Intrathecal gene therapy rescues a model of demyelinating peripheral neuropathy.

Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot-Marie-Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.

Assessing the axonal translocation of CeO2 and SiO2 nanoparticles in the sciatic nerve fibers of the frog: an ex vivo electrophysiological study.

The axonal translocation of two commonly used nanoparticles in medicine, namely CeO2 and SiO2, is investigated. The study was conducted on frog sciatic nerve fibers in an ex vivo preparation. Nanoparticles were applied at the proximal end of the excised nerve. A nerve stimulation protocol was followed for over 35 hours. Nerve vitality curve comparison between control and exposed nerves showed that CeO2 has no neurotoxic effect at the concentrations tested. After exposure, specimens were fixed and then screen scanned every 1 mm along their length for nanoparticle presence by means of Fourier transform infrared microscopy. We demonstrated that both nanoparticles translocate within the nerve by formation of narrow bands in the Fourier transform infrared spectrum. For the CeO2, we also demonstrated that the translocation depends on both axonal integrity and electrical activity. The speed of translocation for the two species was estimated in the range of 0.45-0.58 mm/h, close to slow axonal transportation rate. Transmission electron microscopy provided direct evidence for the presence of SiO2 in the treated nerves.

Oxaliplatin-induced neurotoxicity is mediated through gap junction channels and hemichannels and can be prevented by octanol.

Oxaliplatin-induced neurotoxicity (OIN) is a common complication of chemotherapy without effective treatment. In order to clarify the mechanisms of both acute and chronic OIN, we used an ex-vivo mouse sciatic nerve model. Exposure to 25 µM oxaliplatin caused a marked prolongation in the duration of the nerve evoked compound action potential (CAP) by nearly 1200% within 300 min while amplitude remained constant for over 20 h. This oxaliplatin effect was almost completely reversed by the gap junction (GJ) inhibitor octanol in a concentration-dependent manner. Further GJ blockers showed similar effects although with a narrower therapeutic window. To clarify the target molecule we studied sciatic nerves from connexin32 (Cx32) and Cx29 knockout (KO) mice. The oxaliplatin effect and neuroprotection by octanol partially persisted in Cx29 better than in Cx32 KO nerves, suggesting that oxaliplatin affects both, but Cx32 GJ channels more than Cx29 hemichannels. Oxaliplatin also accelerated neurobiotin uptake in HeLa cells expressing the human ortholog of Cx29, Cx31.3, as well as dye transfer between cells expressing the human Cx32, and this effect was blocked by octanol. Oxaliplatin caused no morphological changes initially (up to 3 h of exposure), but prolonged nerve exposure caused juxtaparonodal axonal edema, which was prevented by octanol. Our study indicates that oxaliplatin causes forced opening of Cx32 channels and Cx29 hemichannels in peripheral myelinated fibers leading to disruption of axonal K(+) homeostasis. The GJ blocker octanol prevents OIN at very low concentrations and should be further studied as a neuroprotectant.

Assessing the permeability of the rat sciatic nerve epineural sheath against compounds with local anesthetic activity: an ex vivo electrophysiological study.

Abstract Studies have shown that the sciatic nerve epineural sheath acts as a barrier and has a delaying effect on the diffusion of local anesthetics into the nerve fibers and endoneurium. The purpose of this work is to assess and to quantify the permeability of the epineural sheath. For this purpose, we isolated the rat sciatic nerve in a three-chamber recording bath that allowed us to monitor the constant in amplitude evoked nerve compound action potential (nCAP) for over 24 h. For nerves exposed to the compounds under investigation, we estimated the IT50 the time required to inhibit the nCAP to 50% of its initial value. For desheathed nerves, the half-vitality time was denoted as IT50(-) and for the ensheath normal nerves as IT50(+). There was no significant difference between the IT50 of desheathed and ensheathed nerves exposed to normal saline. The IT50(-) for nerves exposed to 40 mM lidocaine was 12.1 ± 0.95 s (n=14) and the IT50(+) was 341.4 ± 2.49 s (n=6). The permeability (P) coefficient of the epineural sheath was defined as the ratio IT50(+)/IT50(-). The P coefficient for 40 mM lidocaine and linalool was 28.2 and 3.48, correspondingly, and for 30 mM 2-heptanone was 4.87. This is an indication that the epineural sheath provided a stronger barrier against lidocaine, compared to natural local anesthetics, linalool and 2-heptanone. The methodology presented here is a useful tool for studying epineural sheath permeability to compounds with local anesthetic properties.

