The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria.

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2h and 12h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml(-1) bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2h after treatment but viability decreased up to 8h. Even very low concentrations of bvPLA2 (10(-12) mg ml(-1)) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2h bacterial cultures but bacteriostatic to 12h ones. Minimum bactericidal concentrations were 10(-5)-10(-6) mg ml(-1). The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.

The structure, distribution, and quantitative relationships of the glia in the abdominal ganglia of the horse leech, Haemopis sanguisuga.

The glial cells in abdominal ganglia of the horse leech Haemopis sanguisuga were studied by electron microscopy and analysed quantitatively to evaluate the suitability of this easily obtainable carnivorous species for physiological studies. Each abdominal ganglion contains eight giant glial cells, 12,000-14,000 small glial cells, and approximately 300 neurons. The giant glial cells constituted 44.6% and the small glial cells 6.4% of the ganglion's volume. The giant glial cells contain glycogen and bundles of filaments that are chiefly located in their periphery, close to the neurons into which they send processes. The small glial cells are frequently surrounded by the giant glial cells but also occur around neuronal perikarya and axon tracts, as well as against the basal lamina and connective tissue layers. The small glial cells contain lysosomes and sometimes form a trophospongium with the neurons. A system of extracellular channels, which is continuous with the basal lamina, indents the giant glial cells and extends around parts of the neurons. The extracellular channels contain a matrix that appears very similar to the basal lamina and to the cytoplasm in the processes of the small glial cells. Some of the extracellular channels contain collagen fibrils. Hemidesmosomes join the matrix-filled extracellular channels to both the neurons and the giant glial cells. Ionic lanthanum has a free access to the neurons and glial cells via the extracellular spaces and matrix-filled channels. Areas of synaptic intermingling rarely contain glial cell processes.

Lipofuscin accumulation and its prevention by vitamin E in nervous tissue: quantitative analysis using snail buccal ganglia as a simple model system.

The total numbers and sizes of the lipofuscin granules in buccal ganglia of the pond snail Planorbis corneus were measured by light microscope morphometry of serial sections in different groups of animals. One group, weight range 0.5-5.0 g (age 1-4 years), was maintained on a lettuce diet (low Vitamin E content). Lipofuscin content increased with age by over a 100 times in this group. The lipofuscin deposits accumulate in the glial cells at the periphery of the ganglia. The other group, weight range 0.5-5.0 g was fed a supplemented Vitamin E diet for 4 months before quantitisation of the lipofuscin. This diet prevented lipofuscin appearance in the young animals, and reduced the lipofuscin content by 50% in the old animals. The findings provide direct evidence that Vitamin E reduces lipofuscin accumulation in glial cells in intact nervous tissue. The buccal ganglia of Planorbis provide a useful model system for studying age and dietary related alterations of lipofuscin in nervous tissue.

Incorporation of [3H]2-deoxyglucose into glycogen in nervous tissues.

Mice were injected with [3H]2-deoxyglucose and after 1 h high molecular weight glycogen was extracted from brain, liver and muscle tissues. 1-2% of the total radioactivity in each tissue was recovered in the glycogen fraction. Isolated buccal ganglia of the pond snail, Planorbis, and isolated abdominal ganglia of the horse leech Haemopis, were exposed in vitro to [3H]2-deoxyglucose for 1 h. 1-10% of the total radioactivity in these tissues was located in the high molecular weight glycogen fraction. Treatment of the extracted labelled glycogen fractions with amyloglucosidase caused release of the label in a manner consistent with the breakdown of labelled glycogen. Ganglia of snail and leech were exposed to [3H]2-deoxyglucose, fixed in glutaraldehyde and osmium tetroxide solutions, and prepared for autoradiography using aqueous histological processing. Light and electron microscope autoradiography showed that over 90% of the label was positively associated with glycogen particles (alpha- and beta-particles). Certain previously published reports on the incorporation of 2-deoxyglucose into glycogen are discussed in relation to these findings. It is concluded that [3H]2-deoxyglucose is partially incorporated into glycogen in nervous tissue; the labelled 2-deoxyglycogen withstands aqueous histological processing and can be visualized directly by autoradiography.

Effects of 5,7-dihydroxytryptamine on an identified 5-hydroxytryptamine-containing neurone in the central nervous sytem of the snail Helix pomatia.

