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Significant amounts of aluminium (Al) are commonly present in rivers and lakes, largely in particulate form in neutral waters. Freshwater bivalves, as filter feeders are therefore exposed to both particulate and dissolved metal and are potentially vulnerable to Al. The effect of Al on filtering behaviour of the freshwater mussel Anodonta cygnea L. was investigated during short (1 hour) and long-term (15 days) exposure to environmentally relevant concentrations (250 and 500 microg l(-1)) at neutral pH. Water flow through the outflow siphon was monitored as an indicator of pumping capacity. Short-term (1 hour) exposure to 500 microg l(-1) added Al produced an irreversible decrease in the duration of filtering periods, presumably as an avoidance response to the toxicant. One-hour exposure 250 microg l(-1) Al had no detectable effect. When mussels were exposed to 250 or 500 microg l(-1) added Al for 15 days, siphon activity measured in days 11-15 of exposure was inhibited by 50% and 65%, respectively, compared to pre-exposure levels. Recovery occurred following transfer of mussels to uncontaminated water. Interaction between Al and freshwater bivalves at neutral pH may affect both the performance of the mussels and the chemical speciation of the metal in the natural environment.

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The effect of growth and physical condition on the level of heavy metals accumulated in the organs of common bream (Abramis brama L.) populating Lake Balaton was investigated on samples collected in October 1999 and May 2000 from two well-separable sites regarding their trophic state and pollution impact (western and eastern basins). The average metal concentrations in the organs of fish varied in the following ranges: Cd, 0.39-1.98; Cu, 1.73-57.3; Hg, 0.02-0.13; Pb, 0.39-3.15; and Zn, 12.7-159.3 microg/g dry weight. The highest Cd, Cu, Pb and Zn concentrations were detected in the gill and liver of fish, whereas the highest Hg concentrations were measured in the muscle. The maximum metal concentrations in the muscle of bream were on average below the maximum permissible levels for human consumption. Significant positive correlation was found among the heavy metal load of bream and their instantaneous growth rate; hardly any connection was observed related to the physical condition of samples. The relatively low metal concentrations of the ambient water and their poor correlation with the heavy metal load of bream, indicates that for the mature stages of this fish species the metal uptake from food is predominant, and thus the heavy metal load of fish reflect more the pollution state of the sediment and its biota, rather than that of the ambient water.

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The effect of Cd2+, as one of the most widespread toxic environmental pollutants, was studied on gamma-aminobutyric acid (GABA) evoked responses of identified neurons in the central nervous system of the pond snail, LYmnaea stagnalis L. (Gastropoda). In the experiments, the modulation of the action of GABA both on neuronal activity (current clamp recording) and on the a GABA activated membrane Cl- current (voltage clamp studies) has been shown. It was found that: 1. GABA could evoked three different various types of response in GABA sensitive neurons: i) hyperpolarization with strong inhibition of ongoing spike activity, ii) short depolarization with an increase of spike the activity, iii) biphasic respone with a short excitation followed by a more prolonged long inhibition. 2. In low-Cl- solution the inhibitory action of GABA was reduced or eliminated, but the excitatory one was not or only moderately affected. 3. CdCl2 inhibited the GABA evoked hyperpolarization, but left intact or only slightly reduced the excitation evoked by GABA. 4. The inward Cl- current evoked by GABA at a -75 mV holding potential was slightly augmented in the presence of I micromol/l Cd2+, but was reduced or blocked at higher cadmium concentrations. The effect of Cd2+ was concentration and time dependent. 5. Parallel with reducing the GABA evoked current, cadmium increased both the time to peak and the half inactivation time of the current. 6. CdCl2 alone, in 50 micromol/l concentration, induced a 1-2 nA inward current. The blocking effect of cadmium on GABA activated inhibitory processes can be an important component of the neuro-toxic effects of this heavy metal ion.

