Abstract: We investigated the effect of several acaricides on Varroa destructor by monitoring the rhythmic expansion of the sternum, followed by a strong flexion of the legs, initiated when the mite was placed in a dorsal side-down position, as an indication of a mite’s vitality. The pulses generated by the force of the rhythmic expansions had an average duration of 3.11 s, force (amplitude) of 73 μN, and frequency of 0.228 Hz. These parameters remained constant for the first 10 h of recording, whilst significant changes occurred after 15 h. The rhythmic sternal expansion is an indication of a Varroa mite’s gravitational reflex, or attempt to return to an upright position; this reflex is observed in all invertebrates and vertebrates. The sternal expansion can be recorded for over 20 h, or for as long as the Varroa mite remains alive, and the expansion stops as soon as the mite is placed in a normal, upright position. Proper function of the chain of proprioceptors, interneurons, motorneurons, neuromuscular junctions, and muscles of Varroa is required for the initiation and maintenance of such a behavioural motor pattern. Any deleterious effect of synthetic chemicals or natural compounds (acaricides) may have a direct effect on one or more of these links, thereby disturbing or even inhibiting the reflex. Topical application of 1.81 × 10−3 mg/mite of amitraz completely inhibited the gravitational reflex within 60–70 min for all mites tested. The volatile acaricides formic acid (13.83 mg), thymol crystals (250 mg), and Apiguard® (1000 mg) eliminated the reflex within 10–35 min. This bioassay, based on the gravitational reflex, could be a useful tool for accurate assessment of the acaricidal action of numerous compounds under laboratory conditions, saving money and time necessary to conduct field trials.
Graphical abstract
Highlights
► Varroa demonstrates a constant gravitational reflex when fixed dorsal side-down. ► The rhythmic sternum movement is maintained for over 20 h. ► The parameters of the gravitational reflex can be measured accurately. ► The gravitational reflex was used as an indication of the vitality of Varroa. ► The toxic effect of amitraz, formic acid, thymol, and Apiguard® were tested.
Abstract: The effects of octopamine, the main cardio acceleratory transmitter in insects, were investigated, in the isolated hearts of the honeybee, Apis mellifera macedonica, and the olive fruit fly, Bactrocera oleae. Octopamine induced a biphasic effects on the frequency and force of cardiac contractions acting as agonist, with a strong acceleratory effect, at concentrations higher than 10-12 M for the honeybee and higher than 5x10-9 M for the olive fruit fly. The heart of the honeybee is far more sensitive than the heart of olive fruit fly. This unusual sensitivity t is extended to the blockers of octapaminergic receptors, where phentolamine at 10-5 M ceased completely and permanently the spontaneous contractions of the honeybee heart, while the same blocker at the same concentratio caused only 50% inhibition in the heart of olive fruit fly. Phentolamine and mianserin at low concentration, 10-7 M also block the heart octopaminergic receptors, but for a short period of time, less than 15.0 min, while a partial recovery of the heart contraction started in spite of the presence of the antagonist. The unusual response of the honeybee heart in the presence of phentolamine and/or mianserin suggest excitatory effects of octopamine via two different receptor subtypes. At lower concentrations, 10-14 M, the agonist octopamine was converted to an antagonist inducing a hyperpolarization in the membrane potential of the honeybee cardiac pacemaker cells and inhibiting the firing rate of the heart. The inhibitory effects of octopamine on certain parameters of the rhythmic bursts of the heart of the honeybee, were similar to those of mianserin and phentolamine, typical blockers of octopaminergic receptors. The heart of olive fruit fly was 105 times less sensitive to octopamine, since a persistent inhibition of heart contractions occurred at 10-9 M. In conclusion, the acceleration of insect heart is achieved with increasing the levels of octopamine, while there is a passive but also an active decrease in the heart activity due to the minimization of octopamine.
Abstract: Dichloroacetate (DCA) has been used extensively in the treatment of cancer and genetic mitochondrial diseases, but there have been reports of DCA-induced peripheral neuropathy. In the present study, the acute effects of sodium dichloroacetate on the peripheral nerve fibers were investigated using an ex vivo preparation, the isolated sciatic nerve of the rat. The amplitude of the evoked nerve compound action potential (CAP) was measured in order to confirm the proper functioning of the nerve fibers. The half-vitality time (the time required to decrease the CAP to 50% of its initial value, called here IT50), of the nerve fibers which had been incubated in normal saline was 30.4 ± 0.26 h (n=12). When the nerve fibers were incubated in 10.0 mM of DCA, the IT50 was 29.7 ± 0.34 h (n=8), with no significant difference from the control (P>0.05). The fact that such a high concentration of DCA as 10.0 mM has no effect on the parameters of the evoked CAP is an indication of the high tolerance of peripheral nerve fibers to this compound. When a concentration of 20.0 mM DCA was tested, a 15.2 ± 1.25 % (n=12) inhibition in the CAP amplitude occurred, but although a relatively small population of nerve fibers was inactive, the vitality of the remaining active axons was not affected, with a final IT50 of 28.1 ± 0.64 h (n=12), with no significant difference from the IT50 of the control, which for this group of experiments was 28.1 ± 0.17 h (P>0.05). This moderate effect, with a 15.2 ± 1.25 % decrease in the CAP amplitude, suggests that within the exposure limitation of the sciatic nerve preparation of 28.0 to 30.0 h, there could be the gradual development of certain biochemical changes leading to the early stages of DCA-induced neurotoxicity.
Abstract: Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na+/K+-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na+/K+-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibers. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT50) incubated in normal saline was 24.5 ± 0.40 h (n=5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT50 of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n=5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n=5) and inhibitory concentration to 50% (IC50) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 μM (n=5) and IC50 of 370.0 ± 18.00 µM (incubation 24.5 h). Finally, when the two compounds were combined—e.g. pre-incubation of the nerve fibre in 250.0 µM ouabain for 1 h and then exposure to 1.0 nM PlTX—ouabain offered minor a neuroprotection of 9.1-17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 µM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay— the standard ex vivo test for PlTX toxicity.