1. The effect of 5,7-dihydroxytryptamine (5,7-DHT) on an identified 5-hydroxytryptamine (5-HT)-containing neurone in the CNS of the snail was studied by histochemical, biochemical and electrophysiological methods. 2. Low concentrations of 5,7-DHT decreased the endogenous 5-HT content of the neurone without affecting the amino acids, while relatively large amounts of the drug proportionately lowered 5-HT and in addition slightly decreased the tryptophan and methionine content of the cell. 3. 5,7-DHT blocked the uptake of [3H]-5-HT into the neurone; the close analogue 5,6-DHT was more potent. 4. As well as slightly influencing the accumulation of [3H]-tryptophan by the neurone 5,7-DHT inhibited the metabolism of this amino acid to form 5-HT, probably by affecting the enzyme tryptophan-hydroxylase. 5. 5,7-DHT produced a postsynaptic blockade of transmission from the neurone by blocking the 5-HT receptors of the follower neurones. This effect appeared to be specific for 5-HT receptors. 6. The data support the idea that 5,7-DHT is neurotoxic for indoleamine-containing neurones.

Modulation of glial glycogen metabolism by 5-hydroxytryptamine in leech segmental ganglia.

Leech segmental ganglia (16 out of 23 per animal) were divided into experimental and control groups (4 ganglia per group). The amounts of glycogen in the ganglia were assayed by a specific extraction procedure and fluorimetry, or by liquid scintillation counting following labelling of the glycogen by [(3)H]glucose. Within any individual animal the amounts of glycogen in the ganglia were relatively constant (max. variation 16%). 5-HT (10(?6)-10(?4) M) reduced in a dose-dependent manner the endogenous glycogen (max. 20% reduction), and the [(3)H]glycogen (max. 60% reduction). The glycogenolytic effect was studied by light-microscope autoradiography in serial sections of segmental ganglia previously exposed to [(3)H]glucose. The 5-HT-mediated glycogenolysis was localized principally in the glial cells surrounding the neuron perikarya. 5-HT, in addition to its conventional transmitter role, may regulate the supply of energy substrate from glial cells to neurons within domains defined by the projections of the neurons from which it is released.

Energy utilization and gluconeogenesis in isolated leech segmental ganglia: Quantitative studies on the control and cellular localization of endogenous glycogen.

The isolated segmental ganglia of the horse leech Haemopis sanguisuga were used as a model system to study the utilization and control of glycogen stores within nervous tissue. The glycogen in the ganglia was extracted and assayed fluorimentrically and its cellular localization and turnover studied by autoradiography in conjunction with [(3)H]glucose. We measured the glycogen after various periods of electrical stimulation and after incubation with K(+), Ca(2+), ouabain and glucose. The results for each experimental ganglion were compared to a paired control ganglion and the results analysed by paired t-tests. Electrical stimulation caused sequential changes in glycogen levels: a reduction of up to 67% (5-10 min); followed by an increase of up to 124% (between 15-50 min); followed by a reduction of up to 63% (60-90 min). Values were calculated for glucose utilization (e.g. 0.53 ?mol glucose/gm wet weight/min after 90 min) and estimates derived for glucose consumption per action potential per neuron (e.g. 0.12 fmol at 90 min). Glucose (1.5-10 mM) increased the amount of glycogen (1.5 mM by 30% at 60 min) and attenuated the effects of electrical stimulation. Ouabain (1 mM) blocked the effect of 5 min electrical stimulation. Nine millimolar K(+) increased glycogen by 27% after 10 min and decreased glycogen by 34% after 60 min; 3 mM Ca(2+) had no effect after 10 or 20 min and decreased glycogen by 29% after 60 min. Other concentrations of K(+) and Ca(2+) reduced glycogen after 60 min. Autoradiographic analysis demonstrated that the effects of elevated K(+) were principally within the glial cells. We conclude that (i) the glycogen stores in the glial cells of leech segmental ganglia provide an endogenous energy source which can support sustained neuronal activity, (ii) both electrical stimulation and elevated K(+) can induce gluconeogenesis within the ganglia, (iii) that electrical activation of neurons produces changes in the glycogen in the glial cells which are controlled in part by changes in K(+).

Neuropeptides modulate the transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia.