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This study examined the effect of aluminium (Al) on the filtering behaviour (shell opening or gape) of the freshwater bivalve Anodonta cygnea L in neutral fresh water. Parallel measurements of Al concentration in the soft tissues were made to examine the relationship between changes in behaviour and accumulation of Al. The number of lysosomal granules in the gill, kidney and digestive gland were counted, as lysosomes are known to be involved in the excretion and detoxification of trace metals. The bivalves were exposed to two environmentally relevant concentrations of added Al i.e. 250 and 500 microg l(-1) (9.25 and 18.5 microM l(-1)) at neutral pH for 15 days and shell movement monitored continuously. Aluminium affected the mussels' filtering activity, producing an avoidance reaction whose magnitude was concentration-dependent; 250 microg l(-1) added Al produced no detectable change, while 500 microg l(-1) Al reduced mean duration of shell opening by 50%. This effect was irreversible over a 15 day recovery period. Tissue levels of Al after 15 days exposure were an order of magnitude higher in animals exposed to 250 microg l(-1) added Al than in those exposed to 500 microg l(-1). This was consistent with the inhibition of filtering activity due to valve closure at the higher concentration, which may have prevented uptake of Al. In addition, probable different chemical speciation of Al in the water column (soluble for 250 and colloidal for 500 microg l(-1)) may lead to marked differences in tissue uptake. The kidney and digestive gland were the main sites of accumulation of Al and concentrations remained significantly elevated 15 days after transfer of animals to clean water. It is suggested that mucus plays a role in the exclusion of Al as elevated concentrations were measured in the pseudofaeces of animals during and after exposure. Lysosomal granules may be involved in the intracellular handling and detoxification of Al as numbers increased significantly in all organs during exposure and continued to increase after the animals were transferred to clean water. The present study provides evidence for the bioavailability and toxicity of Al to mussels at neutral pH and at concentrations which are known to enter neutral freshwaters when mobilised by natural or anthropogenic acidity. The changes in behaviour and uptake of Al in the mussel observed in this investigation are, therefore, likely to be reflected in the natural environment and the degree to which Al affects the 'fitness' of the mussel populations and the transfer of Al through the food chain merit investigation.

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As part of a long-term monitoring study of the anthropogenic pollution of the Lake of Balaton (Hungary) the concentrations of toxic metals (Cd2+, Hg2+, Pb2+), as well as of bioelements (Cu2+ and Zn2+) were measured in the muscle, gill and liver of bream (Abramis brama L.) collected in the open water during the autumn of 1999, The highest Cd, Cu, Pb and Zn concentrations were detected in the gill and liver of fish, whereas the highest Hg concentrations were measured in the muscle. The maximum metal concentrations measured in the muscle of bream were generally below maximum permissible levels for human consumption established by the Hungarian Food Directorate. Depending on the sampling site, significantly higher Cd, Hg and Pb concentrations (p < 0.05) were detected in the muscle of fish samples collected from the Western basin, while no significant differences were observed for Cu and Zn. In the muscle and gill an increasing trend of heavy metal (Cd and Cu) load characterizes the individuals of the 2-4 calendar age group (p < 0.05), while for the older specimens the concentration of these elements decreased significantly (p < 0.05). Positive relationships on the whole age scale were found for Cd in the liver, for Pb in the gill, and for Hg both in the muscle and the liver of fish.

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The pH-dependent actions of aluminum(III) hydroxides (Al(III))on the voltage-activated sodium currents (VASCs) in the giant neurons of the pond snail Lymnaea stagnalis L. were studied by means of a conventional two-electrode voltage-clamp technique. The final concentration of Al(III) was 5-500 microM at pH 7.7, 6.9 or 6.0. A significant and concentration-dependent increase in the peak amplitude of the VASCs was recorded over the entire voltage range at pH 7.7 (EC50 = 100.7 +/- 33.7 microM, n = 9), without alteration of the gating properties. A concentration-dependent decrease in the peak amplitude (IC50 = 175.9 +/- 73.6 microM, n = 6) and concomitant increases in the time constants of activation and inactivation of the VASCs were recorded in slightly acidic media (pH 6.0), whereas there were no changes in the investigated parameters at pH 6.9. A significant increase in the V1/2 of the half-maximal current of the steady-state inactivation resulted on Al(III) application at pH 7.7, but not at pH 6.9 or 6.0. These results suggest that Al(III) can differentially up- and down-modulate the sodium current and related physiological functions to extents dependent on the pH-determined speciation of the Al(III) hydroxides present.