Abstract: We studied the effects of five monoterpenoids, viz. 1,8-cineol, fenchone, linalool, p-cymene and α-pinene, on the sciatic nerve fibres of the frog Rana rindibunda (Pallas, 1771) and compared them to that of lidocaine, a standard local anesthetic. The isolated sciatic nerve, with its perineurium intact, was placed in a three-chambered recording bath, which allowed us to monitor the compound action potentials (CAP), stable in amplitude, for over 2 days. The half-vitality time (IT50), which is the time required for the amplitude of the CAP to decrease to 50% of its control value, was 53.5 ± 0.9 h, for a nerve incubated in normal saline, at 26.0o C. The IT50 values for nerves incubated in saline with p-cymene, 1,8-cineol or α-pinene, at 30.0 mM, were 19.9 ± 0.4, 32.9 ± 0.5 and 31.0 ± 0.3 hours, respectively. As the IT50 value for 30.0 mM lidocaine, a standard local anesthetic, was 1.6 ± 0.3 min under the same conditions, these three compounds cannot be considered as having a local anesthetic effect. The IT50 values for 30.0 mM linalool and fenchone were 5.7 ± 0.6 and 15.4 ± 1.1 min, respectively; they were significantly, but not markedly different from the respective value for lidocaine. These results combined with the fast inhibition of the CAP and its fast recovery after the removal of either linalool or fenchone indicate a local anesthetic activity of the two compounds. Linalool retained this activity even at lower concentrations, of 15.0 and 7.5 mM. The local anesthetic effects of lidocaine and linalool were concentration-dependent; this was not the case for fenchone, which had a relatively strong local anesthetic activity at 30.0 mM, but was entirely inactive at 25.0 mM. On the basis of the effects of the five monoterpenoids on the electrophysiological properties of the sciatic nerve fibres of the frog, we conclude that, whereas 1,8-cineol, p-cymene and α-pinene cause only minor effects, linalool and fenchone exhibit acute local anesthetic activity.
Abstract: Imidacloprid (IMI) is widely used systemic insecticide that acts as an agonist on nicotinic acetylcholine receptors (nAChRs). IMI has been reported to be more active against insect nAChRs (EC50 0.86–1 μM) than it is against mammalian nAChRs (EC50 70 μM). The objective of this study was to determine to what extent IMI affects the nAChRs of the stellate cells of mouse cochlear nucleus (CN), using whole-cell patch-clamp recording. Puff application of 1 μM IMI had no significant effect on the membrane properties of the neurons tested, while a concentration of 10 μM caused a significant depolarizing shift in the membrane potential and resulted in increases in the fluctuation of the membrane potential and in the frequency of miniature postsynaptic potentials (mpps) within less than a minute of exposure. IMI at concentrations ≥50 μM caused a significant depolarizing shift in the membrane potential, accompanied by a marked increase in the frequency of action potential. IMI decreased the membrane input resistance and the membrane time constants. Bath application of 50 μM d-tubocurarine (d-TC) reversibly blocked the depolarizing shift of the resting membrane potential and the spontaneous firing induced by IMI application in current clamp and blocked the inward currents through nicotinic receptors induced by IMI application in voltage clamp. Similarly, 100 nM α-bungarotoxin (α-BgTx) blocked the spontaneous firing induced by IMI (n = 3). The amplitude of the 100 μM IMI-induced inward current at −60 mV holding potential was 115.0 ± 16.2 pA (n = 7). IMI at a concentration of 10 μM produced 11.3 ± 3.4 pA inward current (n = 4). We conclude that exposure to IMI at concentrations ≥10 μM for <1 min can change the membrane properties of neurons that have nAChRs and, as a consequence, their function.
Abstract: Organophosphates (OPs) can provoke toxicity by inhibiting acetylcholinesterase (AChE) in non-target organisms, like fish. In a previous pilot study, the anticholinesterase effects of paraoxon on the heart of Sparus aurata were examined [Tryfonos, M., Antonopoulou, E., Papaefthimiou, C., Chaleplis, G., Theophilidis, G., 2009. An in vitro assay for the assessment of the effects of an organophosphate, paraoxon, and a triazine, atrazine, on the heart of the gilthead sea bream (Sparus aurata). Pest. Biochem. Physiol. 93, 40–46]. The objective of the present study was to investigate the effects of the five protoxicant OPs, azinphos-methyl (MeAZP), parathion-methyl (MePS), chlorpyriphos-methyl (MeCCP), methamidophos (MET) and diazinon (DZ), on the spontaneously beating auricle of S. aurata. The results showed that: (1) MeAZP and MET induced exclusively cholinergic effects on auricle contractility. These effects were expressed as a significant decrease in the force and frequency of contractions and were fully reversible (140%) after the application of the muscarinic cholinergic receptor antagonist, atropine (15 μM). MeAZP was found to be the most effective anticholinesterase compound, with an IC50 of 2.19 ± 1.05 μM (n = 6), while MET was less effective, with an IC50 of 72.3 ± 1.2 μM (n = 6). (2) DZ and MePS, although classified as OPs, induced non-cholinergic effects. These effects were observed as an irreversible decrease in force and frequency of the auricle in all the concentrations examined; the depression is retained even after application of 15 μM atropine. (3) MeCCP was halfway between a typical OP and an OP lacking anticholinesterase properties, since there was a partial recovery in the force, but no recovery in the frequency of the auricle contractions. (4) The toxicity order, based on the IC50, was as follows: MeAZP, 2.19 ± 1.05 μM > paraoxon, 3.2 ± 1.5 μM MET, 72.3 ± 1.2 μM > MePS, 80.3 ± 1.03 μM > MeCPP, 93.7 ± 1.01 μM > DZ, 164 ± 1.01 μM. (5) There was a good correlation (r = 0.779, p = 0.04, n = 5) between IC50 and the previously determined log P (octanol:water partition coefficient) values for MeAZP, paraoxon, MeCCP, MePS and DZ. The results indicated that the increase in lipophilicity of MePS, MeCCP and DZ is accompanied by a decrease in their acute cardiotoxic properties in vitro. The non-cholinergic effects of these relatively high lipophilic OPs, might be caused by their tendency to distribute preferentially in the lipid bilayer of cardiac cells, affecting the proper functioning of the ionic channels which regulate the force (Ca2+ channels) and the frequency (K+ channels) of the spontaneous auricle contractions.