The force of the spontaneously contracting zebrafish heart, in the assessment of cardiovascular toxicity: application on adriamycin.

The heart of the zebrafish has been used extensively to assess the cardiotoxic effect of compounds, using the frequency of heart contractions as the main index of cardiac response to drugs. In this study, the force and the frequency generated by the spontaneously contracting zebrafish heart, isolated in saline, were found to be 0.87 ± 0.05 mN and 1.54 ± 0.03 Hz (n=6) respectively within the first hour of recording. Both values of force and frequency remained constant for over 8h. The advantage of prolonged vitality in the assessment of cardiovascular toxicity was shown using the well-known anticancer drug adriamycin, which has severe cardiotoxic side effects. At 10.0 µM there was a 21.05 ± 4.42% (p=0.02, n=4) decrease in the force of contraction, while the frequency was not affected after 3h treatment (p>0.05). At 50.0 and 100.0 µM there was a 33.24 ± 3.0 and 46.6 ± 4.80% irreversible decrease in force (p<0.05, n=4), while a 18.02 ± 4.07% and 16.16 ± 4.07% reversible increase was observed in the frequency (p=0.02, n=4). These contradictory positive chronotropic and negative inotropic responses indicate the strong inhibitory effect of adriamycin on ventricular cardiomyocytes and its excitatory effects on auto-rhythmical pacemaker cells. If heart frequency was the only parameter used to assess the cardiotoxic effect of adriamycin, at the above range of concentrations, this compound would have been classified as non-cardiotoxic.

Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz.

Amitraz is a pesticide targeting the octopaminergic receptors. In a previous study, octopamine, a biogenic amine, was found to induce a biphasic effect on the honeybee heart, inhibition at low concentrations and excitation at high concentrations. Furthermore, the honeybee heart was found to be far more sensitive to octopamine compared to other insect hearts. The objective of the present study was to investigate the effects of amitraz on the electrical and mechanical properties of the honeybee heart ex vivo and on the heart rate in vivo. In ex vivo conditions, amitraz at 10(-12) M caused a significant inhibition in the mechanical (p<0.05, n=4) and electrical properties (p<0.05, n=4). Higher concentrations such as 10(-9) and 10(-6) M induced a biphasic effect, with total inhibition for 7.86±1.26 min (n=7), followed by strong excitation of spontaneously-generated contractions (n=7). The initial elimination of heart activity was caused by strong hyperpolarization, while the subsequent excitation was caused by a depolarization in the membrane potential of pacemaker cells at 10(-9) M (n=8). In the in vivo experiments, abdominal injection or oral application of 0.20 ng of amitraz per bee induced a persistent increase of 134.28±4.07% (p<0.05, n=4) in the frequency of the cardiac action potentials. The above responses clearly show that the heart of the honeybee is extremely vulnerable to amitraz, which is nevertheless still used inside beehives, ostensibly to "protect" the honeybees against their main parasite, Varroa destructor.

Oxaliplatin-induced hyperexcitation of rat sciatic nerve fibers: an intra-axonal study.

Oxaliplatin is an agent that is used extensively in gastrointestinal cancer chemotherapy. The agent's major dose-limiting toxicity is peripheral neuropathy that can manifest as a chronic or an acute syndrome. Oxaliplatin-induced acute neuropathy is purportedly caused by an alteration of the biophysical properties of voltage-gated sodium channels. However, sodium channel blockers have not been successful at preventing acute neuropathy in the clinical setting. We report intra-axonal recordings from the isolated rat sciatic nerve preparation under the effect of oxaliplatin. The depolarization phase of single action potentials remains intact with a duration of 0.52 ± 0.02 ms (n=68) before and 0.55 ± 0.01 ms (n=68) after 1-5 h of exposure to 150 µM oxaliplatin (unpaired t-test, P > 0.05) whereas there is a significant broadening of the repolarization phase (2.16 ± 0.10 ms, n=68, before and 5.90 ± 0.32 ms after, n=68, unpaired t-test, P < 0.05). Apart from changes in spike shape, oxaliplatin also had drastic concentration- and time-dependent effects on the firing responses of fibers to short stimuli. In the intra-axonal recordings, three groups of firing patterns were indentified. The first group shows bursting (internal frequency 90 - 130 Hz, n=88), the second shows a characteristic plateau (at -19.27�2.84 mV, n=31, with durations ranging from 45 - 140 ms depending on the exposure time), and the third combines a plateau and a bursting period. Our results implicate the voltage-gated potassium channels as additional oxaliplatin targets, opening up new perspectives for the pharmacological prevention of peripheral neuropathy.

