Neurocytology of the cerebral ganglion of Fasciola hepatica (Platyhelminthes).

An ultrastructural study of the organization and fine structure of the nervous system of the parasitic flatworm Fasciola hepatica was undertaken. The brain consists of paired cerebral ganglia, located just posterior to the oral sucker, that are connected by a transverse commissure which crosses over the dorsal surface of the pharynx. The cell bodies of the cerebral ganglia are not organized into a clearly defined rind around the neuropile but are loosely scattered around and within the neuropile area. The neuropile consists of two morphologically distinct types of unmyelinated nerve processes. The small nerve processes, with smooth cell membranes, are less than 2 micron in diameter, whereas the "giant" processes are greater than 12 micron in diameter and have extensively invaginated cell membranes. Giant processes make up the bulk of the nerve fibers in the transverse commissure and longitudinal nerve cords. Four morphological types of vesicles are present in the small processes; small clear vesicles (which were found associated with synapses), spheroidal and ellipsoidal dense vesicles, and dense-core vesicles. Two types of synapses are found in the neuropile: simple synapses characterized by pre- and postsynaptic membrane densities, clusters of small clear vesicles, and a clear synaptic cleft; and wedge-shaped synapses or divergent diads each having one presynaptic process synapsing onto two postsynaptic processes. Synaptic contacts were observed only between small processes; no synapses were observed on the cell bodies or on giant processes.

A study of phosphoenolpyruvate carboxykinase from Moniliformis dubius (Acanthocephala).

Procedures have been developed for the extraction and subsequent purification of the enzyme phosphoenolpyruvate carboxykinase (GTP) (PEPCK) from Moniliformis dubius (Acanthocephala), a parasite of the rat small intestine. This is believed to be the first purification of PEPCK from an invertebrate animal. The enzyme, when purified to homogeneity as indicated by electrophoretic criteria, has a molecular weight of 73 700. Kinetic studies indicated that the enzyme had a pH optimum of 5.5 and required Mn2+ as the divalent cation. The apparent Km values determined for the substrates of the carboxylation reaction were low compared with the published values for purified PEPCK from vertebrate sources. Several competitive inhibitors were found and their Ki values determined. The possible regulation of PEPCK activity in M. dubius is discussed with reference to the observed kinetic parameters.

Effect of 5-hydroxytryptamine (5-HT; serotonin) on in vitro glucose uptake and glycogen reserves in Hymenolepis diminuta.

Glucose uptake by Hymenolepis diminuta was linear for up to 60 min following in vitro incubation in 10 mM glucose. Following the addition of 1 mM 5-HT, with a 95% O2/5% CO2, gas phase, glucose uptake by H. diminuta was significantly (P less than 0.001) enhanced, remaining linear and parallel to that of control groups between 15 and 60 min incubation. Under air, the rates of glucose uptake were higher, but were only significantly increased by 5-HT during the first 30 min of incubation. In further experiments, in the absence of glucose in the incubation media worm glycogen reserves decreased by over 50% after 60 min. With the addition of 1 mM 5-HT, the reduction in glycogen content was significantly (P less than 0.025) greater, exceeding 65% after 60min. When glucose was added to the incubation media, worm glycogen reserves were not significantly depleted irrespective of the presence or absence of 5-HT. Incubations under a 95% O2/5% CO2 gas phase did not significantly influence glycogen content compared to corresponding groups incubated in air. The results suggest that 5-HT stimulates glycolysis in H. diminuta through increased glucose uptake by the worm or by a reduction in glycogen reserves in the absence of external glucose.

The behavior of juvenile Fasciola hepatica.

The behavioral repertoire of the infective stage of Fasciola hepatica was qualitatively characterized. During activation, a primary activity was the emptying of the ceca by peristaltic-like contractions. Emergence behavior comprised coordinated patterns of body movement and sucker activity specifically directed at disruption of the ventral plug. The stimulus specificity of the emergence response for glycine-conjugated cholic acid and the log dose-effect relationship of this response with glycocholic acid suggested a receptor-mediated sensory recognition. Extracts from the duodenum (departure organ) and the liver (arrival site) significantly affected the rate of locomotion and the orientation of the migratory stage. The evidence for orientation in the migrating stages is unequivocal, but the mechanisms by which they orient are unclear.

Ultrastructural morphology of the neuropile of the cerebral ganglion of Moniliformis moniliformis (Acanthocephala).