Abstract: The results of this study have shown that N-acetyl-l-cysteine (NAC), a compound used for protection of tissues or cell cultures against the deleterious effects of various environmental pollutants, has certain unusual effects on the contraction of the spontaneously beating atria of the frog isolated in saline (ex vivo): (1) NAC, 6.0 and 10.0 mM, eliminated, in a concentration-dependent manner, the contractile properties of the atria (force and frequency) within minutes, without affecting its electrical properties; (2) the IC50 of NAC for the force was 5.09 ± 1.01 mM (n = 6) [4.98–5.19 mM, 95% confidence interval (CI)], significantly lower than the IC50 for the frequency, 6.15 ± 1.01 mM, (6.02–6.29 mM, 95% CI), indicating that working atria cells are more sensitive to NAC than autorhythmic cells. The no-observed-effect concentration (NOEC) was 1–2 mM; (3) the pattern of NAC-induced inhibition of electromechanical activity was similar to that of verapamil, an indication that NAC possibly affects L-type voltage-gated calcium channels; (4) NAC at 2 mM protected against cadmium-induced inhibition of atria contraction. The IC50 for cadmium was 17.9 ± 1.1 μM (n = 6) (16.9–19.0 μM, 95% CI), while in the presence of 2 mM NAC, it became 123.3 ± 1.0 μΜ (n = 6) (114.8–132.4 μM, 95% CI). The same concentration of NAC failed to exert any protective effects against rotenone (5 μM)-induced inhibition of atria contraction. The protective effects of NAC are probably due to chelation of cadmium, rather than scavenging of oxidants.
Abstract: On the basis of computer prediction of biological activity by PASS and toxicity by DEREK, the most promising 32-alkylaminoacyl derivatives of 3-aminobenzo[d]isothiazole were selected for possible local anaesthetic action. This action was evaluated using an in vitro preparation of the isolated sciatic nerve of the rat and compared with lidocaine which was used as a reference compound. QSAR studies showed that the polarizability, polarity and molecular shape of molecules have a positive influence on their local anaesthetic activity, while contributions of aromatic CH and singly bonded nitrogen are negative. Since the estimated PASS probabilities to find local anaesthetic activity in the most active compounds are less than 50%, these compounds may be considered to be possible NCEs.
On the basis of computer prediction of biological activity by PASS and toxicity by DEREK, the most promising 32 alkylaminoacyl derivatives of 3-aminobenzo[d]isothiazole were selected for possible local anaesthetic action which was evaluated using an in vitro preparation of the isolated sciatic nerve of the rat.
Abstract: The effects of paraoxon and atrazine on the spontaneously beating auricle, isolated from the heart of Sparus aurata, were assessed. Paraoxon, 5 μM, eliminated the atria contraction within 28.4 ± 2.8 min, an effect which was fully reversed by 15 μM atropine, an antagonist of muscarinic cholinergic receptors. The IC50 was estimated to be 3.2 ± 1.5 μΜ. Atrazine, 50 and 100 μM, induced a 22.5 ± 3.2 and 32.9 ± 2.3% increase in the force of auricle contraction, caused by excitation of sympathetic synaptic terminals releasing adrenaline. This effect was reversed by 50 μM propranolol, a blocker of β-adrenoreceptors. The results have shown that both sympathetic and parasympathetic nerve terminals are activated by atrazine. Also, the auricle contraction is mainly under sympathetic control, while the frequency is dominated by cholinergic system. Finally, the detailed parameters of the auricle contraction estimated during exposure to specific pesticides, force, frequency, time-response curves and electromechanical coupling can be further used to assess and compare the toxic effects of other compounds, anticholinesterases for example, on the heart of the fish.
Abstract: Oxaliplatin is a novel chemotherapeutic agent which is effective against advanced colorectal cancer, but at the same time causes severe neuropathy in the peripheral nerve fibres, affecting mainly the voltage-gated sodium (Na+) channels (VGNaCs), according to literature. In this study the effects of oxaliplatin on the peripheral myelinated nerve fibres (PMNFs) were investigated in vitro using the isolated sciatic nerve of the adult rat. The advantage of this nerve-preparation was that stable in amplitude evoked compound action potentials (CAP) were recorded for over 1000 min. Incubation of the sciatic nerve fibres in 25, 100 and 500 μM oxaliplatin, for 300–700 min caused dramatic distortion of the waveform of the CAP, namely broadening the repolarization phase, repetitive firing and afterhyperpolarization (AHP), related to the malfunction of voltage-gated potassium (K+) channels (VGKCs). At a concentration of 5 μM, oxaliplatin caused broadening of the repolarization phase of the CAP only, while the no observed effect concentration was estimated to be 1 μM. These findings are indicative of severe effects of oxaliplatin on the VGKCs. In contrast, the amplitude and the rise-time of the depolarization of the CAP did not change significantly, a clear indication that the VGNaCs of the particular nerve preparation were not affected by oxaliplatin. The effects of oxaliplatin on the PMNFs were similar to those of 4-aminopyridine (4-AP), a classical antagonist of VGKCs. These similarities in the pattern of action between oxaliplatin and 4-AP combined with the fact that the effects of oxaliplatin were more pronounced and developed at lower concentrations suggest that oxaliplatin acts as a potent VGKCs antagonist.