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Due to the relative simplicity of their nervous system, invertebrate animals were widely used in the past decades for studying the processes of excitability at membrane level, as well as the mechanisms of neuronal events and interneuronal communication. Parallel with investigating basic questions of neurobiology, lower animals have also been the object of toxicological studies, because simple invertebrate preparations with well-known physiological, biochemical and pharmacological characteristics proved to be excellent models for testing the action of natural and synthetic compounds important to human pharmaceutical research as well as in searching suitable chemicals for pest control. In the last ten-fifteen years with the growing interest towards environmental protection, a new field was opened for the application of invertebrates, namely, testing and monitoring the presence and harmful effects of anthropogenic toxic substances. Invertebrates are used today both as passive and as active biomonitors to detect and evaluate the level of pollution in a given ecosystem, and to study the effects and mechanisms of action of pollutants. Invertebrate nervous systems are suitable objects in clarifying the mechanisms of action of toxic chemicals at various levels of the neural regulation. Toxic influences can be reflected in behavioural alterations, by the modification of the function of different organs as well as the neural regulation, presented by examples on mussels and snails. In case of neurotoxicity, the targets of action are the elements of the nervous system. Alterations can occur in the permeability (ion channels) of the neuronal membrane influencing excitability, potential generation and propagation of nerve impulse, in the transmitter system (synthesis, release, elimination and binding to the receptors), in the interneuronal and neuroeffector connections responsible for co-ordinated and adequate responses to the internal and external challenges. For the future, it can be predicted that neurotoxicological research with new compounds cannot be effective without using invertebrate preparations, since, due to animal protection and restrictions in animal experimentation, the permission of the use of vertebrates is much more limited in most of the developed countries, and this trend is certainly to be expanded.

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1. Inorganic tin and organotin compounds, occurring in aquatic ecosystems, are toxic and can cause behavioral abnormalities in living organisms. To determine the possible neuronal basis of these actions, the effects of both forms of Sn were studied on identified neurones of the mollusk, Lymnaea stagnalis L. 2. SnCl2 caused a dose-dependent decrease in the acetylcholine (Ach)-induced inward current. The effective threshold concentration, measured by a two microelectrode voltage clamp technique, was 0.1 microM, and the maximal effect occurred at 5 microM SnCl2. The depression of the inward current was greater after a 10 min preapplication (20%) than after 3 min treatment (7%). 3. The next series of experiments compared the actions of inorganic or organic tin compounds. In whole cell clamp experiments both (CH3)2SnCl2 and (CH3)3SnCl, like inorganic Sn, decreased the amplitude of Ach-induced current. Increasing the duration of the preapplication time resulted in an increase in the effect, but the action was not reversible. SnCl2 treatment caused a concentration-dependent alteration (initial potentiation followed by depression) of the amplitude of I(Na(V)) over the whole voltage range and slightly shifted the I-V curves to the left. In contrast, trimethyl tin decreased the amplitude of I(Na(V)) only at high concentration (100 microM). The activation time course of I(Na) was increased (tau = 0.43 ms in control and 0.55 ms in Sn), but Sn did not alter the inactivation parameters (tau = 3.43 and 3.41 ms). 4. These results support earlier findings that agonist- and voltage-activated channels are direct targets of toxic metals. We conclude that tin in both inorganic and organic forms acts at neuronal membranes to modulate synaptic transmission through direct actions on agonist-activated ion channels, and suggest that these actions may be the basis of the altered behavior of animals in tin-polluted environments.

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Recording of the mollusc osphardium nerve activity revealed the osphardium sensitivity to hyperosmotic pressure, sodium chloride, and aminoacidosis. The osphardium was found to detect quality of the water where the animals were kept. Pont snail osphradium preserves ancestors' multisensority which unites perception of different chemical and physical signals. Patch-clamp method revealed membrane currents in certain ganglia and receptor cells which are sensitive to Na+ and L-aspartate increase in the solution around the osphardium. Inward components of these currents are, probably, of the sodium and/or calcium nature, whereas the outward components--of the potassium one.

