Two kinds of peripheral afferent neurons in the earthworm reflex arc

• 1.1. Two kinds of neurons were identified in the body-wall longitudinal muscle layer of the earthworm, Amynthas hawayanus, by the simultaneous potential recording and Lucifer Yellow-CH injection method with a single microelectrode.
• 2.2. Both kinds of neurons have their somata, neuntes and longitudinal processes imbedded in the longitudinal muscle layer. Those with two circular processes extending into the third segmental nerve trunk are tentatively named “intra-nerve-trunk” neurons and those with four circular processes extending into four setae shafts are tentatively named “intramural” neurons.
• 3.3. Both kinds of neurons responded to electrical and mechanical stimuli applied in an afferent direction to them.
• 4.4. The “intra-nerve-trunk” neuron decreased its response amplitudes to these stimuli after the third nerve trunk was sectioned in correlation to the response amplitude decrease recorded from the nerve trunk after it was sectioned.
• 5.5. The response amplitude decrease due to denervation implies a nonlinear structure of the earthworm reflex circuits.
• 6.6. The “intramural” neurons are believed to be primary sensory neurons connected to the mechanoreceptors in the setae.

     

The ultrastructure and distribution of 5-HT-containing neurons in the intestine of a teleost fish,Aldrichetta forsteri

Summary The intestine of Aldrichetta forsteri was examined ultrastructurally for evidence of 5-HT-containing intrinsic neurons. A population of nerve cell bodies and processes characterized by the presence of amine-containing vesicles was found after the destruction of adrenergic processes with 6-hydroxydopamine. The distribution of the neurons matched that of the 5-HT-containing neurons previously seen with formaldehyde-induced fluorescence and 5-HT immunohistochemistry in A. forsteri and other teleosts. The axons made close contacts with the circular smooth muscle layer and the muscularis mucosae. The axons also lay exposed to the epithelial cells of the mucosa but did not make contact with neurons or with the longitudinal smooth muscle layer.     

人胎大肠氮能神经元发育的研究

By using histochemical methed of NADPH-diaphorse, the development of the nitrergic neurons in the large intestine of human fetus were studied. The results showed: At the fifth month of gestation, weak positive reaction of nitric oxide synthase (NOS) appeared in part of the round cells of intermuscular ganglia. The round cells differentiated into the nitrergic nerve cells. At the sixth month, the bodies of nitrergic neurons were obviously enlarged, the processes of which were lengthened. The nitrergic nerve fibers were seen in the muscle layer, the submucosa and the base of the intestinal gland. The growth and development of nitrergic neurons reached its peak at the seventh month. The number of intermuscular ganglionic cells was increased. The density of nitrergic nerve fibers was increased in the inner circular muscle layer, and have bead-like structures. At the eighth to tenth month, the staining intensity of nitrergic neurons was increased. The myenteric plexus was densely distributed with nitrergic nerve cell bodies, whereas the submucosa and the inner circular muscle layers contained only a few neurons. The nitrergic nerve fibers were observed in all layer of large intestine, the density of the distribution of nitrergic nerve fibers was by far the highest in the inner circular muscle layer, less in the submucosa and outer longitudinal muscle layer, and only a few were found in the mucous layer. To our knowledge, it is the first time that the development of nitrergic neurons in the large intestine of human fetus was demonstrated.     

人胎大肠氮能神经元发育的研究

用NADPH-d组织化学法对人胎大肠氮能神经元的发育进行了观察.结果表明第5个月胎龄时,肌间神经节处圆形细胞中部分细胞出现一氧化氮合酶(NOS)阳性反应,并分化成氮能神经细胞.第6个月胎龄时,氮能神经元胞体增大,突起伸长,在肌层、粘膜下层和肠腺基部出现氮能神经纤维分布.第7个月胎龄时,氮能神经元生长发育达到高峰,肌间神经节细胞数目增多,环肌层神经纤维分布密度增加,膨体结构明显.第8-10个月胎龄时,氮能神经元染色强度加深,其胞体分布以肌间神经节最多,粘膜下层和内环肌层较少.氮能神经纤维的分布密度以内环肌层最高,粘膜下层和外纵肌层次之,粘膜层较低.本研究揭示了大肠氮能神经元发育的变化规律.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the presence of enterochromaffin cells