Abstract: N-acetyl-l-cysteine (NAC), at a concentration of 1-60mM, has been previously used extensively for protection in a variety of cell cultures against the deleterious effects of various compounds. The results of this in vitro study show that NAC has certain unusual effects on the evoked compound action potential (CAP) of the rat sciatic nerve fibers. Firstly, at concentrations of 5.0, 3.5 and 2.5mM, concentrations used by others as a protectant for cell cultures, NAC inhibits the action potentials of the sciatic nerve fibers completely in a concentration-dependent manner within a few minutes or hours (2.5mM). Secondly, the acute inhibitory action of NAC on the CAP of the nerve fibers was not spontaneously reversible, but as soon as NAC was replaced with saline there was a partial ( approximately 75%) recovery in the function of the nerve fibers. Thirdly, the no observed effect concentration for NAC was estimated to be 1mM. The paradox is that NAC at 1mM not only had no effect on the nerve fibers, but it became an excellent neuroprotective compound, giving almost 100% neuroprotection against cadmium-induced neurotoxicity. The results show a possible effect of NAC on voltage-gated sodium and potassium channels. The observed neuroprotective-neurotoxic properties of NAC require careful reconsideration of its use in either in vitro studies or in vivo pharmaceutical applications.
Abstract: Deciphering the electrical activity of individual neurons from multi-unit noisy recordings is critical for understanding complex neural systems. A widely used spike sorting algorithm is being evaluated for single-electrode nerve trunk recordings. The algorithm is based on principal component analysis (PCA) for spike feature extraction. In the neuroscience literature it is generally assumed that the use of the first two or most commonly three principal components is sufficient. We estimate the optimum PCA-based feature space by evaluating the algorithm's performance on simulated series of action potentials. A number of modifications are made to the open source nev2lkit software to enable systematic investigation of the parameter space. We introduce a new metric to define clustering error considering over-clustering more favorable than under-clustering as proposed by experimentalists for our data. Both the program patch and the metric are available online. Correlated and white Gaussian noise processes are superimposed to account for biological and artificial jitter in the recordings. We report that the employment of more than three principal components is in general beneficial for all noise cases considered. Finally, we apply our results to experimental data and verify that the sorting process with four principal components is in agreement with a panel of electrophysiology experts.
Abstract: Abstract
Two pesticides, the insecticide imidacloprid and the herbicide acetochlor,
were evaluated for their insecticidal and genotoxic effects in Drosophila
melanogaster. Their insecticidal effects were assessed by calculating LC50
values after acute or chronic exposure of larvae and adults to different
concentrations of the test compounds. After acute exposure, the LC50 of
imidacloprid was 7.59 · 10-5 M for larvae and 1.43 · 10-4 M for adults,
and after chronic exposure, it was 2.67 · 10-5 M and 6.09 · 10-5 M for
larvae and adults, respectively. On the contrary, the herbicide acetochlor
showed no acute or chronic insecticidal effect against either larvae or
adults, even at very high concentrations (8 · 10-3 M). For the evalua-
tion of genotoxic properties of the two pesticides, the Somatic Mutation
and Recombination Test in D. melanogaster was used. Our results suggest
that neither imidacloprid nor acetochlor exhibits mutagenic or recombi-
nogenic activity at applicable concentrations.
Abstract: The effects on nerve tissue of three dental impression pastes were compared in this study. Two of the impression pastes, Examix and Express 3M, contained vinyl polysiloxane while the other, Xanthopren, did not. An in vitro model based on the isolated sciatic nerve of the frog and rat was used. As an indication of the proper functioning of the fibres in the nerve, the amplitude of evoked compound action potential (CAP) was monitored continuously. The results clearly showed that the number of active nerve fibres in the isolated sciatic nerves of either rat or frog exposed directly to impression pastes containing vinyl polysiloxane, decreased much faster than those of the nerves in contact to impression material without vinyl polysiloxane. When the nerve of the frog was exposed to Xanthopren there was a decrease in the CAP to 50% of the control values within 56.87+/-2.42 h (n=6). This value was called inhibition time to 50%, IT(50) and for Examix it was found to be 9.97+/-1.53 h. When the nerve of the rat was exposed to Xanthopren, the IT(50) was 15.34+/-2.97 h (n=6) for the Xanthopren and only 2.86+/-1.20 h for Examix and 2.76+/-0.48 h for Express 3M (n=6). There was no significant difference between the action of the last two compounds (P=0.85). This fast nerve fibre inactivation could be caused either by the chemical used for the synthesis of the two impression pastes, Examix and Express 3M, or by the unusual constriction of the nerve when it is embedded in the materials with vinyl polysiloxane. There is strong evidence to support the first case, since the incubation of the nerve in the presence of Examix, Express 3M and Xantopren in a way so the nerve was not in contact with the impression pastes, shows a much faster decrease of the CAP in the presence of the first two pastes. The decrease is caused by the death of nerve fibres, since there is no recovery in the CAP after the removal of Examix from the incubating saline.
