Structure of Nerve Cells and Fibers, Neuroglia, and Sheaths of the Ganglion Chain in the Cockroach (Periplaneta americana)

The abdominal nerve cord of Periplaneta americana was studied utilizing light and electron microscopes. In the nerve cells, delicate granules, similar to those probably responsible for cytoplasmic basophilia, are evenly distributed in "dark" cells and clumped in "light" cells. Neuroglial cells are stained metachromatically by cresyl violet. The neuroglial cells have many processes which ramify extensively and are enmeshed to form overlapping layers. These imbricated processes ensheath the nerve cells; the inner layer of the sheath penetrates into the neuron and is responsible for the appearance of the trophospongium of Holmgren. Nerve fibers are embedded within glial cells and surrounded by extensions of the plasma membrane similar to mesaxons. Depending on their size, two or several nerve fibers may share a single glial cell. Nerve fibers near their terminations on other nerve fibers contain particles and numerous, large mitochondria. The ganglion is ensheathed by a thick feltwork of connective tissue and perilemmal cells. The abdominal connective has a thinner connective tissue sheath which is without perilemmal cells. The nerve fibers and sheaths in the connective become thinner as they pass through ganglia.     

The fine structure of nerve cells and fibers,neuroglia, and sheaths of the ganglion chain in the cockroach (Periplaneta americana)

The abdominal nerve cord of Periplaneta americana was studied utilizing light and electron microscopes. In the nerve cells, delicate granules, similar to those probably responsible for cytoplasmic basophilia, are evenly distributed in "dark" cells and clumped in "light" cells. Neuroglial cells are stained metachromatically by cresyl violet. The neuroglial cells have many processes which ramify extensively and are enmeshed to form overlapping layers. These imbricated processes ensheath the nerve cells; the inner layer of the sheath penetrates into the neuron and is responsible for the appearance of the trophospongium of Holmgren. Nerve fibers are embedded within glial cells and surrounded by extensions of the plasma membrane similar to mesaxons. Depending on their size, two or several nerve fibers may share a single glial cell. Nerve fibers near their terminations on other nerve fibers contain particles and numerous, large mitochondria. The ganglion is ensheathed by a thick feltwork of connective tissue and perilemmal cells. The abdominal connective has a thinner connective tissue sheath which is without perilemmal cells. The nerve fibers and sheaths in the connective become thinner as they pass through ganglia.     

The ultrastructure of the cardiac ganglion of the desert scorpion,Paruroctonus mesaensis (Scorpionida: Vaejovidae)

Light and electron microscopy of the pacemaker ganglion of the scorpion heart indicate that it is about 15 mm long and 50 μm in diameter and extends along the dorsal midline of the heart. The largest cell bodies (30–45 μm in diameter) occur in clusters along the length of the ganglion. The ganglion appears to be innervated with fibers from the subesophageal and first three abdominal ganglia. The cardiac ganglion is surrounded by a neurilemma and a membranous sheath. The latter is apparently derived from connective tissue cells seen outside the ganglion. Nerve fibers other than those in the neuropil areas are usually surrounded by membrane and cytoplasm of glial cells. Often there are several layers of glial membrane, forming a loose myelin. The cardiac nerves to the heart muscle are also surrounded by a neurilemma, and the axons are surrounded by glia. The motor nerves contain lucent vesicles 60–100 nm and opaque granules 120–180 nm in diameter. In the cardiac ganglion, some nerve cell bodies have complex invaginations of glial processes forming a peripheral trophospongium. In the neuropil areas, nerve cell processes are often in close apposition. The septilaminar configuration typical of gap junctions is common, with gap distances of 1–4 nm. In tissues stained with lanthanum phosphate during fixation, we found gaps with unstained connections (1–2 nm diameter) between nerve-nerve and glial-nerve cell processes. Annular or double-membrane vesicles in various stages of formation were also seen in some nerve fibers in ganglia stained with lanthanum phosphate. Nerve endings with electron-lucent vesicles 40–60 nm in diameter are abundant in the cardiac ganglion, suggesting that these contain the excitatory transmitter of intrinsic neurons of the ganglion. Less abundant are fibers with membrane-limited opaque granules, circular or oblong in shape and as much as 330 nm in their longest dimension. Also seen were some nerve endings with both vesicles and granules.     