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To investigate the functional organisation and information processing in Lymnaea neuronal networks, artificial spike trains were elicited in one of the main respiratory interneurons (RPeD1) and the modulation of the firing patterns of postsynaptic cells was examined. This was performed by precisely timing the action potential generation of the presynaptic cell using a computer-controlled voltage clamp amplifier (pattern clamp technique). Induced oscillation (0.1-0.4 Hz) in the firing pattern of RPeD1 spread to a large number of postsynaptic cells, as clearly demonstrated by Fourier power spectra. At the same time, no signs of precise (millisecond) spike timing was observed in the cells studied. The results confirm that the neurons used in our experiments process information as pure rate-coders.

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In isolated single neurone or in whole ganglia preparation, SnCl2 caused a decrease in the acetylcholine-induced inward current in a dose-dependent way. Threshold concentration was 0.1 mkM and saturated at 5 mkM SnCl2. After 10-min. pre-treatment with SnCl2 the effect was more obvious (by 20%) than after 3-min pre-treatment. Sn(CH3)2 decreased the amplitude of Ach-induced current similarly to the inorganic Sn. The effect of Sn(CH3)2 was irreversible. The findings support earlier data that the agonist-activated channels are important targets for toxic metals. Direct action upon neuronal membrane seems to be an important event in modulation of synaptic transmission.

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1. The effect of a herbicide (paraquat) and an insecticide (methidation) on the periodic activity of mussel in relation to serotonin and dopamine levels and their release and uptake in nervous system was investigated. 2. LC50 values of methidation in 24-, 48-, and 96-hr treatments, respectively, were 50% lower than those of paraquat. 3. Serotonin and dopamine levels decreased in all three ganglia by 20% on average upon 24- and 48-hr in vivo treatments with paraquat. In the 96-hr treatment, however, both monoamines showed a 20-40% increase. With methidation treatment, the level of monoamines tested did not change significantly. 4. After paraquat treatment, both the average lengths of resting periods and their total duration significantly increased. Parallel to this, the average length of active periods and their total duration dramatically decreased. Methidation increased the average length and total duration of resting periods during the first 2 days at the expense of active periods. On the fourth day, however, the effect reversed: average length and total duration of active periods increased of the expense of parameters characteristic of the resting state.

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1. The effects of inorganic Hg2+ and methylmercuric chloride in the ionic currents of cultured hippocampal neurons were studied and compared. We examined the effects of acute exposure to the two forms of mercury on the properties of voltage-activated Ca2+ and Na+ currents and N-methyl-D-aspartate (NMDA)-induced currents. 2. High-voltage activated Ca2+ currents (L type) were inhibited by both compounds at low micromolar concentrations in an irreversible manner. Mercuric chloride was five times as potent as methylmercury in blocking L-channels. 3. Both compounds caused a transient increase in the low-voltage activated (T-type) currents at low concentrations (1 microM) but blocked at higher concentrations and with longer periods of time. 4. Inorganic mercury blockade was partially use dependent, but that by methylmercury was not. There was no effect of exposure of either form of mercury on the I-V characteristics of Ca2+ currents. 5. Na(+)- and NMDA-induced currents were essentially unaffected by either mercury compound, showing only a delayed nonspecific effect at a time of overall damage of the membrane. 6. We conclude that both mercury compounds show a relatively selective blockade of Ca2+ currents, but inorganic mercury is more potent than methylmercury.

