Method for Detecting Hyaluronan in Isolated Myenteric Plexus Ganglia of Adult Rat Small Intestine.

The cellular components of the enteric nervous system (ENS), namely enteric neurons and glia, display plasticity and respond to environmental cues deriving from growth factors, extracellular matrix (ECM) molecules, and cell-surface molecules, both in physiological and pathological conditions. ECM, in particular, provides an important framework for the enteric microenvironment and influences the homeostasis of myenteric neuronal circuitries. Isolation of pure myenteric plexus preparations from adult tissue permits to investigate changes in the ENS involving specific ECM, such as hyaluronan. This approach is based upon the possibility to isolate myenteric ganglia from the intestinal wall of either adult animals or humans, after microdissection and subsequent enzymatic digestion of the tissue. Enteric ganglia are free of connective tissue, extracellular collagen, and blood vessels, and thus treatment of intact intestinal segments with highly purified collagenases permits ganglia isolation from the surrounding smooth muscle cells. In this chapter, we describe methods for visualizing HA in isolated primary cultures of adult rat small intestine myenteric ganglia.

Coati Bodies: Cytoplasmic Hyaline Globules in the Ganglionic Neurons of Aging Captive Coatis.

Intensely eosinophilic and glassy intracytoplasmic inclusions were present in the neurons of the peripheral autonomic ganglia, Meissner's and Auerbach's plexus, and spinal ganglia in 20 aged white-nosed coatis ( Nasua narica, 7-19 years old) and in 4 of 7 brown-nosed coatis ( Nasua nasua, 2-21 years old) from multiple zoological institutions. Inclusions were single to numerous, sometimes distorting the cell. Pheochromocytomas were present in 5 of 16 white-nosed and 2 of 6 brown-nosed coatis, although no inclusions were present in the adrenal glands. Histochemically, immunohistochemically, and ultrastructurally, these inclusions were consistent with dense neurosecretory granules. Although similar inclusions have been reported sporadically in the adrenal medulla of humans and several other mammalian species as both incidental and pathologic findings, ganglionic inclusions reported herein appear to be unique and related to age in these species.

Presence of Ganglia and Telocytes in Proximity to Myocardial Sleeve Tissue in the Porcine Pulmonary Veins Wall.

Ganglia and telocytes were identified inside the porcine pulmonary veins wall near myocardial sleeve tissue at the atriopulmonary junction. These structures are reported to play a role in the initiation of pulses from outside the heart, which potentially can cause cardiac conduction disorders such as atrial fibrillation. In-depth knowledge on the fine structure of the pulmonary vein wall is a pre-requisite to better understand the underlying pathophysiology of atrial fibrillation and the origin and conduction of ectopic pulses. The importance of pulmonary vein myocardial sleeves as triggering foci for atrial fibrillation has been shown in human patients. In this context, the fine structure of the pulmonary vein wall was investigated qualitatively by light and transmission electron microscopy in the pig, which is a frequently used animal model for development of new treatment strategies. Additionally, intra and extramural ganglia, containing telocytes that create a network near the neurone cell bodies, were identified in pigs. Detailed illustration of the distribution and organization of tissues and cell types, potentially involved in the origin and propagation of ectopic stimuli originating from the pulmonary veins, might lead to a better insight on the actual composition of the tissues affected by ablation as studied in pigs.

Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS.

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms.

Immunohistochemical characteristics of neurons in the ganglia of the greater splanchnic nerve of the pig.

INTRODUCTION: Greater splanchnic nerve (GSN) is by far the largest of the splanchnic nerves and connects the paravertebral and prevertebral ganglia to transmit the majority of nociceptive information from the viscera. Despite its importance, the immunohistochemical features of the porcine GSN neurons have not yet been examined. Therefore, the aim of the study was to investigate the neurochemistry of the porcine GSN neurons and to compare their neurochemical coding with those of the paravertebral and prevertebral ganglia. MATERIAL AND METHODS: Four gilts of Large White Polish breed were examined in this study. Antibodies to tyrosine hydroxylase (TH), dopamine b-hydroxylase (DBH), choline acetyltransferase (ChAT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), methionine-enkephalin (MET), calcitonin gene-related peptide (CGRP), and substance P (SP) were used for immunohistochemical detection of classical neurotransmitters marker enzymes and neuropeptides in neuronal cell bodies of the GSN. RESULTS: Double-labeling immunofluorescence revealed that virtually all GSN neurons exhibited the presence of catecholamine-synthesizing enzymes (TH/DBH-positive) and subpopulations of neurons contained immunoreactivity to NPY, VIP, SOM, GAL and MET. However, CGRP and SP-immunoreactivity were not observed in neuronal somata. CONCLUSIONS: Our data strongly suggest that the general immunohistochemical characterization of ganglion cells in the porcine greater splanchnic nerve is similar to that of the prevertebral ganglia (e.g. celiacomesenteric ganglion).