Multicellular spheroid formation and evolutionary conserved behaviors of apple snail hemocytes in culture

A hemocyte primary culture system for Pomacea canaliculata in a medium mimicking hemolymphatic plasma composition was developed. Hemocytes adhered and spread onto culture dish in the first few hours after seeding but later began forming aggregates. Time-lapse video microscopy showed the dynamics of the early aggregation, with cells both entering and leaving the aggregates. During this period phagocytosis occurs and was quantified. Later (>4 h), hemocytes formed large spheroidal aggregates that increased in size and also merged with adjacent spheroids (24–96 h). Large single spheroids and spheroid aggregates detach from the bottom surface and float freely in the medium. Correlative confocal, transmission electron and phase contrast microscopy showed a peculiar organization of the spheroids, with a compact core, an intermediate zone with large extracellular lacunae and an outer zone of flattened cells; also, numerous round cells emitting cytoplasmic extensions were seen attaching to the spheroids' smooth surface. Dual DAPI/propidium iodide staining revealed the coexistence of viable and non-viable cells within aggregates, in varying proportions. DNA concentration increased during the first 24 h of culture and stabilized afterward. BrdU incorporation also indicated proliferation. Spontaneous spheroid formation in culture bears interesting parallels with spheroidal hemocyte aggregates found in vivo in P. canaliculata, and also with spheroids formed by tumoral or non-tumoral mammalian cells in vitro.     

Structure of hematopoietic nodules in the ridgeback prawn,Sicyonia ingentis: Light and electron microscopic observations

The architecture and fine structure of the epigastric hematopoietic nodules of the ridgeback prawn, Sicyonia ingentis, are described. The nodules consist of a highly branched series of tubules that contain the maturing hemocytes within a connective tissue stroma. Hemocytes can exit the hematopoietic nodules by penetrating through fenestrations in the endothelial cell layer into the central hemal space or by migrating through the outer later of capsular cells and associated collagen fibrils. Four hemocyte categories were observed: agranular, small granule with cytoplasmic deposits, small granule without cytoplasmic deposits, and large granule hemocytes. This classification was based upon the presence, size, and type of cytoplasmic granules and the presence of cytoplasmic deposits. Only agranular cells and small granule hemocytes without cytoplasmic deposits appeared capable of division. Intermediate stages were observed between agranular hemocytes and small granule hemocytes with deposits and between small granule hemocytes without deposits and large granule hemocytes, suggesting existence of two distinct hemocyte lines.     

Electron Microscopy of a Plant-Pathogenic Virus in the Nervous System of Its Insect Vector

The central nervous system of wound tumor virus (WTV)-infected Agallia constricta was studied by electron microscopy to obtain information concerning the virus distribution in the nervous system. Wound tumor virions were mostly found in the cytoplasm of the ganglion cells and less frequently in the glial cells. WTV was occasionally observed in the perineurium cells, nerve axons, tracheoblasts, and lateral nerves. In the ganglion cells, virions appeared as individual isolated particles (V(1)), in tubular formation (V(2)), and occasionally in aggregates (V(3)). In the glial cells, the virions were mostly seen in the V(3) formation, and very seldom in the V(1) and V(2) formations. In the perineurium cells and tracheoblasts, only small V(3) formations were observed. The isolated virions were usually surrounded with polyribosomes, and often appeared around the foci of the viroplasm. Sometimes degenerating ganglion cells infected with the WTV were encountered. These damaged cells strongly indicated that WTV exerted a cytopathogenic effect on the nerve cells.     

Glial cells in peripheral nerves of the cockroach, Periplaneta americana

The ultrastructural organization and the junctional complexes of peripheral nerves have been investigated in the cockroach Periplaneta americana. Nerve 5 is surrounded by a layer of connective tissue, the neural lamella, beneath which is a layer of perineurial glial cells wrapping the axons. Adjacent perineurial cells are joined to one another by septate, gap and tight junctions. Septate and gap junctions were observed in freeze-fracture replicas of main trunk nerve 5. Septate junctions were found as rows of PF particles mainly in perineurial cell membranes. Gap junctions exhibited EF macular aggregates in perineurial and subperineurial glial cells. During incubations in vivo with extracellularly applied ionic lanthanum, the lanthanum did not penetrate beyond the perineurium. Where nerve 5 branches and contacts the muscle, lanthanum penetrated freely between the muscle fibres and the nerve branches. In small peripheral branches where the axons are surrounded by single a glial layer, lanthanum is unable to penetrate to the axolemma.     