The effects of four neuropeptides (AKH, FMRFamide, proctolin, VIP) on the alterations in glycogen levels induced by other transmitters were studied in isolated leech segmental ganglia. With the exception of FMRFamide, which produced a small increase (14%) in glycogen, the peptides were by themselves without effect on the glycogen levels. Proctolin abolished the glycogenolytic effects of 5-HT, dopamine and octopamine but not histamine. AKH in combination with ACh produced glycogenolysis although each by themselves were ineffective. AKH modified the effects of other transmitters in different ways i.e. by reduction or reversal of effect. VIP and noradrenaline produced an increase in glycogen (cf. noradrenaline alone which decreased glycogen), but did not modify the effects of the other transmitters. FMRFamide produced a complex variety of modulatory effects on the other transmitters. It is concluded that the glycogen stores in leech ganglia, which are localized principally in the glial cells, may be controlled in complex ways by the different combinations of monoamines, amino acids and neuropeptides.

Transmitter mediated regulation of energy metabolism in nervous tissue at the cellular level.

The Monoamines 5-hydroxytryptamine (5-HT), noradrenaline (NA) and histamine, and the peptide Vasoactive Intestinal Polypeptide (VIP), regulate energy metabolism in nervous tissue, in addition to producing excitation and/or inhibition. These transmitters induce glycogen hydrolysis in a concentration dependent manner. The glycogen breakdown is brought about by increased cyclic AMP formation, or translocation of calcium ions to activate phosphorylase, and is partially localized in glial cells. Data from a diversity of nervous systems, including leech and snail ganglia, and rodent cortex, point towards important roles for neurons containing these transmitters in the regulation of the glycogen turnover. It is proposed that energy metabolism may be controlled within domains defined by the geometric arrangements of the neurons releasing these transmitters. The different domains may overlap temporally and spatially to coordinate energy metabolism in relation to increases in neuronal activity. The non-myelin forming glial cells, which contain glycogen whose turnover rate is altered by the transmitters, appear to be important in the local supply of energy substrate to neurons.

Quantitative analysis of neuron-glial relationships in the buccal ganglion of Planorbis: life constancy in the absence of changes in functional output.

The numbers and volumes of the neurons and glial cells in the buccal ganglia of Planorbis were analyzed by a new technique (computer image analysis of serial-section autoradiograms). The nerve-glia relationship are remarkably constant in the ganglia throughout the adult life of the animal (4-5 years), which correlates with their unchanging functional output to control the feeding cycle of the animal.

Royal Society of Tropical Medicine and Hygiene Meeting at Manson House, London, 19 May 1994. Trypanosomiasis and the nervous system. Pathology and immunology.

Damage to the nervous system occurs in both African and American trypanosomiases, but it differs considerably in form and extent in each disease, and with different strains and disease stages. With Trypanosoma brucei infections there is a progressive central nervous system (CNS) pathology which involves the meninges, choroid, blood-brain barrier, and immunopathological changes including perivascular infiltrations, astrocyte activation and alterations in the cytokine/mediator network. These changes underly the altered behaviour in the late or secondary disease stages, prevalent in the chronic gambian form, characterized by hypersomnia leading, if untreated or if treatment is followed by reactive changes, to coma and death. T. cruzi infections can be divided into 3 stages; acute, intermediate and chronic. Each stage has a different neurological involvement. In the acute stage the parasite produces direct destructive and inflammatory changes in the CNS which can be life-threatening, but which normally resolve, giving way to an intermediate period with effective parasite suppression and little or no perpetuation in the nervous system. The chronic stage is characterized by alteration to a progressive peripheral neuroimmunopathology, with autoimmune destruction of many nerve components, especially the autonomic innervation of the heart and gut.

The somnogenic T lymphocyte suppressor prostaglandin D2 is selectively elevated in cerebrospinal fluid of advanced sleeping sickness patients.

To help to elucidate the changes induced by Trypanosoma brucei gambiense in the central nervous system (CNS) in advanced sleeping sickness patients, levels of interleukin-1 (IL-1) and prostaglandins D2 (PGD2) and E2 (PGE2) were measured by radioimmunoassay in the cerebrospinal fluid (CSF) from 24 patients diagnosed on the criteria of CSF protein, leucocyte count and parasite presence as having CNS (i.e. late stage) involvement, and from 12 patients without CNS involvement. PGD2 concentrations were selectively and markedly elevated in the late stage patients. The increased PGD2 may in part account for the increased somnolence and the immunosuppression within the CNS. Measurement of PGD2 levels in CSF may be a useful criterion for CNS involvement.

Antioxidant defense against antidepressants in C6 and 1321N1 cells.