Abstract: To assess the relative toxicity of the herbicides acetochlor and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the nervous system, the sciatic nerve of the frog (Rana ridibunda) nerve was incubated in saline inside a specially designed recording chamber. This chamber permits monitoring of the evoked compound action potential (CAP) of the nerve, a parameter that could be used to quantify the vitality of the nerve in normal conditions as well as when the nerve was exposed to the compounds under investigation. Thus, when the nerve was exposed to acetochlor, the EC(50) was estimated to be 0.22mM, while for 2,4,5-T the EC(50) was 0.90mM. Using the identical nerve preparation, the EC(50) of 2,4-D was estimated to be 3.80mM [Kouri, G., Theophilidis, G., 2002. The action of the herbicide 2,4-dichlorophenoxyacetic acid on the isolated sciatic nerve of the frog (Rana ridibunda). Neurotoxicol. Res. 4, 25-32]. The ratio of the relative toxicity for acetochlor, 2,4,5-T and 2,4-D was found to be 1:4:17.2. However, because it is well-known that the action of 2,4-D is dependent on the pH, the relative toxicity of the three compounds was tested at pH 3.3, since it has been found that the sciatic nerve of the frog is tolerant of such a low pH. Under these conditions, the EC(50) was 0.77mM (from 0.22mM at pH 7.2) for acetochlor, 0.20mM (from 0.90mM) for 2,4,5-T and 0.24mM (from 3.80mM at pH 7.2) for 2,4-D. Thus, the relative toxicity of the three compounds changed drastically to 1:0.25:0.31. This change in the relative toxicity is due not only to the increase in the toxicity of 2,4,5-T and 2,4-D at low pH levels, but also to the decrease in the toxicity of acetochlor at pH 3.3.
Abstract: We investigate the spontaneous contraction generated by the atria of a frog's heart isolated in a physiological solution. In the relaxation phase, the recorded time series for two different sampling rates possesses an intermittent component similar to the dynamics of the order parameter's fluctuations of a thermal critical system belonging to the mean field universality class. This behavior is not visible through conventional analysis in the frequency space due to the presence of Brownian noise dominating the corresponding power spectrum.
Abstract: Two different test systems, one based on the isolated sciatic nerve of an amphibian and the other on a microbial eukaryote, were used for the assessment of herbicide toxicity. More specifically, we determined the deleterious effects of increasing concentrations of herbicides of different chemical classes (phenoxyacetic acids, triazines, and acetamides), and of 2,4-dichlorophenol (2,4-DCP), a degradation product of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), on electrophysiological parameters and the vitality of the axons of the isolated sciatic nerve of the frog (Rana ridibunda) and on the growth curve of the yeast Saccharomyces cerevisiae based on microtiter plate susceptibility assays. The no-observed-effect-concentration (NOEC), defined as the maximum concentration of the tested compound that has no effect on these biological parameters, was estimated. In spite of the different methodological approaches and biological systems compared, the NOEC values were identical and correlated with the lipophilicity of the tested compounds. The relative toxicity established here, 2,4-DCP > alachlor, metolachlor >> metribuzin > 2,4-D, 2-methyl-4-chlorophenoxyacetic acid (MCPA), correlates with the toxicity indexes reported in the literature for freshwater organisms. Based on these results, we suggest that the relatively simple, rapid, and low-cost test systems examined here may be of interest as alternative or complementary tests for toxicological assessment of herbicides.
Abstract: Imidacloprid is an insecticide which has the nicotinic acetylcholine receptors (nAChRs) as its primary site of action; acetylcholine is the major excitatory neurotransmitter in the insect central nervous system (CNS). In this study, the action of imidacloprid was tested using the synapses of the respiratory central pattern generator of the beetle Tenebrio molitor. The no observed effect concentration (NOEC) for imidacloprid was estimated to be between 0.001 and 0.010 microM. A concentration of 0.10 microM caused hyperexcitation in firing of the respiratory motoneurons, while the concentration of 1.00 microM caused an abrupt increase in their frequency and then a complete inhibition of the activity of the respiratory motoneurons. The possible implication of the action of such low concentrations of imidacloprid in the contraction of the respiratory muscles is also demonstrated and discussed.
Abstract: On the basis of computer prediction of biological activity by PASS and toxicity by DEREK, the most prospective 18 alkylaminoacyl derivatives of 3-amino-benzo-[d]-isothiazole were selected. Their local anesthetic action was assessed using an in vitro preparation of the isolated peroneal nerve of the frog. The local anesthetics action of the compounds was assessed according to the time required for each compound to reduce the amplitude of the evoked compound action potential (CAP). Lidocaine was used as the control compound. The results show that the tested compounds can be divided into three groups: (a) compounds with action similar to lidocaine, (b) compounds with action lower than lidocaine and (c) compounds which block completely the evoked CAP, but after the compound was removed and replaced with normal saline showed no recovery of the potential at all. QSAR studies showed that polarizability, polarity and presence of five-membered rings in molecules have a positive influence on local anesthetic activity, while contributions of aromatic CH and singly bonded nitrogen are negative. Since estimations from PASS probabilities to find local anesthetic activity in the most active compounds were less than 50%, these compounds may be considered as new chemical entities (NCEs).
Abstract: Three triazine herbicides, atrazine, simazine and metribuzine, and some of their major metabolites (cyanuric acid and 6-azauracil) were investigated for their action on synaptic terminals using three different isolated tissue preparations from the atria of the frog, Rana ridibunda, the heart of the honeybee, Apis mellifera macedonica, and the ventral nerve cord of the beetle, Tenebrio molitor. The results indicate that triazines facilitate the release of neurotransmitters from nerve terminals, as already reported for the mammalian central nervous system. The no observed effect concentration, the maximum concentration of the herbicide diluted in the saline that has no effect on the physiological properties of the isolated tissue, was estimated for each individual preparation. According to their relative potency, the three triazines tested can be ranked as follows: atrazine (cyanuric acid), simazine>metribuzine (6-azauracil). The action of these compounds on the cholinergic (amphibians, insects), adrenergic (amphibian) and octopaminergic (insects) synaptic terminals is discussed.