The cerebral ganglion of Moniliformis moniliformis consists of an outer single layer of cell bodies and an inner core of neuropile occupied by cellular processes. Morphological characteristics of 5 types of cellular processes have been described. Most of the processes in the neuropile were of large size, although sites containing numerous small processes (neurites) were also observed. Small processes in the neuropile were the sites of synaptic interaction. The majority of presynaptic membranes were well defined by the presence of electron-dense material at the inner leaflet, while postsynaptic membranes appeared to lack marked postsynaptic density. Round or flattened lucent vesicles were concentrated around the presynaptic membranes. Electron-dense and dense-cored vesicles were usually separated from the clear vesicles and situated at some distance from synaptic sites.

Effect of electrical vagal stimulation on migration of hymenolepis diminuta.

Stimulation of the left efferent vagal nerve (2 msec, 5 V, 10 Hz), for up to 60 min in rats infected with 11-day-old Hymenolepis diminuta induced anteriad migration of worm scolex and biomass in the small intestine. Ligation at the pyloric sphincter prior to vagal stimulation shortened the length of time before significant relocation occurred and increased the migrational response. The results support the hypothesis that work migration is mediated via vagal stimulation of gastrointestinal function, and is unrelated to actual feeding.

Effects of cholinergic drugs on muscle contraction in Moniliformis moniliformis (Acanthocephala).

In whole Moniliformis moniliformis spontaneous muscle contractions were rhythmic; longitudinal contractions were measured with a force transducer. The cholinergic agonists levamisole and nicotine significantly increased muscle tension in whole worms; these contractions were tonic and were antagonised by the ganglionic blocker pentolinium and by piperazine. In addition, levamisole-induced contractions were inhibited by gallamine, hexamethonium, and norepinephrine. In worm segments, where drugs in solution were injected through the worms, acetylcholine (ACh) and nicotinic agonists were effective in causing contractions, whereas muscarinic agonists in concentrations up to 1 mM had no effect. Although muscle contraction in M. moniliformis was induced by nicotinic agonists, these contractions were effectively antagonised by a range of chemicals that block ganglionic, skeletal, and muscarinic sites in vertebrates. The presence of ACh in M. moniliformis and the effects of nicotinic agonists on muscle contraction suggest that ACh is a putative excitatory neurotransmitter.

Effect of size of Trichinella spiralis (Nematode) infections on glucose and ion transport in the rat intestine.

An in vivo perfusion technique, using 3 intestinal loops representing the anterior, mid and posterior regions of the rat small intestine, was used to determine intestinal glucose uptake 5 days after infection with Trichinella spiralis. At high levels of infection (3,000 and 6,000 larvae/rat) net glucose absorption by the intestinal mucosa was significantly impaired in all regions of the small intestine when compared to uninfected controls. At low levels of infection (50 larvae/rat) glucose uptake by the mucosa was significantly enhanced in all 3 regions of the small intestine. Intermediate levels of infections (200-1,000 larvae/rat) also enhanced glucose uptake, but only in the anterior regions of the small intestine. When washings from the small intestine of rats infected with 50 larvae/rat were added to the perfusion fluid used on uninfected rats, glucose uptake was also significantly enhanced. These results suggest that at low levels of infection the intestinal lumen contains a metabolite which may affect the mucosal transport of glucose and the related fluxes of H2O, Na+, Cl-, and K+, in the rat intestine. Luminal [H+] and pCO2 decreased from the proximal to distal regions of the small intestine following perfusion; pO2 was significantly decreased in the proximal and distal regions.

Purification and properties of phosphoenolpyruvate carboxykinase from Hymenolepis diminuta (Cestoda).

A method is described for the purification of the enzyme phosphoenolpyruvate carboxykinase (PEPCK) from the cestode Hymenolepis diminuta. When purified to electrophoretic homogeneity, the enzyme had a molecular weight of 70,600 and an isoelectric point of 7.5. Kinetic studies indicated that the pH 5.6 was optimal for the carboxylation reaction and that Mn++ was the preferred divalent cation; there was no activity of the enzyme in the presence of Mg++. Apparent Km values for the carboxylation reaction were determined; those for GDP (20.6 muM) and PEP (38.9 muM) were lower than the values previously reported. GTP, GMP, ITP, IMP, fumarate, succinate and alpha-ketoglutarate were found to be competitive inhibitors and their Ki values determined.

The effects of bile on the locomotory cycle of Fasciola hepatica.

The rate of production of locomotory cycles in juvenile Fasciola hepatica was significantly decreased following treatment with conjugated chenodeoxycholic acids and significantly increased following treatment with dehydrocholic acid or whole bovine bile. Deoxycholic acid caused death and lysis of the parasites in less than 30 min. These results suggest that bile components act as a specific stimulus for a fixed action pattern (the locomotory cycle) in this parasite.

Serotonin and distribution of radiocarbon from D-[14C-U]-glucose in Schistosoma mansoni.