The effects of pretreatment with the antioxidants reduced glutathione (GSH), ascorbate (ASC), Trolox (TROL), and combined ascorbate and Trolox (ASC/TROL) exposure on the acute (24 h) toxicities (EC50 value) of the antidepressants amitriptyline, imipramine (tricyclic antidepressants), fluoxetine (a selective serotonin reuptake inhibitor; SSRI), and tranylcypromine (a monoamine oxidase inhibitor; MAOI) were determined in the rat (C6) glioma and human (1321N1) astrocytoma cell lines using the neutral red uptake assay. The effects of pretreatment with buthionine-[S, R]-sulfoximine (BSO), and manipulation of intracellular cyclic AMP (cAMP) using isoproterenol (beta-receptor agonist), 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor), and dibutyryl cyclic AMP (dBcAMP; cAMP analogue) on antidepressant toxicity were also determined. Protective responses were observed after antioxidant treatments and manipulation of cAMP in both C6 cells pretreated with dBcAMP (+dBcAMP) and 1321N1 cells not pretreated with dBcAMP (-dBcAMP), with a few exceptions in 1321N1 cells (-dBcAMP). Some protective responses occurred in C6 cells (-dBcAMP) and 1321N1 cells (+dBcAMP) after isoproterenol and combined IBMX/isoproterenol pretreatment but not after just IBMX pretreatment. Pretreatment with BSO enhanced toxicity with the exception of fluoxetine. The antidepressants caused increases in intracellular GSH in the C6 cells at subcytotoxic concentrations, with decreases in GSH occurring at higher concentrations. Cytotoxicity of the antidepressants may be partly mediated through oxidative stress with alterations in signal transduction pathways.

Alterations in the glial fibrillary acidic protein content of primary astrocyte cultures for evaluation of glial cell toxicity.

Astrocyte-enriched primary glial cell cultures from the rat cerebral cortex were exposed to a range of neurotoxic compounds and the effects on three proteins were examined by enzyme-linked immunosorbent assay (ELISA). The most consistent marker for astrocyte toxicity was the intermediate filament protein glial fibrillary acidic protein (GFAp). Many toxicants reported to have specificity for astrocytes (e.g. mercuric chloride, aluminium chloride, toluene or ethanol) produced a similar dose-response curve: increases in the GFAp content of the cells at sub-cytotoxic concentrations with attenuation at higher concentrations. Some of the concentrations required to increase GFAp were extremely low, indicating that this is a sensitive and consistent marker of cellular damage, as has been shown in vivo. Toxicants that have neuronal specificity (such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium or acrylamide) had no effects on GFAp levels, indicating that the system measures the susceptibility of astrocytes to specific toxicants rather than neurotoxicity. The levels of S-100 protein and vimentin were measured for a smaller number of toxicants. S100 was a less sensitive and inconsistent marker in comparison with GFAp, whilst vimentin levels were not seen to increase with any tested compound. These results suggest that astrocytes have a valuable place in culture models for neurotoxicity testing.

Hypertrophy and increased glial fibrillary acidic protein are coupled to increased protection against cytotoxicity in glioma cell lines.

This study examined the associations of increased glial fibrillary acidic protein (GFAP) levels and hypertrophie changes with susceptibility to toxic insult in C6 rat glioma cells. The cells were treated with serial pulses of dibutyryl-cAMP (dBcAMP) (each 48 hr) which increased levels of GFAP approximately twofold and the surface to volume ratio by approximately 1.7 times after the third pulse. This treatment reduced cell number by 22% and increased total protein by 10%. The cells were then exposed to different toxic substances [tin chloride, lead tetraacetate, chloroquine, cadmium chloride, aluminium chloride, mercuric chloride, acrylamide, triethyltin bromide, ethylenediaminetetraacetatic acid (EDTA)] and toxicity to the cells measured by the neutral red (NR) assay. With the exceptions of aluminium chloride and EDTA, 50% effective concentration (EC(50)) values for the toxic substances were increased up to 10,000 times (for cadmium chloride). Very similar increases in protection against the substances following dBcAMP treatment were found with the 1321N1 human astrocytoma cell line. We conclude that two components of gliosis, hypertrophy and increased GFAP, are associated with increased protection against a range of known neurotoxicants.

Protective roles of glutathione in the toxicity of mercury and cadmium compounds to C6 glioma cells.