Viral Spread to Enteric Neurons Links Genital HSV-1 Infection to Toxic Megacolon and Lethality.

Herpes simplex virus 1 (HSV-1), a leading cause of genital herpes, infects oral or genital mucosal epithelial cells before infecting the peripheral sensory nervous system. The spread of HSV-1 beyond the sensory nervous system and the resulting broader spectrum of disease are not well understood. Using a mouse model of genital herpes, we found that HSV-1-infection-associated lethality correlated with severe fecal and urinary retention. No inflammation or infection of the brain was evident. Instead, HSV-1 spread via the dorsal root ganglia to the autonomic ganglia of the enteric nervous system (ENS) in the colon. ENS infection led to robust viral gene transcription, pathological inflammatory responses, and neutrophil-mediated destruction of enteric neurons, ultimately resulting in permanent loss of peristalsis and the development of toxic megacolon. Laxative treatment rescued mice from lethality following genital HSV-1 infection. These results reveal an unexpected pathogenesis of HSV associated with ENS infection.

Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.

Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enablede novostudies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatwormSchmidtea mediterraneaat different time points during cephalic ganglia regeneration. A protocol was developed to makeS. mediterraneatissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.

[TOPOGRAPHIC ANATOMY OF ASCENDING AND DESCENDING NEURONS OF SUPRAESOPHAGEAL, MESO- AND METATHORACIC GANGLIA IN PALEO- AND NEOPTEROUS INSECTS].

Topographic anatomy of ascending (AN) and descending (DN) neurons of the supraesophageal and thoracic ganglia in the nervous system of winged insects (Pterygota) - representatives of infraclasses Plaeoptera (Odonata, Aeschna grandis, dragonfly) and Neoptera (Blattoptera, Periplaneta americana, cockroach) was studied. These insects are different in ecological niches, lifestyles, sets of behavioral complexes, levels of locomotor system development, evolutionary age and systematic position. Neuronal bodies and processes of ANs and DNs were stained with nickel chloride (NiC2), their topography was studied on total prerapations of the supraesophageal and thoracic ganglia. Unlike cockroaches, in dragon- fly protocerebrum DNs sending their processes to ocelli were found. Dragonfly DN processes show a spe- cific type of arborization in thoracic ganglia, with collaterals directed both ipsi- and contralaterally. In cockroaches collaterals of DN processes are arranged ipsilaterally. AN bodies in meso- and metathoracic ganglia of dragonfly lie both ipsi- and contralaterally in respect to the ascending process whereas in cock- roaches AN bodies in the same ganglia are predominantly localized contralaterally. Substantial differences in allocation of DNs and ANs in insects dissimilar in locomotor manner reflect a different extent of supraesophageal ganglion control over activity of segmental centers. It seems related to neither the evolu- tionary age of insects, nor the antiquity of origin, nor their systematic position. Probably, a different de- gree of locomotion control depends on the way of getting food - catching prey in air by <> dragonflies unlike <> maneuverable walking or running across a solid substrate by <> cockroaches.

Armored kinorhynch-like scalidophoran animals from the early Cambrian.