The bite of the honeybee: 2-heptanone secreted from honeybee mandibles during a bite acts as a local anaesthetic in insects and mammals.

Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation-the isolated rat sciatic nerve-the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

In quest of the missing neuron: spike sorting based on dominant-sets clustering.

Spike sorting algorithms aim at decomposing complex extracellular signals to independent events from single neurons in the electrode's vicinity. The decision about the actual number of active neurons is still an open issue, with sparsely firing neurons and background activity the most influencing factors. We introduce a graph-theoretical algorithmic procedure that successfully resolves this issue. Dimensionality reduction coupled with a modern, efficient and progressively executable clustering routine proved to achieve higher performance standards than popular spike sorting methods. Our method is validated extensively using simulated data for different levels of SNR.

Assessing the neurotoxic effects of palytoxin and ouabain, both Na⁺/K⁺-ATPase inhibitors, on the myelinated sciatic nerve fibres of the mouse: an ex vivo electrophysiological study.

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 fibres. 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.

High concentrations of dichloroacetate have minor effects on the vitality of the mammalian nerve fibers: an ex-vivo electrophysiological study.

Dichloroacetate has been used extensively in the treatment of cancer and genetic mitochondrial diseases, but there have been reports of dichloroacetate-induced peripheral neuropathy. In this study, the acute effects of sodium dichloroacetate on the peripheral nerve fibers were investigated, using an ex-vivo preparation, in the isolated sciatic nerve of the rat. The amplitude of the evoked nerve compound action potential (CAP) was measured 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, here called inhibitory time 50% (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 mmol/l of dichloroacetate, 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 dichloroacetate as 10 mmol/l had 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 mmol/l of dichloroacetate 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 28-30 h, there could be a gradual development of certain biochemical changes leading to the early stages of dichloroacetate-induced neurotoxicity.

Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.

The effects of octopamine, the main cardioacceleratory 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 effect on the frequency and force of cardiac contractions acting as an agonist, with a strong acceleratory effect, at concentrations higher than 10(-12)M for the honeybee and higher than 50×10(-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 is extended to the blockers of octopaminergic receptors, where phentolamine at 10(-5)M stopped the spontaneous contractions of the honeybee heart completely and permanently, while the same blocker at the same concentration caused only 50% inhibition in the heart of the olive fruit fly. Phentolamine and mianserin at low concentrations of 10(-7)M also blocked the heart octopaminergic receptors, but for a short period of time, of less than 15.0 min, while a partial recovery in 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 suggests 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 the olive fruit fly was 10(5) times less sensitive to octopamine, since a persistent inhibition of heart contractions occurred at 10(-9)M. In conclusion, the acceleration of the insect heart is achieved by increasing the levels of octopamine, while there is a passive but also an active decrease in heart activity due to the minimization of octopamine.

NASS: an empirical approach to spike sorting with overlap resolution based on a hybrid noise-assisted methodology.

Background noise and spike overlap pose problems in contemporary spike-sorting strategies. We attempted to resolve both issues by introducing a hybrid scheme that combines the robust representation of spike waveforms to facilitate the reliable identification of contributing neurons with efficient data learning to enable the precise decomposition of coactivations. The isometric feature mapping (ISOMAP) technique reveals the intrinsic data structure, helps with recognising the involved neurons and, simultaneously, identifies the overlaps. Exemplar activation patterns are first estimated for all detected neurons and consecutively used to build a synthetic database in which spike overlaps are systematically varied and realistic noise is added. An Extreme Learning Machine (ELM) is then trained with the ISOMAP representation of this database and learns to associate the synthesised waveforms with the corresponding source neurons. The trained ELM is finally applied to the actual overlaps from the experimental data and this completes the entire spike-sorting process. Our approach is better characterised as semi-supervised, noise-assisted strategy of an empirical nature. The user's engagement is restricted at recognising the number of active neurons from low-dimensional point-diagrams and at deciding about the complexity of overlaps. Efficiency is inherited from the incorporation of well-established algorithms. Moreover, robustness is guaranteed by adaptation to the actual noise properties of a given data set. The validity of our work has been verified via extensive experimentation, using realistically simulated data, under different levels of noise.

