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.

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.

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.

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.

Effects of 5-hydroxytryptamine, cyclic AMP, AMP, and fructose 2,6-bisphosphate on phosphofructokinase activity in Hymenolepis diminuta.

1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK 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.

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.

Schistosoma mansoni: effects of in vitro serotonin (5-HT) on aerobic and anaerobic carbohydrate metabolism.

The effect of Serotonin on carbohydrate metabolism, excreted end products, and adenine nucleotide pools in Schistosoma mansoni was determined following 60 min in vitro incubations under air (= 21% O2) and anaerobic (95% N2:5% CO2) conditions. In the presence of 0.25 mM Serotonin, glucose uptake increased by 82-84% and lactate excretion increased by 77-78%; levels of excreted lactate were significantly higher under aerobic than under anaerobic conditions. The tissue pools of glucose, hexosephosphates, fructose 1,6-bisphosphate, pyruvate, and lactate were significantly increased under anaerobic conditions compared to air incubation; the presence of Serotonin decreased tissue glucose pools and increased the size of the pyruvate and lactate tissue pools. The glycolytic carbon pool was significantly greater under anaerobic than under aerobic conditions, irrespective of Serotonin. Serotonin increased adenosine 5'-diphosphate and adenosine 5'-monophosphate levels under aerobic conditions; neither Serotonin nor gas phase significantly affected total adenine nucleotide levels or the adenylate energy charge. Serotonin increased energy requirements by S. mansoni due to increased muscle contractions; demand was met by enhanced rates of carbohydrate metabolism. Irrespective of gas phase, 74-78% of available carbohydrate was converted to lactate. In the presence of Serotonin, conversion of glucose to lactate was reduced to 63-67%. In view of the requirements by S. mansoni for an abundant supply of glycoprotein and glycolipid precursors for surface membrane renewal, it is suggested that carbohydrate (glucose and glycogen) that was not converted to lactate may have been incorporated into biosynthetic processes leading to membrane synthesis.

Heligmosomoides polygyrus: inoculum size and glucose, ion, and gas fluxes in the small intestine of mice.

Female CDI mice were inoculated with 10, 50, 100, 250, or 500 larvae of Heligmosomoides polygyrus. At Days 7, 9, and 12 after infection, the anterior third of the small intestine was perfused using an in vivo technique. The distribution of worms in the mouse intestine was determined after 7, 9, and 12 days. All worms that were recovered were from the proximal half of the small intestine. When compared to uninfected controls, there was a significant increase (+56%) in glucose absorption of the small intestine at Day 7 after infection with inocula of 50 and 100 larvae; at Day 9, glucose absorption was significantly increased with a 10-larvae inoculum. A decrease in glucose absorption occurred at Days 7 and 9 after infection with a 500-larvae inoculum. Net water absorption was significantly increased (+183%) with the 50- and 100-larvae inocula at Day 7, but was significantly reduced at Day 9 after infection with the 50-, 100-, 250-, and 500-larvae inocula. Both Cl- and Na+ absorption were significantly increased with the 50-, 100-, and 250-larvae inocula at Day 7 after infection; at 9 and 12 days, there was significant net secretion of both ions. In control mice, there was net secretion of K+, while with the 50-, 100-, and 250-larvae inocula on Day 7 there was significant net absorption of K+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Neuromuscular physiology of Hymenolepis diminuta and H. microstoma (Cestoda).

The physiology of the neuromuscular systems in Hymenolepis diminuta and H. microstoma was studied in vitro using intact, adult worm and strips of worm body wall. Intact worms were insensitive to ionic changes in the in vitro buffering system. However, strips of body wall containing longitudinal muscles were extremely sensitive to ionic manipulation. In intact worms tension generated in the strobila had two components; small brief tension peaks up to 500 mg amplitude are superimposed on larger, longer peaks of up to 1200 mg amplitude. Removal of the scolex and neck region either failed to show significant changes in tension, or showed a reduction in amplitude but not of frequency. Muscle contraction of both H. diminuta and H. microstoma were qualitatively similar. In split-worm preparations the concentration of Ca2+ in the bathing solution significantly affected both spontaneous and evoked contractions in H. diminuta and H. microstoma; the addition of CaCl2 greatly reduced the amplitude and frequency of the contractions. The chloride salts of cobalt, barium, cadmium and manganese elicited prolonged contractions of the longitudinal musculature of both H. diminuta and H. microstoma. While CoCl2 was the most effective in stimulating muscle contraction, the magnitude of the response varied with the concentration of Ca2+ in the bath. The results indicate that peripheral inhibition is extremely important in cestode motor control and that extracellular calcium ions may regulate the peripheral inhibitory mechanisms.

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.

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.

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.

Migrational responses of Hymenolepis diminuta to surgical alteration of gastro-intestinal secretions.

The effects of the direction of gut flow, of injections of glucose and saline into different regions of the small intestine and of surgical re-routing or ligature of gastric, biliary and pancreatic secretions into the small intestine have been correlated with changes in the migratory response of the rat tapeworm Hymenolepis diminuta. Reversing the normal anterior to distal flow of luminal contents in the small intestine did not affect worm migration following feeding. Injections of a glucose-saline solution into the duodenum did not initiate a migratory response; similar injections into the mid- and posterior regions of the small intestine resulted in migrational responses similar to those following intragastric glucose feeding. Re-routing gastric secretions to the distal duodenum inhibited anterior migration of the worms beyond the new point of entry of gastric juices. Results following re-routing and ligation of the biliary and pancreatic secretions suggest that there is a potent cue to anteriad migration in the pancreatic secretions. Biliary secretions also appear to contain an additional migratory cue to worm migration. In order of importance the factors stimulating/inhibiting worm migration are pancreatic greater than gastric greater than biliary greater than glucose. The results support the hypothesis that the factors affecting worm distribution in the small intestine are interactive and synergistic, involve other luminal factors, such as 5-hydroxytryptamine and the physico-chemical gradients, and are of a regional nature such that the migratory response of a particular worm is directly related to its position in the small intestine when the cues to relocation are received.