Morphology-based phylogenetic analyses support the monophyly of the Scalidophora (Kinorhyncha, Loricifera, Priapulida) and Nematoida (Nematoda, Nematomorpha), together constituting the monophyletic Cycloneuralia that is the sister group of the Panarthropoda. Kinorhynchs are unique among living cycloneuralians in having a segmented body with repeated cuticular plates, longitudinal muscles, dorsoventral muscles, and ganglia. Molecular clock estimates suggest that kinorhynchs may have diverged in the Ediacaran Period. Remarkably, no kinorhynch fossils have been discovered, in sharp contrast to priapulids and loriciferans that are represented by numerous Cambrian fossils. Here we describe several early Cambrian (~535 million years old) kinorhynch-like fossils, including the new species Eokinorhynchus rarus and two unnamed but related forms. E. rarus has characteristic scalidophoran features, including an introvert with pentaradially arranged hollow scalids. Its trunk bears at least 20 annuli each consisting of numerous small rectangular plates, and is armored with five pairs of large and bilaterally placed sclerites. Its trunk annuli are reminiscent of the epidermis segments of kinorhynchs. A phylogenetic analysis resolves E. rarus as a stem-group kinorhynch. Thus, the fossil record confirms that all three scalidophoran phyla diverged no later than the Cambrian Period.

An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid, Doryteuthis pealeii.

Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.

Morphological study of the sinus node and its artery in yak.

The sinus node of yak has been studied by the histological methods and transmission electron microscopy. The sinus node artery of yak was also determined by the injection-corrosion casting technique, the angiography, and histological methods. The results showed that the sinus node of yak contained an extensive framework of collagen and two main type cells: pacemaker cells (P cells) and transitional cells (T cells). The P cells had a perinuclear clear zone, contained less myofibrils, and appeared smaller mitochondria than T cells. The T cells were longer and slender than P cells, and had a variety of shapes. At the periphery of sinus node there were many nerve fibers and ganglions. Gap junction did not reveal reaction with anti-connexin43, but it was detected by electron microscopy in the central part of sinus node of yak. The sinus node artery of yak originated from left coronary artery more frequently (98%) than by right (2%). The artery located at the periphery of sinus node. It had an internal elastic membrane throughout its course, and a large nerve bundle was found running in a longitudinal direction.

The primary cilium: A relevant characteristic in interstitial cells of rat duodenum enteric plexus.

Studies in vitro have permitted the identification of enteric neural progenitor cells. Now the question arises as to where these progenitor cells are located in vivo. The purpose of this paper is to identify possible candidate cells by means of transmission electron microscopy (TEM). We have located three interstitial cellular types around the rat duodenum myenteric plexus. Type I cells have been identified as Interstitial Cells of Cajal (ICCs). These cells present well defined ultrastructural characteristics, including the triple connexion IC- nervous trunk- blood vessels. Type II cells show characteristics of immature cells, emphasizing the presence of a single cilium with the structure (9+0). To analyse this nanostructure, we have elaborated a reconstruction on ultrathin sections. The two previously described cellular types could be considered to be different functional states of the same cell. Type III cells present ultrastructural characteristics of fibroblast-like cells. This study suggests that Type II cells could be a source of neural progenitor cells.

Immunohistochemical demonstration of cholinergic structures in central ganglia of the slug (Limax maximus, Limax valentianus).

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans.

The morphology and fine structure of the giant interneurons of the wood cricket Nemobius sylvestris.

The structural and ultrastructural characteristics of giant interneurons in the terminal abdominal ganglion of the cricket Nemobius sylvestris were investigated by means of cobalt and fluorescent dye backfilling and transmission electron microscopy. The projections of the 8 eight pairs of the biggest ascending interneurons (giant interneurons) are described in detail. The somata of all interneurons analyzed are located contralateral to their axons, which project to the posterior region of the terminal ganglion and arborise in the cercal glomerulus. Neuron 7-1a is an exception, because its arborisation is restricted to the anterior region of the ganglion. The fine structure of giant interneurons shows typical features of highly active cells. We observed striking indentations in the perineural layer, enabling the somata of the giant interneurons to be very close to the haemolymph. The cercal glomerulus exhibits a high diversity of synaptic contacts (i.e. axo-dendritic, axo-axonic, dendro-axonic, and dendro-dendritic), as well as areas of tight junctions. Electrical synapses seem to be present, as well as mixed synapses. The anatomical organization of the giant interneurons is finally discussed in terms of functional implications and on a comparative basis.

A new organellar complex in rat sympathetic neurons.