Following a 60 min in vitro incubation of Schistosoma mansoni with D-[14C-U]-glucose 76% of the radiocarbon was incorporated into metabolic end products and excreted back into the medium. In the presence of 5-HT uptake of glucose increased 61%; excreted end products accounted for 87% of the radiocarbon, indicating increased levels of energy utilization. Substantial amounts of radiolabelled carbon from D-[14C-U-]-glucose were incorporated into glycogen, lipids, amino acids and proteins, suggesting the utilization of glucose carbon in the biosynthetic processes of the parasite. Incorporation of glucose carbon was diminished in the presence of 5-HT, indicating the priority of energy generation over biosynthesis to meet the demands of increased muscular activity.

Effects of cholinergic drugs on longitudinal muscle contractions of Fasciola hepatica.

Acetylcholine, cholinergic agonists and acetylcholinesterase inhibitors significantly decrease the amplitude and frequency of spontaneous longitudinal muscle contractions in Fasciola hepatica. In order of their effects on the inhibition of muscle contractions, the cholinergic agonists can be ranked as nicotine greater than carbachol greater than acetylcholine. High calcium ion concentration also causes a significant inhibition of contractions. Atropine, a cholinergic antagonist that acts on muscarinic receptors, significantly increases the amplitude and frequency of spontaneous contractions and completely reverses the effects of cholinomimetic drugs, including nicotine. In adult F. hepatica, the levels of acetylcholine and its precursor choline are 3.14 +/- 0.55 and 13.75 +/- 3.72 pmol/mg wet weight, respectively. The activities of choline acetyltransferase, specific acetylcholinesterase and the nonspecific cholinesterase are 1.25 +/- 0.19, 238.0 +/- 13.0, and 83.0 +/- 33.0 nmol/hr/mg protein, respectively.

Histochemical localization of acetylcholinesterase in the cerebral ganglia of Fasciola hepatica, a parasitic flatworm.

Acetylcholinesterase activity was found in the cell bodies and extracellularly in the neuropile of the cerebral ganglia of the adult trematode parasite, Fasciola hepatica. Within neuronal cell bodies of the cerebral ganglion, acetylcholinesterase reaction product was found in the endoplasmic reticulum, in the cisternae of the Golgi apparatus, and in secretory vesicles near the inner (releasing face) cisternae. Acetylcholinesterase reaction product was not seen intracellularly within any nerve processes. The reaction product was found around the somatic cell membranes and in the extracellular space between closely apposed nerve processes in the neuropile. Acetylcholinesterase reaction product was associated with synaptic endings that contained clear spheroidal synaptic vesicles, and the reaction product was localized at the site of synaptic contact between the zone of apposition of the pre- and postsynaptic terminals. This intracellular and extracellular distribution of the enzyme is consistent with its function as the degrading enzyme in cholinergic transmission.

Ultrastructural morphology of the nerve cells in the cerebral ganglion of the Acanthocephalan Moniliformis moniliformis.

The morphology of the cerebral ganglion of the Acanthocephalan Moniliformis moniliformis was studied in serial sections using electron microscopy. The organization of the cerebral ganglion was typical of other invertebrates with the cell bodies forming a rind, 1 cell thick, and their processes forming the central core of the neuropile. The ganglion was surrounded by a connective tissue capsule composed of collagen-like fibrils. Externally, the free surface of the cell bodies was covered by an electron-dense extracellular lamina. Seventy-six cells were identified in every ganglion examined and, on the basis of their cellular characteristics, they were divided into 5 distinct cell types, classified as type A, B, C, D and E cells. The characteristic morphological features of each cell type have been described, and the distribution of the different cell types in the cerebral ganglion was mapped.

Hymenolepis diminuta: behavioral effects of 5-hydroxytryptamine, acetylcholine, histamine and somatostatin.

Consistent in vitro behavioral patterns were found in the scolex and strobila of adult Hymenolepis diminuta. These patterns were measured with a force transducer and the behavior analyzed with a slow motion closed circuit T.V. Varying concentrations of serotonin (5-HT), acetylcholine (Ach), histamine and somatostatin, in the range of 10(-3) to 10(-9) M, were tested for their influence on the rhythmic patterns of behavior. High concentrations of 5-HT and of Ach decreased scolex motility. While 5-HT significantly increased motility in the anterior-, mid- and posterior regions of the strobila at 10(-3) M, Ach inhibited motility in all 3 regions of the strobila at the same concentrations. At high concentrations, somatostatin had a smaller stimulatory effect on worm motility in the anterior and mid-regions; histamine only significantly affected worm motility in the posterior region of the strobila. Depending on concentration, the action of 5-HT, Ach and histamine can be reversed, particularly in the anterior and posterior regions of the strobila. The in vivo assay for worm migrational responses suggests that the action of the neuromuscular stimulators and inhibitors on worm migration is indirect.