Summary The formaldehyde-induced fluorescence technique had shown 5-hydroxytryptamine-containing enteric neurons in the intestine of the teleost Platycephalus bassensis, but did not reveal such neurons in the intestine of Tetractenos glaber or Anguilla australis. Re-examination of these animals with 5-hydroxytryptamine immunohistochemistry showed immunoreactive enteric neurons in the intestine of all three teleost species. The 5-hydroxytryptamine-containing enteric neurons showed essentially the same morphology in all species examined: the somata were situated in the myenteric plexus, extending down into the circular muscle layer, but none were found in the submucosa; processes were found in the myenteric plexus, the circular muscle layer and the lamina propria. It was concluded that the neurons may innervate the muscle layers or the mucosal epithelium, but were unlikely to be interneurons. In a range of teleosts, enterochromaffin cells were found in the intestine of only those species in which the formaldehyde technique did not visualize neuronal 5-hydroxytryptamine. Available evidence suggests that, in vertebrates, 5-HT-containing enterochromaffin cells are lacking only where there is an innervation of the gut mucosa by nerve fibres containing high concentrations of 5-HT.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Ultrastructure of the nerve-muscle junction in the pharynx of the earthworm,Lumbricus terrestris

Summary InLumbricus terrestris the wall of the pharynx is built up from obliquely striated longitudinal and circular muscle layers. The occurrence of perikarya and nerve bundles showing green fluorescence suggests the presence of aminergic innervation in the pharynx. A significant number of chemical synapses were detected in the neuropil among axon terminals. The junctional gap is generally 100–300 nm wide in type I junctions which resemble the cholinergic motor endplates of vertebrate skeletal muscle. A narrow junctional gap of about 25 nm is characteristic of the close contacts in the type II neuromuscular junction. Agranular spherical vesicles, together with small and large dense-cored granules, fill in these axon terminals.     

Predicting exotic earthworm distribution in the northern Great Lakes region

Identifying influences of earthworm invasion and distribution in the northern Great Lakes is an important step in predicting the potential extent and impact of earthworms across the region. The occurrence of earthworm signs, indicating presence in general, and middens, indicating presence of Lumbricus terrestris exclusively, in the Huron Mountains located in the Upper Peninsula of Michigan were modeled using generalized linear models and stepwise regression to identify important environmental variables. Models were then applied to earthworm occurrence data from Seney National Wildlife Refuge, also located in the Upper Peninsula of Michigan to validate results. Occurrence of earthworm signs was associated with high soil pH, high basal area of earthworm preferred overstory species, and north facing aspects. Middens of L. terrestris were associated with high soil pH, high basal area of preferred species, and close proximity to roads. The resulting model for L. terrestris was incorporated into a geographic information system (GIS) to map the expected distribution, both current and potential, across the study area. Results indicate that L. terrestris has not yet fully saturated its potential habitat, as it is currently found close to roads and has yet to establish in most interior forests sampled. Comparing field measured data to GIS layers revealed limitations in the precision of publicly available spatial data layers that should be addressed in future attempts to predict the extent of earthworm invasion across the larger Great Lakes region. However, within the Huron Mountains, it is predicted that the distribution of L. terrestris will cover, at minimum, 41 % of the area.     

Thyroglobulin-like immunoreactivity in the nervous system of Eisenia foetida (Annelida,Oligochaeta)

Summary Immunohistochemical studies were performed by use of specific rabbit antisera and purified antibodies to human Tg on cephalic and body sections of Eisenia foetida and on cephalic sections of Lumbricus terrestris. Secondary antisera, either fluorescein- or peroxidase-conjugated, were used to identify the immunoreaction. Immunoreactive perikarya and some immunoreactive nerve fibres were detected in both the cerebral ganglion and the ventral nerve cord of E. foetida. From 8 to 19 Tg-like positive neurons per frontal section were observed in the brain, mainly in the dorsal zone. From 2 to 4 positive perikarya per ganglion were found in sagittal sections of the ventral nerve cord with a repetitive distribution. Numerous positive neurons were also found in the cephalic segments of L. terrestris. The present results indicate that a substance immunologically related to mammalian Tg is synthesized in earthworms. This suggests that some conservative sequences of Tg structure arose very early in evolution and supports the idea of a common evolutionary origin for endocrine and nervous systems.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.     

