Neuron-microglia communication in the CNS of the freshwater snail Planorbarius corneus

The aim of the present study was to identify molecules that may be involved in neuron-microglia communication in the CNS of freshwater snail Planorbarius corneus. Messenger molecules are exchanged in normal and pathological conditions and we tried to identify some of them by immunocytochemistry on whole ganglia and cell cultures. In particular, we examined neurons and microglia for the expression of some cytokines, IL-1alpha, IL-1beta, IL-6 and TNF-alpha and the neurotransmitter glutamate. These substances may be released by suffering or injured neurons and communicate to microglia the damaging event. Even microglia, on own turn, once activated, express and released the same or other substances in order to reestablish the system homeostasis, depending on modalities and times of activation. We discuss the possibility that hyperactivated microglia can shift from neuroprotective to neurodegenerative. Moreover, we examined in neuron-microglia co-coltures the direct interaction effects in terms of neuronal survival and improved neurite regeneration.     

Immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata).

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius corneus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of alpha 1-antichymotrypsin-bombesin-, calcitonin-, CCK-8 (INC)-, CCK-39-, gastrin-, glucagon-, Met-enkephalin-, neurotensin-, oxytocin-, somatostatin-, substance P-, VIP-, and vasopressin-immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti-bombesin, anticalcitonin, anti-CCK-8 (INC), anti-CCK-39, anti-neurotensin, anti-oxytocin, anti-substance P and anti-vasopressin antibodies. No immunostaining was observed with anti-CCK-8 (Peninsula), anti-insulin, anti-prolactin, anti-thyroglobulin and anti-thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.     

淡水蜗牛视觉系统的输入与输出通路(英文)

通过神经生物素在视神经上的逆行性传输对淡水蜗牛(Planorbarius corneus)视网膜及中央神经节的输入、输出神经元进行标记。由于没有发现突触联系,所以至少一部分光感受细胞的轴突可被视为直接参与形成视神经。这些神经元的轴突进入大脑神经节形成密集的细传入神经纤维束-视神经堆。传出神经元则存在于除颊部以外的所有神经节。一些上行轴突在大脑神经节处分叉,通过脑-脑联合,到达对侧眼并在眼杯处形成分枝。部分传出神经元的轴突也投射于不同的外周神经,如:n.n.intestinalis,pallialis dexter,pallialis sinister internus et externus。五羟色胺能纤维和FMRF-酰胺能纤维均存在于视神经上,且这些纤维隶属于只投射在同侧眼的中央神经元。它们形成了位于眼杯处的丰富曲张结构及视网膜核心层,并且可能有助于调节视网膜对光的敏感性。     

Neuronal mechanisms of defense reaction to statocyst receptor stimulation in the freshwater snail Planorbarius corneus

Tilting of the freshwater snailPlanorbarius corneus triggering dynamic statocyst receptor response resulted in defense reaction attended by rapid lowering of the shell over the head, foreshortening of the foot, and inhibited locomotion and buccal apparatus operation. Large numbers of neurons from different ganglia were found to take part in this reaction in isolated nervous system preparations. The response usually followed an "all or none" pattern and did not depend on which statocyst receptors had been stimulated. Each successive response arose no sooner than 10–20 sec after the previous reaction to tilting the preparation. It is deduced that defense reaction to statocyst receptor stimulation takes the form of a "fixed action" governed by a special central mechanism. It was found during the process of investigating interaction between response to statocyte receptor and cutaneous nerve stimulation that the same central mechanism serves to produce defense reactions evoked by presentation of different stimuli.     

Microglia proliferation as a response to activation in the freshwater snail Planorbarius corneus: a BrdU incorporation study

Invertebrate microglia constitute a class of cells resident in the ganglionic nervous system which are activated after tissue injury or by the presence of pathogens. The microglia activation response includes graduated morpho-functional and biochemical changes and cell proliferation. In this study we verified in the freshwater snail Planorbarius corneus that an activation caused by a traumatic event may induce microglia division. Cell proliferation was assessed immunocytochemically using BrdU incorporation technique and documented on both ganglionic sections and microglia cultured cells at different experimental conditions and times after activation. In addition, we studied the possibility of increasing microglia proliferation by adding to the cultured medium the Macrophage-Colony Stimulating Factor (M-CSF) that has been shown to stimulate specifically this process in vertebrates.     

Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical, immunocytochemical and ultrastructural studies

All stages of regeneration in hamster tracheal epithelium were studied following a denuding mechanical injury. At 1 h all the cells had sloughed from the wound site leaving a bare and sometimes disrupted basal lamina. Viable cells at the wound margins rapidly changed shape, flattened and migrated to cover the denuded lesion by 12 h. In addition, epithelial cells that remained viable demonstrated sublethal changes that included the rapid discharge of mucous granules from secretory cells, internalization of cilia by ciliated cells and evidence of heterophagy in both cell types. By 24 h a wave of epithelial cell divisions occurred, primarily by secretory cells. This produced a multilayered epidermoid metaplasia that was best developed at 48 h. The metaplastic epithelium was largely composed of cells with both secretory (mucous granules) and epidermoid (tonofilament bundles and numerous desmosomes) characteristics. The peroxidase-antiperoxidase (PAP) method demonstrated a few keratin-positive cells in the wound as early as 12 h post-wounding and keratin was demonstrated in more cells by 24 h. All cells in the metaplastic wound epithelium were keratin-positive by 48 h. Following 48 h some of the most superficial keratinized cells sloughed from the epithelium and the keratin content of the remaining cells began to decline. At 72 h pre-ciliated and pre-secretory cells were seen in the wound. Pre-ciliated cells were characterized by an abundant electron-lucent cytoplasm, large pale nucleus, filiform apical microvilli and evidence of ciliogenesis, similar to that seen during fetal development. Pre-ciliated cells often contained apical mucous granules, apparently carried over from the parent secretory cells. With the appearance of these columnar cells the normal mucociliary morphology was restored in small wounds by 120 h, but some persistent epidermoid metaplasia remained in the large wounds through 168 h post-wounding. These data provide further evidence for the important role of secretory cells in the histogenesis of epidermoid metaplasia and the regeneration of normal morphology following injury. The implications of these findings in understanding the histogenesis of other lesions in the tracheo-bronchial epithelium are discussed.     

Histochemical and ultrastructural observations on feeding and digestion in Reighardia sternae (Pentastomida: Cephalobaenida)

Histological, histochemical and ultrastructural methods have been used to study gut structure and function in the cephalobaenid pentastomid Reighardia sternae (Diesing, 1864).
R. sternae feeds exclusively on blood. Haemolysis of ingested erythrocytes is thought to be initiated by non-specific esterase secreted by cells lining the posterior oesophagus. Only one other enzyme, acid phosphatase, was demonstrated; its activity is confined to the microvillar layer of the gastrodermis.
Digestion appears to be largely extracellular, as large volumes of haematin form in the gut lumen after haemolysis. However, this is supplemented by a significant amount of intracellular digestion: accumulations of iron-positive granules occur in approximately 20% of gastrodermal cells.
All gastrodermal cells are alike, and all can undertake the same intracellular digestive process. This is characterized by a number of morphologically distinct stages which have been studied using the electron microscope. The iron-positive particles distinguished under the light microscope were easily identifiable ultrastructurally. Spherical, weakly iron-positive bodies form within the cisternae of the endoplasmic reticulum. Phagosomes containing haemoglobin, or some breakdown product of haemoglobin, apparently bud off from the termini of a labyrinth system of tubules under the microvilli and fuse with the ER to form these phago-lysosomes. Intensely iron-positive heterolysosomes, also present in the cytoplasm of these cells, accumulate haemosiderin particles which are thought to be released into the cytoplasm during phago-lysosomal digestion.
The ensuing complex changes in cell morphology are, apparently, related to the elimination of spent phago-lysosomes. It has been suggested that the purpose of intracellular digestion is to provide a specific metabolite which the extracellular degradation of haemoglobin cannot supply.     