Microscopic localization of cholinesterases in the nervous systems of the lobsters, Panulirus argus and Homarus americanus

Using acetylthiocholine as substrate, microscopically localizable cholinesterase (ChE) activity is demonstrated in neural and glial elements of central and peripheral nervous systems of the lobsters, Panulirus argus and Homarus americanus. Moderate to very intense ChE activity occurs in all synaptic regions of the central ganglia and stomatogastric ganglion, in glial sheaths around neuron somata and peripheral nerve axons, and in cytoplasm of a few nerve cell bodies. Axons, identified as motor, contain extremely little ChE. The principal reaction in peripheral nerves occurs in sheath elements of sensory fibres; in most cases, much of the reaction is lost as the nerves lose the sheaths at the point of entry into brain.     

Structure and anatomical relationships of the synganglion in the American dog tick,Dermacentor variabilis (Acari: Ixodiadae)

The synganglion of Dermacentor variabilis Say is a single nerve mass, condensed around the esophagus and within the periganglionic sinus of the ciculatory system. Protocerebral, cheliceral (including stomodeal bridge), and pedipalpal ganglia lie in the pre-esophageal portion of the nerve mass and bear optic, cheliceral, and pedipalpal nerves respectively. The unpaired stomodeal and the recurrent nerve which forms the hyper-esophageal ganglion arise from the stomodeal bridge. Paired primary and accessory nerves to the retrocerebral organ complex have mixed protocerebral-cheliceral origins. Pedal ganglia (including ventral olfactory lobes of pedal ganglia I) and composite opisthosomal ganglion lie in the post-esophageal nerve mass and bear pedal nerve trunks and two pairs of opisthosomal nerves respectively. Internally, the synganglion consists of cellular rind and fibrous core. A welldefined neurilemma with a laminar matrix covers nerve mass and peripheral nerves. The rind contains the somata of ganglionic neurons and ensheathing glial cells and is restricted to the synganglion mass. It is limited by two specialized glial layers, the external perineurium and internal subperineurium. Discrete glomerular formations are present within the protocerebrum and olfactory lobes. Olfactory glomeruli located in pedal ganglia I are associated with a pair of globuli cell groups. Possible physiological relationships between anatomical specializations of the synganglion, extraneural sinuses and circulating hemocytes are considered. The evolutionary significances of condensation in the stomatogastric neuropile regions and throughout the synganglion, together with the simplification and loss of glomerular formations, are discussed.     

Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation

Summary The presence of neurofilament (NF)-like and glial fibrillary acidic protein (GFAP)-like immunoreactivities was studied in sympathetic ganglia of adult rats and guinea pigs during normal conditions and after perturbation. In the superior cervical ganglion (SCG) of normal rats, many ganglion cells and nerve fibers show NF immunoreactivity. Some of these nerve fibers disappear after preganglionic decentralization of SCG; this indicates the presence of a mixture of preand postganglionic NF-positive nerves in the ganglion. Cuts in both preand postganglionic nerves result in a marked increase in GFAP immunoreactivity in SCG, whereas NF immunoreactivity increases in nerve cell bodies after preganglionic cuts. Only a few ganglion cells show NF immunoreactivity in the normal SCG of guinea pig. All intraganglionic NF-positive nerves are of preganglionic origin; decentralization abolishes NF immunoreactivity in these nerve fibers. The inferior mesenteric ganglion, the hypogastric nerves and colonic nerves in guinea pigs contain large numbers of strongly NF-immunoreactive nerve fibers.When the SCG of adult rat is grafted to the anterior eye chamber of adult rat recipients, both ganglionic cell bodies and nerve fibers, forming on the host iris from the grafted ganglion, are NF-positive. As only the perikarya of these neurons normally exhibit NF immunoreactivity, and the terminal iris arborizations are NF-negative, it appears that the grafting procedure causes NF immunoreactivity to become more widespread in growing SCG neurons.     

Fine Structure of the Hemopoietic Tissues in the Mealworm Beetle, Tenebrio molitor

The fine structure of the hemopoietic tissue and its detailed reticular organization in the mealworm beetle, T. molitor were examined using light and scanning electron microscopes. The major hemopoietic tissues in the abdomen were located on the upper surface of the dorsal diaphragm which continuous over the ventral wall of the heart. Histologic characteristics of this hemopoietic tissues are dense clusters of cells. They are irregular in outline and are not surrounded by any connective tissue sheath. The hemopoietic tissue of this insect is consisted of three cellular components which are the reticular cells, hemocytic stem cells and several kinds of mature hemocytes. The reticular cells had numerous cytoplasmic processes and forming a complex network. The stem cells give rise to differentiating hemocytes of the different cell lineages. Mature hemocytes within this hemopoietic tissue are originated from the stem cells and differentiated into several types of hemocytes including prohemocytes, plasmatocytes, and granulocytes.     