The neurotoxic effects of mercury (II) chloride, methylmercury (MeHg) and cadmium chloride on astrocytes were modelled using C6 glioma cell cultures. All three compounds were cytotoxic to these cells with an order of potency of cadmium > MeHg > mercurychloride. Addition of reduced glutathione (GSH) to the media protected the cells in all three cases, whereas depletion of GSH with l-buthionine-S,R-sulfoximine enhanced the toxicity of cadmium and mercury chloride but not MeHg. The effects of subcytotoxic concentrations of these compounds on intracellular GSH levels were assessed using chlorobimane staining. All three showed a similar type of effect-an initial depletion of GSH followed by an increase to levels greater than in untreated cells. For mercury chloride-exposed cells (0.37-3.7 muM), the initial depletion occurred over 4 hr with the cells recovering by 7 hr and increases in the GSH content seen after 24 hr. With cadmium or MeHg (0.04-4 muM), the initial depletion was more protracted, with treated cells having less GSH than control cells for 4-7 hr. Glutathione S-transferase activity in the cells was increased after 24 hr of exposure to all three metals to about 200% of control values. These results show that some components of the glutathione system in C6 glioma cells are activated after exposure to heavy metal compounds.

Use of the MTT assay for estimating toxicity in primary astrocyte and C6 glioma cell cultures.

Primary cultures of rat cortical astrocytes were exposed to 25 neurotoxic and non-neurotoxic compounds for 24 hr over a concentration range of 0.001 to 1000 mug/ml and the effects were quantified using the MTT assay. EC(50) values were obtained for nine of the compounds in the tested range, while increases in MTT conversion were seen at sub-cytotoxic concentrations for 12 compounds. The concentrations causing activation of this system were found, with some exceptions (notably organotin compounds) to be similar to those previously reported to cause increases in expression of the astrocyte marker glial fibrillary acidic protein. MTT conversion was also measured in the C6 cell line and, with some exceptions, the response to toxicants was found to be the same as that in the primary cultures of astrocytes. However, this was only the case when the C6 cells were pretreated for 48 hr with 0.5 mM dibutyryl cyclic AMP. This study represents the first direct comparison between primary astrocytes and C6 cells using a broad range of chemical neurotoxicants.

Use of astrocytes for in vitro neurotoxicity testing.

The use of primary cultures of astrocytes as indicators of toxic potential was assessed using 20 selected compounds. Multiple endpoints were used to evaluate astrocyte reactions. Trypan blue dye exclusion and total cellular protein content of the cells were used as general indices. EC(50) values from the trypan blue experiments could be used to rank toxicity of compounds in a manner that correlates well with known toxicity for compounds that have specific astrocyte toxicities. Neurone specific neurotoxicants had no measurable effects on astrocytes indicating that this system differentiates gliotoxicity from neurotoxicity. Protein content, and content of the astrocyte specific glial fibrillary acidic protein (GFAp), were seen to increase at lower doses of gliotoxic compounds. This phenomenon appears to be similar to reactive gliosis in vivo, as assessed by immunostaining, and is an extremely sensitive indication of cellular damage. Support studies using astrocyte uptake of 2-deoxy glucose showed a similar pattern of activation in the cells as protein increases. This has been confirmed using the nonisotopic technique of MTT reduction.

Potentiation of dopaminergic transmission by phosphodiesterase inhibitors and cyclic nucleotides.

Experiments were made to determine whether cyclic AMP plays a role in transmission at identified dopaminergic synapses in the water snail Planorbis corneus. Intracellular stimulation of a specific dopamine neuron produces direct inhibitory postsynaptic potentials (ipsps) in a number of other neurons. These ipsps, which are mediated by dopamine, were potentiated by as much as 120% by caffeine, theophylline or dibutyryl cyclic AMP, although they were unaffected by cyclic AMP and prostaglandin E1. Caffeine and theophylline also potentiated the inhibitory response to dopamine, applied to the postsynaptic neurons by perfusion or iontophoresis, but the effects were generally much smaller (maximum potentiation 30%). The results provide evidence that postsynaptic cyclic AMP is involved in transmission at these synapses, but that the phosphodiesterase inhibitors may also have a presynaptic effect.

Anatomical studies of simple invertebrate synapses utilizing stage rotation election microscopy and densitometry.

The radial nerve cords of starfish and the central ganglia of a gastropod mollusc were examined for the presence of chemical synapses. No structures with the degree of specialization of synapses in the vertebrate CNS were observed. Presumed chemical synapses, which possessed slight but variable paramembrane densities, were examined with a tilting stage. This showed that such densities were frequently due to overlap of vesicle and axon membrane within the section, which were resolved at the correct angle of tilt. Many structures resembled demonosomes. The necessity for care in interpreting the structure of chemical synapses in vertebrates is emphasized.