Assessing the local anesthetic effect of five essential oil constituents.

We studied the effects of five monoterpenoids, viz. 1,8-cineole, fenchone, linalool, p-cymene and α-pinene, on the sciatic nerve fibers of the frog Rana ridibunda (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 (IT(50)), 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 26.0 °C. The IT(50) values for nerves incubated in saline with p-cymene, 1,8-cineole, 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 IT(50) 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 IT(50) 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 fibers of the frog, we conclude that, whereas 1,8-cineole, p-cymene and α-pinene cause only minor effects, linalool and fenchone exhibit acute local anesthetic activity.

Assessing the effects of the neonicotinoid insecticide imidacloprid in the cholinergic synapses of the stellate cells of the mouse cochlear nucleus using whole-cell patch-clamp recording.

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 (EC(50) 0.86-1 microM) than it is against mammalian nAChRs (EC(50) 70 microM). 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 microM IMI had no significant effect on the membrane properties of the neurons tested, while a concentration of 10 microM 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 >or=50 microM 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 microM 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 alpha-bungarotoxin (alpha-BgTx) blocked the spontaneous firing induced by IMI (n=3). The amplitude of the 100 microM IMI-induced inward current at -60 mV holding potential was 115.0+/-16.2 pA (n=7). IMI at a concentration of 10 microM produced 11.3+/-3.4 pA inward current (n=4). We conclude that exposure to IMI at concentrations >or=10 microM for <1 min can change the membrane properties of neurons that have nAChRs and, as a consequence, their function.

Comparing the inhibitory effects of five protoxicant organophosphates (azinphos-methyl, parathion-methyl, chlorpyriphos-methyl, methamidophos and diazinon) on the spontaneously beating auricle of Sparus aurata: an in vitro study.

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 microM). MeAZP was found to be the most effective anticholinesterase compound, with an IC(50) of 2.19+/-1.05 microM (n=6), while MET was less effective, with an IC(50) of 72.3+/-1.2 microM (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 microM 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 IC(50), was as follows: MeAZP, 2.19+/-1.05 microM>paraoxon, 3.2+/-1.5 microM"MET, 72.3+/-1.2 microM>MePS, 80.3+/-1.03 microM>MeCPP, 93.7+/-1.01 microM>DZ, 164+/-1.01 microM. (5) There was a good correlation (r=0.779, p=0.04, n=5) between IC(50) and the previously determined logP (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 (Ca(2+) channels) and the frequency (K(+) channels) of the spontaneous auricle contractions.

Inhibitory vs. protective effects of N-acetyl-l-cysteine (NAC) on the electromechanical properties of the spontaneously beating atria of the frog (Rana ridibunda): an ex vivo study.

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 10.0mM, eliminated, in a concentration-dependent manner, the contractile properties of the atria (force and frequency) within minutes, without affecting its electrical properties; (2) the IC(50) 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 IC(50) 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 1-2mM; (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 2mM protected against cadmium-induced inhibition of atria contraction. The IC(50) for cadmium was 17.9+/-1.1 microM (n=6) (16.9-19.0 microM, 95% CI), while in the presence of 2mM NAC, it became 123.3+/-1.0 microM (n=6) (114.8-132.4 microM, 95% CI). The same concentration of NAC failed to exert any protective effects against rotenone (5 microM)-induced inhibition of atria contraction. The protective effects of NAC are probably due to chelation of cadmium, rather than scavenging of oxidants.

Evaluation of the local anaesthetic activity of 3-aminobenzo[d]isothiazole derivatives using the rat sciatic nerve model.

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.

The effects of oxaliplatin, an anticancer drug, on potassium channels of the peripheral myelinated nerve fibres of the adult rat.

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 1000min. Incubation of the sciatic nerve fibres in 25, 100 and 500microM oxaliplatin, for 300-700min 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 5microM, oxaliplatin caused broadening of the repolarization phase of the CAP only, while the no observed effect concentration was estimated to be 1microM. 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.

Performance evaluation of PCA-based spike sorting algorithms.

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.