Abstract: The action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the isolated heart of the frog (Rana ridibunda) and two insects, the honeybee (Apis mellifera macedonica) and the beetle (Tenebrio molitor), was investigated using basic electrophysiological methods. The results of this study showed that a concentration of 1 μM 2,4-D was required to reduce the force and the frequency of the isolated heart of the honeybee to about 70% of the initial contraction in less than 20 min. To cause the same effects on the atria of the frog, 45 μM 2,4-D was required and on the isolated heart of the beetle, over 1000 μM of 2,4-D. The presence of an extensive system of gap junctions found in the honeybee is most probably the cause of the unusual sensitivity of its heart to 2,4-D, compared with the heart of the beetle, where no gap junctions were identified.
Abstract: The isolated sciatic nerve of the frog was used to assess the effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the peripheral nervous system. For each experiment, both sciatic nerves were used. The evoked compound action potentials of the nerves were monitored for over 48 h as an indication of their viability. The viability of nerve incubated in control saline was compared to the viability of the nerve incubated in saline where 2,4-D was diluted. The minimum effective concentration (minEC) of 2,4-D was estimated to be between 2 and 4 mM.
Abstract: A recording chamber for monitoring the electrophysiological properties of the isolated heart of adult Drosophila melanogaster has been developed. Spontaneously generated field potentials of constant amplitude can be recorded for 6-8 h (n = 14); in very few cases, records were maintained stable for over 10 h (n = 4), and in some cases below 6 h (n = 5). The chamber consists of the tip of a micropipette, which allows for monitoring the field potential generated by the spontaneously contracting heart. The method can produce accurate information about the heart rate and the amplitude of the cardiac action potential. The preparation can be used for pharmacological studies on the heart of D. melanogaster since it responds, with an increase in the heart rate, to unusually low concentrations of octopamine, 1 nM, a compound with cardioaccelerating properties for insect heart. The recording system can be easily modified for experiments on the heart of other insects. Finally, the isolated heart of D. melanogaster provides a simple method for identifying mutations that affect heart physiology.
Abstract: The contraction of the isolated heart of the bee in physiological solution can be monitored for hours, making this preparation suitable for the investigation of the cardiotoxic action of certain compounds. The results of this study have shown that exposure of the semi-isolated heart of the bee to 1, 0.1, and 0.01 μM deltamethrin causes a temporal increase in the frequency and the force of spontaneously generated contractions, which is followed by a decrease in both parameters. The decrease is dose dependent. The action of deltamethrin was not reversible. The fungicide prochloraz applied at the same concentration levels as deltamethrin has an immediate chronotropic and inotropic effect on the semi-isolated heart of the bee, but its effects are more intense than those caused by deltamethrin. Comparison of the dose-response curves clearly shows that prochloraz is more cardiotoxic than deltamethrin. When prochloraz and deltamethrin are combined there is an increase of over 100 times in the cardiotoxicity of deltamethrin and an increase of 10 times in the toxicity of prochloraz. Our suggestion is that this synergistic action could be caused by the action of the two compounds on the same target site, which in the heart of the bee may be gap junctional intercellular communication, a vital physiological mechanism for the functioning of the heart in both vertebrates and invertebrates
Abstract: Combined intracellular and extracellular recordings from various parts of the isolated dorsal vessel of Tenebrio molitor revealed some of the following electrophysiological properties of the heart and the aorta. (i) The wave of depolarization causing forward pulsation of the dorsal vessel was always transmitted from posterior to anterior, with a conduction velocity of 0.014 m s(-1) in the heart and 0.001 m s(-1) in the aorta when the heart rate was 60 beats min(-1). (ii) There was no pacemaker activity in the aorta. (iii) The duration of the compound action potential in the aortic muscle depended on the duration of the pacemaker action potential generated in the heart. (iv) Isolated parts of the heart continued to contract rhythmically for hours, indicating powerful pacemaker activity in individual cardiac segments. (v) There was a direct relationship between action potential duration and the length of the preceding diastolic interval. (vi) The rhythmic wave of depolarization was dependent on the influx of Ca(2+). (vii) The recovery of the electrical properties of myocardial cells that had been disrupted by sectioning was rapid. (viii) In hearts sectioned into two halves, the rhythmic pacemaker action potentials recorded simultaneously from the two isolated halves eventually drifted out of phase, but they had the same intrinsic frequency. In the light of these data, we discuss two alternative models for the generation of spontaneous rhythmic pumping movements of the heart and aorta.
Abstract: In an isolated preparation which consists of the buccal ganglia and the salivary nerve of Helix lucorum, neuron B2 can be identified by simultaneously recording the soma spike intracellularly and the axon spike extracellularly from the peripheral salivary nerve. The convulsant drug pentylenetetrazol (PTZ) eliminates the axon spike of the neuron within 1-5 min after its application while epileptic activity is induced in the soma of the same neuron for hours. It is interesting that almost all nerve activity in the peripheral nerve is blocked by PTZ.
Abstract: The use of adriamycin (ADR) in cancer chemotherapy has been limited due to its cumulative cardiovascular toxicity. Earlier observations that ADR interacts with mitochondrial cytochrome c oxidase (COX) and suppresses its enzyme activity led us to investigate ADR's action on the cardiovascular functions and heart mitochondrial morphology in Balb-c mice i.p. treated with ADR for several weeks. At various times during treatment, the animals were assessed for cardiovascular functions by electrocardiography and for heart tissue damage by electron microscopy. In parallel, total RNA was extracted from samples of dissected heart and analyzed by Northern blot hybridization to determine the steady-state level of three RNA transcripts encoded by the COXII, COXIII, and COXIV genes. Similarly, samples obtained from the liver of the same animals were analyzed for comparative studies. Our results indicated that 1) treatment of mice with ADR caused cardiovascular arrhythmias characterized by bradycardia, extension of ventricular depolarization time (tQRS), and failure of QRS at high concentrations (10-14 mg/kg body weight cumulative dose); 2) the heart mitochondria underwent swelling, fusion, dissolution, and/or disruption of mitochondrial cristae after several weeks of treatment. Such abnormalities were not observed in the mitochondria of liver tissue; and 3) among the three genes of COX enzyme examined, only COXII gene expression was suppressed by ADR treatment, mainly after 8 weeks in both heart and liver. Knowing that heart mitochondria represent almost 40% of heart muscle by weight, we conclude that the deteriorating effects of ADR on cardiovascular function involve mitochondrial structural and functional impairment.