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal–cytoskeletal, membrane–cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.     

The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila

During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC). We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.     

Structure of the intramural nerves of the rat bladder

The bladder of adult female rats receives ～16,000 axons (i.e., is the target of that many ganglion neurons) of which at least half are sensory. In nerves containing between 40 and 1200 axons cross-sectional area is proportional to number of axons; >99% of axons are unmyelinated. A capsule forms a seal around nerves and ends abruptly where nerves, after branching, contain ～10 axons. A single blood vessel is present in many of the large nerves but never in nerves of <600 axons. The number of glial cells was estimated through the number of their nuclei. There is a glial nucleus profile every 76 axonal profiles. Each glial cell is associated with many axons and collectively covers ～1,000 μm of axonal length. In all nerves a few axonal profiles contain large clusters of vesicles independent of microtubules. The axons do not branch; they alter their relative position along the nerve; they vary in size along their length; none has a circular profile. All the axons are fully wrapped by glial cells and never contact each other. The volume of axons is larger than that of glial cells (55%–45%), while the surface of glial cell is twice as extensive as that of axons; there are ～2.27 m² of axolemma and ～4.60 m² of glial cell membrane per gram of nerve. Of the mitochondria of a nerve ～3/4 are in axons and ～1/4 in glial cells.     

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.     

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

Peripheral nerve connections influence the appearance of neurosecretary material in neural sheath of ventral ganglion of the fly Sarcophaga bullata: an immunocytochemical study

This study examined the role of the brain and peripheral connections with the target organs in the appearance of neurosecretary material within the dorsal neural sheath of the ventral ganglion of the fly S. bullata. Specifically, the accumulation of the neuropeptide FMRFamide (the neurosecretary material) was examined by immunocytochemistry. Immunoreactions were performed on: (1) a normal intact ventral ganglion, (2) an isolated ventral ganglion that was cultured in vivo, and (3) a ventral ganglion that was isolated by transection from the brain, but retained its peripheral nerve connections. The results demonstrate that (a) the neurons of the ganglia survive and exhibit FMRFamide immune reaction independent of their peripheral connections, and (b) the accumulation of neuropeptide in the dorsal neural sheath is controlled by intact peripheral nerve connections with the ganglion. It is suggested that in the absence of their peripheral connections, the axons of FMRFamide immunoreactive neurons fail to invade the neural sheath resulting in the accumulation of neurosecretary material.     

THE FINE STRUCTURAL ORGANIZATION OF NERVE FIBERS, SHEATHS, AND GLIAL CELLS IN THE PRAWN, PALAEMONETES VULGARIS

In view of reports that the nerve fibers of the sea prawn conduct impulses more rapidly than other invertebrate nerves and look like myelinated vertebrate nerves in the light microscope, prawn nerve fibers were studied with the electron microscope. Their sheaths are found to have a consistent and unique structure that is unlike vertebrate myelin in four respects: (1) The sheath is composed of 10 to 50 thin (200- to 1000-A) layers or laminae; each lamina is a cellular process that contains cytoplasm and wraps concentrically around the axon. The laminae do not connect to form a spiral; in fact, no cytoplasmic continuity has been demonstrated among them. (2) Nuclei of sheath cells occur only in the innermost lamina of the sheath; thus, they lie between the sheath and the axon rather than outside the sheath as in vertebrate myelinated fibers. (3) In regions in which the structural integrity of the sheath is most prominent, radially oriented stacks of desmosomes are formed between adjacent laminae. (4) An ~200-A extracellular gap occurs around the axon and between the innermost sheath laminae, but it is separated from surrounding extracellular spaces by gap closure between the outer sheath laminae, as the membranes of adjacent laminae adhere to form external compound membranes (ECM's). Sheaths are interrupted periodically to form nodes, analogous to vertebrate nodes of Ranvier, where a new type of glial cell called the "nodal cell" loosely enmeshes the axon and intermittently forms tight junctions (ECM's) with it. This nodal cell, in turn, forms tight junctions with other glial cells which ramify widely within the cord, suggesting the possibility of functional axon-glia interaction.