Increase in number and size of kidney concretions as a result of PCP exposure in the freshwater snail Planorbarius corneus (Gastropoda, Pulmonata)

Molluscan kidneys are able to excrete solids in the urine in the form of concretions. It is thought that increased formation of these concretions occur under pollutant, environmentally or reproductive induced stress. This study examined the formation of concretions in the kidney of the freshwater snail Planorbarius corneus L. experimentally exposed to pentachlorophenol (PCP). Light microscopic histopathological analysis of the PCP-exposed P. corneus revealed significantly enhanced production of the kidney concretions when compared to the kidneys of control individuals. Measurements of the number of kidney concretions, the apparent area of the concretions, and the epithelial area filled with concretions indicated an increase in the number and size of concretions in all treated snails. Lipofuscin content of excretory cell concretions was detected.     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Presence of ACTH and beta-endorphin immunoreactive molecules in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) and their possible role in phagocytosis

The presence of ACTH and beta-endorphin immunoreactive molecules in the cell-free hemolymph and in the hemocytes of the freshwater snail Planorbarius corneus were demonstrated by immunocytochemistry and RIA tests. Only spreading phagocytic hemocytes were positive, in contrast with other hemocytes devoid of phagocytic activity, i.e., round hemocytes. These data were confirmed by flow cytometry. Another cell type with marked phagocytic activity, i.e., digestive cells of digestive gland, were also positive to anti-ACTH. Corticotropin-releasing factor immunoreactive molecules were found in the cell-free hemolymph and hemocytes, by RIA. Our data suggest that cells with phagocytic activity, the oldest immune response, may represent a suitable model to unravel the tangled web of the common ancestor of the immune and the neuroendocrine systems.     

Argyrophilic proteins in Dinoflagellates: An ultrastructural,cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR-like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non-synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo-granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reaction's temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti-B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co-locating with end-products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23-like protein is probably a DNA partner.     

Coordination between locomotor and respiratory rhythms in the great ramshorn snail Planorbarius corneus: Transmitter-dependent modifications

In a reduced preparation of Planorbarius corneus consisting of the CNS and mantle complex, both the dopamine precursor L-DOPA and the serotonin precursor 5-HTP have been found to be able to induce and maintain rhythmic pneumostome (PN) movements phase-coupled to fictive cyclic locomotion in a neurotransmitter-specific manner. After the transection of pedal commissures, pharmacologically induced PN movements were coordinated with the locomotor activity rhythm generated by the left pedal ganglion, as in Lymnaea regardless of spatial inversion of its CNS. Nevertheless, in Planorbarius during the 5-HTP-induced fictive muscular locomotion, the PN was never opened, but cuddled up to the mantle at the same phase of the locomotor cycle corresponding to close down the PN in Lymnaea.     

Actions of acetylcholine on the salivary gland cells of the pond snail, Planorbis corneus

Intracellular recordings have been made from salivary gland cells of the pond snail Planorbis corneus. Gland cells produced a dose-dependent biphasic response to the bath application of acetylcholine (ACh), an initial depolarization being followed by a hyperpolarization. Nicotine and the nicotinic agonist tetramethylammonium had an excitatory action on the gland cells. The muscarinic agonists acetyl-beta-methyl choline and arecoline were also stimulants, but muscarine, bethanechol and pilocarpine produced no response from gland cells at 10(-3) M. A number of cholinergic antagonists, including atropine, hexamethonium and curare, effectively blocked the response to ACh. The depolarizing phase of the ACh response resulted from an increased membrane permeability to Na+ ions, though the participation of other ionic species cannot be ruled out. The hyperpolarizing phase of the ACh response was produced by the activity of an electrogenic Na+/K+ pump.     

A novel granular cell type of locust Malpighian tubules: ultrastructural and immunocytochemical study

Summary A novel secretory cell type in the initial segment of the Malpighian tubules of the locusts Schistocerca gregaria and Locusta migratoria is described ultrastructurally and studied by means of immunocytochemical techniques. The cells show abundant rough endoplasmic reticulum with interspersed Golgi zones. The richness of the cell secretory machinery and the presence of apical dense plemorphic granules suggest a role in secretion of proteinaceous material to the tubule lumen. The surprising finding of ACTH (1–24)-, -MSH-, and 7B2-like immunoreactivity for this cell is discussed.