Abstract: An in vitro model for the study of the axonal transport of herpes simplex virus-1 (HSV-1) in the nerve fibres of the sciatic nerve of the frog Rana ridibunda, has been developed. The nerve was placed along a three-chambered bath consisting of three isolated chambers arranged in series: the stimulating, perfusion and recording chambers. The HSV-1 inoculum was placed in the stimulating chamber, where the proximal part of the isolated sciatic nerve was immersed. HSV-1 was detected after 24-36 h in the recording chamber, where the distal part of the nerve was immersed in Dulbecco's Modified Eagle Medium (DMEM), indicating an axonal transport speed of 46-60 mm/day. The evoked maximum compound action potentials generated in the stimulating chamber was monitored continuously in the recording chamber as an indication of the viability of the nerve during axonal transport. The in vitro method presented here is a useful tool for the pharmacological study of various parameters, e.g. drugs diluted in the perfusion chamber, ionising radiation and temperature, which may affect the axonal transport or other properties of HSV-1.
Abstract: BACKGROUND: In order to investigate the aetiology of uraemic neuropathy, we evaluated the neurotoxic activity of plasma from uraemic patients. To this end we prepared a concentrate (1:1000) of 2-60 kDa MW compounds from paired filtration dialysis ultrafiltrate and evaluated its activity on peripheral nerve conduction in vivo and in vitro. METHODS: The in vivo neurotoxicity was tested on rat sciatic nerve by intraneural injection of the uraemic concentrate, followed, 1 to 6 days later, by electrophysiological assessment of motor response and maximum conduction velocity. In vitro experiments were performed on isolated frog sciatic nerve in the presence of uraemic concentrate, and the neurotoxicity was evaluated from the rate of the decrease in the amplitude of the evoked maximal action potential. RESULTS: In the in vivo experiments, the sciatic nerves injected with the uraemic concentrate showed a decrease in maximum conduction velocity and a progressive impairment in evoked motor response. In the in vitro experiments uraemic concentrate induced a dose-dependent neurotoxic effect. CONCLUSIONS: Our study demonstrates the presence in plasma of uraemic patients of a compound of 2-60 kDa MW with neurotoxic activity.
Abstract: The neurotoxic action of six pyrethroid insecticides, four type II, (flucythrinate, deltamethrin, fenvalerate, fluvalinate) and two type I (cis- and trans-permethrin) was compared on the isolated sciatic nerve of frog. The nerve was exposed to pyrethroids for 30 min and action potentials were recorded for more than 45 hr after exposure. From the plots of the amplitude of the compound action potential vs time, it was possible to estimate, for each compound, the minimum effective concentration, the concentration which is required to reduce the amplitude of the compound action potential to 50% of its control value (mEC50). Flucythrinate was the most toxic compound, while toxicity decreased in the value: deltamethrin > fenvalerate > fluvalinate >> cis-permethrin > trans-permethrin. Low neurotoxicity of cis-permethrin and trans-permethrin (type I pyrethroids) was expected. The neurotoxicity of type I pyrethroids is mainly due to an action at the synapse, which are not present in the frog sciatic nerve preparation. The relative potencies of the four type II compounds agree with their acute toxicity estimated using the LD50.
Abstract: The neurotoxic action of six pyrethroid insecticides, four type II, (flucythrinate, deltamethrin, fenvalerate, fluvalinate) and two type I (cis- and trans-permethrin) was compared on the isolated sciatic nerve of frog. The nerve was exposed to pyrethroids for 30 min and action potentials were recorded for more than 45 hr after exposure. From the plots of the amplitude of the compound action potential vs time, it was possible to estimate, for each compound, the minimum effective concentration, the concentration which is required to reduce the amplitude of the compound action potential to 50% of its control value (mEC50). Flucythrinate was the most toxic compound, while toxicity decreased in the value: deltamethrin > fenvalerate > fluvalinate >> cis-permethrin > trans-permethrin. Low neurotoxicity of cis-permethrin and trans-permethrin (type I pyrethroids) was expected. The neurotoxicity of type I pyrethroids is mainly due to an action at the synapse, which are not present in the frog sciatic nerve preparation. The relative potencies of the four type II compounds agree with their acute toxicity estimated using the LD50.
Abstract: The stretching of the isolated distal part of the sciatic nerve of the frog causes the excitation of a number of its nerve fibres. The excitation lasts as long as the force is applied, indicating a slow adapting mechanoreceptor system. The fact that in experiments in situ stretching of the sciatic nerve causes the reflex activation of the femoral muscles is a clear indication that the activated nerve fibres belong to primary afferent fibres. According to the results of this study either some of the primary afferent fibres in the sciatic nerve of the frog also have the potential of stimulus transduction (mechanical tension) or the sciatic nerve is equipped with nerve terminals which are sensitive to stimulus energy. The implication of this sensory mechanism in the proprioception of the leg is discussed further.