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Bioindicator methods were used to study the effects of the pyrethroid insecticide deltamethrin on the filtering behavior of the freshwater mussel Anodonta cygnea L in the laboratory, during short-term (30 min) and long-term (1 week) exposure. In the course of the short-term treatment, the water flow through the outflow syphon was monitored. It was found that 1 and 5 micrograms/liter deltamethrin caused an increase, whereas from 10 to 50 micrograms/liter deltamethrin resulted in a depression, in the open time of the outflow syphon and the water outflow. The inhibitory response was concentration dependent. In the long-term experiments, the valve movement representing the activity of the adductor muscles was recorded. At 1 microgram/liter, deltamethrin had no effect on the active period, but reduced the rest time, causing an increase in the filtration; all higher concentrations (10-50 micrograms/liter) caused inhibition of the filtration activity by reducing the active periods and lengthening the rest periods. The active periods were shortened on increase in deltamethrin concentration, the reduction being 80% of the control at 50 micrograms/liter. Lengthening of the rest periods led to a maximum (207% as compared with the control) at 15 micrograms/liter, with a subsequent decline to levels close to the control at 30 and 50 micrograms/liter. Nevertheless, due to the strong reduction of the active periods, the filtering activity remained depressed at higher concentrations. The results provide evidence that deltamethrin pollution may have an adverse effect on the functioning of the bivalve community in the aquatic environment, which should be considered when this chemical is used in agricultural areas near aquatic ecosystems.

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1. We have used biochemical, immunocytochemical, and electrophysiological techniques to evaluate the role of opioid peptides in the central nervous system of the marine mollusc, Aplysia californica. 2. Binding studies using 3H-D-Ala2, met-enkephalinamide (3H-DAMA) showed a single class of high-affinity binding sites with a Kd of 1.3 nM and a binding density of 45 pmol/g. 3. HPLC extracts of ganglia revealed multiple peaks with immunoreactivity for either leu (LEU-IR)- or met-enkephalin (MET-IR), but the amounts were not uniformly distributed in all ganglia. 4. LEU-IR and MET-IR neurons were demonstrated immunocytochemically in all ganglia, but MET-IR neurons were more frequent and were concentrated in pedal and pleural ganglia. While absorption control studies abolished MET-IR, LEU-IR was only partially abolished in the neuropil. 5. In electrophysiological studies, both depolarizing and hyperpolarizing responses were found to D-Ala2-leu-enkephalin (DALEU) and D-Ala2-met enkephalin (DAMET) on some and different neurons. 6. HPLC fractions from regions with retention times corresponding to authentic leu- or met-enkephalin showed physiologic responses similar to those of DALEU and DAMET, respectively. 7. These studies suggest that a variety of endogeneous opioid peptides play physiologically important roles in the nervous system of Aplysia, including but not necessarily limited to leu- and met-enkephalin.

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Using the concentration clamp technique the effects of inorganic mercury (HgCl2) on gamma-aminobutyric acid (GABA) activated Cl- currents were studied on Lymnaea neurons. Hg2+ at concentrations of 0.01-1 microM caused a significant increase in the GABA induced chloride permeability of the cell membrane. With simultaneous application of GABA and 0.1 microM Hg2+ the amplitudes of the GABA activated inward Cl- currents were increased to a mean of 168%. The threshold concentration for potentiation was 0.01 microM Hg2+, while the GABA induced Cl- current was suppressed at concentrations higher than 100 microM. HgCl2 was also effective in decreasing the time-to-peak value and the time of inactivation of the GABA evoked Cl- current. In the presence of 0.1 microM mercury chloride the peak reached the maximum 1.6 times faster than without mercury. The effect was independent of the membrane potential, and mercury did not change the reversal potential of the GABA activated Cl- current. Application of mercury alone at concentrations effective to enhance GABA evoked Cl- permeability caused no or only a very small sustained inward current. However, higher mercury concentrations or repeated mercury application greatly increased the membrane permeability, and this effect could not be reversed by a 15 min wash. The results suggest, that low mercury concentrations act on GABA-ergic synapses at a postsynaptic site, and the potentiation of the ligand gated Cl- current may be an important factor in the mechanism of Hg2+ neurotoxicity.

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1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl2) on acetylcholine-, carbachol-, and glutamate-activated currents on Aplysia neurons. Hg2+ was applied with microperfusion. 2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl- dependent and reversed at about -60 mV, while the slower component was K+ dependent and reversed at greater than -80 mV. 3. Hg2+ (0.1-10 microM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl- current in medial neurons. With only a 1-min preapplication of Hg2+, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 microM Hg2+. 4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1-10 microM Hg2+ caused a complete and irreversible blockade of K(+)-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl2 that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 microM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl2 as well. 5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg2+ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg2+ toxicity.

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