Membranous compartments of neurons such as axons, dendrites and modified primary cilia are defining features of neuronal phenotype. This is unlike organelles deep to the plasma membrane, which are for the most part generic and not related directly to morphological, neurochemical or functional specializations. However, here we use multi-label immunohistochemistry combined with confocal and electron microscopy to identify a very large (approximately 6 microns in diameter), entirely intracellular neuronal organelle which occurs singly in a ubiquitous but neurochemically distinct and morphologically simple subset of sympathetic ganglion neurons. Although usually toroidal, it also occurs as twists or rods depending on its intracellular position: tori are most often perinuclear whereas rods are often found in axons. These 'loukoumasomes' (doughnut-like bodies) bind a monoclonal antibody raised against beta-III-tubulin (SDL.3D10), although their inability to bind other beta-III-tubulin monoclonal antibodies indicate that the responsible antigen is not known. Position-morphology relationships within neurons and their expression of non-muscle heavy chain myosin suggest a dynamic structure. They associate with nematosomes, enigmatic nucleolus-like organelles present in many neural and non-neural tissues, which we now show to be composed of filamentous actin. Loukoumasomes also separately interact with mother centrioles forming the basal body of primary cilia. They express gamma tubulin, a microtubule nucleator which localizes to non-neuronal centrosomes, and cenexin, a mother centriole-associated protein required for ciliogenesis. These data reveal a hitherto undescribed organelle, and depict it as an intracellular transport machine, shuttling material between the primary cilium, the nematosome, and the axon.

Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

[Experimental modeling and the discussion on the syncytial connections in the nervous system].

To solve the problem of the possibility of syncytial connection in the nervous system, this paper for the first time presents the evidence of experimental syncytial fusion of neurons. Neurons, isolated from the ganglia of the mollusc Lymnaea stagnalis and freed from the surrounding glia by pronase treatment, were drawn together by centrifugation and were kept in the culture medium for two days in the aggregated state. The neurons preserved the ability to generate normal processes. At the borders of adjacent cells, contacting mutual protrusions (feet) were formed that were separated from each other by vacuole-like enlargements of the intercellular clefts. Using the electron microscope, it was shown that at the borders of contacting feet the external cell membranes were destroyed. Only residual fragments of the destroyed membranes were detected. The cytoplasm of one adjacent cell was continuous with the cytoplasm of the other. Thus, the experiments confirm once more the correctness of the cell theory concerning the common main properties of all the cells and expand the concepts of the neuronal theory by the statement that in the nervous system, along with chemical synapses and electrical membrane contacts, the syncytial interneuronal connections are also possible.

Neuronal elasticity as measured by atomic force microscopy.

A cell's form and function is determined to a great extent by its cellular membrane and the underlying cytoskeleton. Understanding changes in the cellular membrane and cytoskeleton can provide insight into aging and disease of the cell. The atomic force microscope (AFM) allows unparalled resolution for the imaging of these cellular components and the ability to probe their mechanical properties. This report describes our progress toward the use of AFM as a tool in neuroscience applications. Elasticity measurements are reported on living chick embryo dorsal root ganglion and sympathetic neurons in vitro. The neuronal cellular body and growth cones regions are examined for variations in cellular maturity. In addition, cellular changes due to exposure to various environmental conditions and neurotoxins are investigated. This report includes data obtained on different AFM systems, using various AFM techniques and thus also provides knowledge of AFM instruments and methodology.

[Effect of temperature stress on activities of nitric oxide synthase and tyrosine hydroxylase in the central nervous system of bivalve molluscs].

Effects of temperature stress on nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) activities in CNS of two species of bivalve molluscs Mizuchopecten yessoensis and Chlamys farreri nipponensis (Pectinidae) were studied using NADPH-diaphorase histochemistry and ummunocytochemistry. General and specific peculiarities in distribution and relative content ofNOS- and TH-positive neurons in nervous ganglia were revealed in norm and under stress at 30 degrees C for 10, 30, and 60 min. The initial stress stage (for 10 min) has been shown to be accompanied by an increase of the relative content of TH-positive neurons in some CNS areas of both mollusc species. In Chlamys farreri nipponensis under normal conditions, the presence of NOS in the CNS and its significant activation under temperature stress might have possibly been an important neuroprotective component of stress reaction in some mollusc species.