Abstract: The oviducts of the female Decticus albifrons (Orthoptera: Tettigonidae) are innervated by six bilaterally paired neurones, while those of the female Calliptamus sp. (Orthoptera: Catantopidae) are innervated by three bilaterally paired neurones, located in the seventh abdominal ganglion. Using intracellular recording and staining, five of the six oviductal neurones of D. albifrons and the three oviductal neurones of Calliptamus sp. were physiologically and morphologically identified. All three oviductal neurones of Calliptamus sp. have a motor function. In D. albifrons, however, there is evidence for motor function in only three of the five identified oviductal neurones that appear to participate in the generation of the oviductal contractions. The remaining two identified neurones of D. albifrons have a branching pattern similar to that of motor neurones, but their physiological characteristics, large overshooting soma action potentials (30­40 mV) with a long afterhyperpolarising phase, are similar to those of the oviductal unpaired median neurones, which are known to modulate the oviductal contractions. The oviductal muscle exhibits two different modes of contractions: (a) fast and slow myogenic contractions, the fast contractions being produced by spontaneous potentials (30­40 mV) generated by some oviductal muscle fibres; and (b) neurogenic contractions caused by the rhythmic spiking of the oviductal motor neurones. This motor pattern is produced by the oviductal central pattern generator, a neural network residing in the last two abdominal ganglia (seventh and terminal abdominal ganglia) of the species examined here. When isolated both anteriorly and posteriorly, the seventh abdominal ganglion generates rhythmic oviductal contractions of lower frequency and amplitude than those recorded when the connectives between the genital ganglia are intact. The oviductal pattern generator is activated through release from descending inhibition, which originates, in Calliptamus sp., from the compound metathoracic ganglion (fused metathoracic and first three abdominal neuromeres) and in, D. albifrons, from the first free abdominal ganglion (fused second and third abdominal neuromeres).
Abstract: We have studied the action of deltamethrin on respiratory modulated hypoglossal motoneurons in a brain stem slice from newborn mice. Deltamethrin depolarized the hypoglossal motoneurons, increased the background synaptic noise and reduced the frequency and amplitude of current elicited action potentials. Deltamethrin transiently increased the frequency of the respiratory rhythm. Inspiratory potentials in hypoglossal motoneurons were decreased in amplitude and increased in duration. In conclusion, deltamethrin perturbs the respiratory output from the hypoglossal nucleus through postsynaptic actions on hypoglossal motoneurons and by affecting the inspiratory synaptic drive.
Abstract: The analgesic effects of dimethylsulfoxide (DMSO) on the sensory neurones and the sensory axons of an insect mechanoreceptor, the femoral chordotonal organ of Locusta migratoria, were examined. The metathoracic femur was dissected, to expose the chordotonal organ and its sensory nerve, in a chamber containing oxygenated physiological solution where the DMSO was gradually added. The tibia movement was used for the mechanical stimulation of the chordotonal organ while neural activity was recorded and measured using standard electrophysiological methods. For the chordotonal organ, the blocking concentration of DMSO, the concentration which eliminates the response of the sensory neurones to mechanical stimulation by 50% in 18-20 min, was estimated to be 0.85 +/- 0.11% (n = 6) v/v. On the contrary, the blocking concentration of the DMSO for the sensory axons was 4.6 +/- 0.7% (n = 6) v/v, approximately five times higher than the concentration required to inactivate the sensory neurones. The implications of this difference in the physiological mechanisms involved for the DMSO-induced analgesia are discussed.
Abstract: Most abdominal sternites of the cricket Gryllus bimaculatus and the bushcricket
Decticus albifrons are bridged by a transverse muscle (TM) which supports expiratory movements. In the cricket, ventilatory contractions are controlled both within each segment, by a bilateral pair of excitatory motoneurones in the abdominal ganglion supplying the left and right halves of the TM independently, and intersegmentally, by peripheral collaterals of homologous motoneurones from adjacent segments. The axons of these motoneurones run in the ipsilateral paramedian nerve. This unique divergence of excitatory motoneurones to different muscles also results in massive convergence of excitatory inputs from different ganglia, especially on the TMs of the middle abdominal segments. TM contraction rates are increased by this intersegmentally divergent and convergent motor supply, especially in the middle abdominal segments. In bushcrickets, each transverse muscle in segments 3–7 is innervated bilaterally by four pairs of neurones: (i) two pairs of contralateral excitatory motoneurones with axons that diverge, supplying two adacent muscles; (ii) one pair of contralateral excitatory neurones found in the second anterior ganglion and (iii) a pair of median inhibitory neurones in the segmental ganglion. Transverse muscles 2 and 8 receive reduced
innervation. The excitatory motoneurones generate slow excitatory postsynaptic
potentials (EPSPs), which must sum to cause muscle contractions. During ventilation, contralateral paired transverse motoneurones fire at similar frequencies, thus sychronizing the contractions of the left and right halves of the muscle so that the whole muscle acts as a single unit.
Abstract: The system is designed for data-acquisition and computation of a relatively large number of parameters of a single twitch or tetanic contraction generated by an electrically stimulated skeletal muscle. The parameters that can be computed using the program facilities are: (i) the rise time (Tc), 50% relaxation time (50% Tr) and the maximum tension (Pt) of the twitch contraction; (ii) the approximate number of motor units innervating the muscle and the fatigue index; (iii) the fusion frequency of the tetanic contraction and the twitch to tetanus ratio; (iv) the maximum tetanic tension (P0) and its Tc and 50% Tr; (v) the P0 versus muscle length diagram; (vi) the fatigue-time course.
Abstract: Some numbers of the family of 2-(aminoacetylamino)thiazoles were synthesized. The structure of these compounds was identified both by elemental as well as by spectroscopic analysis (UV, IR, H-NMR, MS). The possible local anaesthetic action of these compounds was also tested using the sciatic nerve of the frog. None of the tested compounds were found to have local anaesthetic action in on in vitro preparations as lidocaine, but one of the compounds showed a similar anaesthetic action to procaine.
Abstract: A method which allows the intracellular staining of physiologically identified muscle fibres is described. The possible applications of this method have been discussed.
Abstract: A simple and easily constructed unit was designed using a buffer amplifier, a programmable transconductance amplifier and an active filter. The unit allows extracellular recording and stimulation through the same electrode. Thus in experiments where it is required to stimulate a nerve and record the effects of the stimulation from the same nerve, the stimulating and the recording electrodes can be combined in one.