The tyrosinase activity of melanosomes from the harding-passey melanoma: The absence of a peroxidase component in vitro

Melanosomes were isolated from the Harding-Passey melanoma with a density gradient technique. Using the Pomerantz radioassay for tyrosinase activity it was found that these isolated melanosomes could hydroxylate tyrosine in the presence of catalase sufficient to deny the enzyme any hydrogen peroxide. It was further found that the rate of hydroxylation was unaffected by the presence of exogenous hydrogen peroxide. Tyrosinase activity could be suppressed by preincubation in diethyldithiocarbamate followed by removal of this inhibitor before enzyme assay. Attempts to regain enzymatic activity, however, by addition of copper II ions were unsuccessful. No peroxidase activity could be detected on the isolated granules, and indeed evidence for a peroxidase inhibitor on the granules was found. It was also found that the peroxidase activity present in a 20% homogenate of mouse muscle did not demonstrate any tyrosinase activity with the Pomerantz assay even in the presence of hydrogen peroxide. It is concluded from these studies that there is tyrosinase on these melanosomes which is capable in vitro of hydroxylating tyrosine without any contribution from an active peroxidase.     

Endogenous peroxidase in the lacrimal gland of the rat and its differentiation against injected catalase and horseradish-peroxidase

Summary The localization of endogenous peroxidase was studied in the glandula orbitalis (lacrimalis) externa of the rat by the method of Graham and Karnovsky (1966). Reaction product is visible in all cisternae of the rough endoplasmic reticulum including the perinuclear cisternae, in condensing vacuoles, and in all secretion granules. The Golgi cisternae seldom are peroxidase positive. Intercalated duct cells rarely contain reaction product in a few scattered cisternae of the rough endoplasmic reticulum and in secretion granules.After the injection of beef liver catalase reaction product is found in the capillary lumen. Both the injected catalase and the endogenous peroxidase are completely inhibited by 10^–2M aminotriazole, while the pseudoperoxidatic activity within the erythrocytes persists. After injection of horseradish-peroxidase reaction product is visible within the capillary lumen and also in the intercellular spaces between lacrimal gland cells. 10^–2M aminotriazole completely inhibits the endogenous peroxidase while the exogenous horseradish-peroxidase remains unaffected. The inhibitory effect of aminotriazole is not specific for catalase since lacrimal gland peroxidase is also inhibited.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.     

Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence

The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.     

Dopamine melanin‐loaded PC12 cells: a model for studies on pigmented neurons

The most conspicuous feature in idiopathic parkinsonism is the degeneration of pigmented neurons in the substantia nigra. A major problem for the study of the significance of neuromelanin for the development of parkinsonism is that common experimental animals lack neuromelanin in substantia nigra. The aim of this study was to develop an in vitro model that could be used to study the role of neuromelanin in chemically induced toxicity in dopaminergic cells. Cultured neuron‐like PC12 cells were exposed to synthetic dopamine melanin (0–1.0 mg/ml) for 48 h, resulting in uptake of dopamine melanin particles into the cells. The intracellular distribution of dopamine melanin granules was similar to that found in neuromelanin‐containing neurons. Dopamine melanin, up to 0.5 mg/ml, had negligible effects on ultrastructure, induction of the endoplasmic reticulum‐stress protein glucose regulating protein 78, activation of caspase‐3 and cell viability. The decreased cell viability in response to the cytotoxic peptide amyloid‐β_25?35 was similar in melanin‐loaded cells and in control cells without melanin. The results of the studies suggest that melanin‐loaded PC12 cells can serve as an in vitro model for studies on the role of neuromelanin for the toxicity of chemicals, in particular neurotoxicants with melanin affinity, in pigmented neurons.     

Hepatoprotective activity of <Emphasis Type="BoldItalic">Tribulus terrestris</Emphasis> extract against acetaminophen-induced toxicity in a freshwater fish (<Emphasis Type="BoldItalic">Oreochromis mossambicus</Emphasis>)

The potential protective role of Tribulus terrestris in acetaminophen-induced hepatotoxicity in Oreochromis mossambicus was investigated. The effect of oral exposure of acetaminophen (500 mg/kg) in O. mossambicus at 24-h duration was evaluated. The plant extract (250 mg/kg) showed a remarkable hepatoprotective activity against acetaminophen-induced hepatotoxicity. It was judged from the tissue-damaging level and antioxidant levels in liver, gill, muscle and kidney tissues. Further acetaminophen impact induced a significant rise in the tissue-damaging level, and the antioxidant level was discernible from the enzyme activity modulations such as glutamate oxaloacetic transaminase, glutamate pyruvic transaminase, alkaline phosphatase, acid phosphatase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, lipid peroxidase and reduced glutathione. The levels of all these enzymes have significantly (p < 0.05) increased in acetaminophen-treated fish tissues. The elevated levels of these enzymes were significantly controlled by the treatment of T. terrestris extract (250 kg/mg). Histopathological changes of liver, gill and muscle samples were compared with respective controls. The results of the present study specify the hepatoprotective and antioxidant properties of T. terrestris against acetaminophen-induced toxicity in freshwater fish, O. mossambicus.     

EDTA reduces heavy metal impacts on Tribulus terrestris photosynthesis and antioxidants

The effects of EDTA application to heavy metal-polluted soil on phytoextraction of heavy metals, leaf anatomy, gas exchange parameters, enzyme activities of C₄ carbon cycle, antioxidant defense, and active compounds of Tribulus terrestris L. were evaluated. The addition of EDTA to the soil polluted with Cd and Pb markedly increased dry weight and Pb, Zn, and Cd contents in shoots. Plants responded to the action of EDTA by an increased stomatal conductance, photosynthetic and transpiration rates, water use efficiency, chlorophyll and carotenoid contents. The activities of C₄ carbon cycle enzymes simultaneously increased, thus concentrating CO₂ for enhanced CO₂ assimilation and providing NADPH for the antioxidant system. Antioxidants, such as ascorbate, reduced glutathione, and flavonoids, increased more in the shoots of T. terrestris after the addition of EDTA. The activities of guaiacol peroxidase, catalase, and the enzymes of the ascorbate-glutathione cycle enhanced significantly in the presence of EDTA. Increased activities of antioxidant enzymes suggest that they have some additive functions in the mechanism of metal tolerance. EDTA application lowered the activity of phenylalanine ammonia-lyase and the content of total phenols, MDA, hydrogen peroxide, dehydroascorbate, and lipid-soluble antioxidant capacity expressed as α-tocopherol. Increased levels of total radical-scavenging activity are in correspondence with the activity of water-soluble antioxidant compounds in T. terrestris tissues. The content of furostanol saponins protodioscin, prototribestin, and rutin increased as a result of EDTA addition. The results obtained allowed us to assume that applied EDTA reduced a negative heavy metal impact on puncture vine photosynthesis and antioxidant potential.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

Relationship between interstitial cells of Cajal and enteric motor neurons in the murine proximal colon

Interstitial cells of Cajal (ICC) are interposed between enteric neurons and smooth muscle cells in gastrointestinal (GI) muscles. The specific relationships between these cells in the murine proximal colon were studied with conventional and immunoelectron microscopy and immunohistochemistry. Intramuscular interstitial cells (IC-IM) formed discrete networks within the circular muscle layer of the murine proximal colon. Nerve trunks ran in close association with IC-IM and individual nerve trunks came into close contact with multiple IC-IM. Conventional electron microscopy revealed very close (< or = 20 nm) associations between nerve fibers and IC-IM. Processes of IC-IM also formed close contacts with neighboring smooth muscle cells. At the points of close association between neurons and IC-IM, areas of membrane densification in both pre- and postjunctional cells were present, suggesting specialized contacts or synaptic-like structures. Similar points of contact between neurons and smooth muscle cells were extremely rare. Immunoelectron microscopy demonstrated that IC-IM formed close associations with neurons containing nitric oxide synthase-like immunoreactivity (NOS-LI) or vesicular acetylcholine transporter-like immunoreactivity (vAChT-LI), suggesting innervation by both inhibitory and excitatory motor neurons. IC-IM were also labeled with anti-NOS antibodies. These observations suggest that IC-IM are an integral part of the neuromuscular junction in the colon. These cells may be the primary site of innervation, and neural regulation of the musculature may occur via IC-IM.