A B S T R A C T Histrionicotoxin, a toxin isolated from skin secretions of a Colombian arrow poison frog, Dendrobates histrionicus, decreased the amplitude and timecourse of the endplate current, and altered the voltage dependence of the halfdecay time. In addition, the toxin produced a characteristic nonlinearity in the current-voltage relationship of the endplate current when 3-s voltage conditioning steps were used. Reduction in time of the conditioning steps to 10 ms made the current-voltage relationship linear. The decrease in peak amplitude of the endplate current (epc) produced by histrionicotoxin measured during long hyperpolarizing conditioning steps was fitted by a single exponential function. The calculated rate constants ranged from 0.03 to 0.14 s -1 and varied with membrane potential at hyperpolarizing levels. The voltage-and time-dependent action of histrionicotoxin does not require an initial activation of receptors by acetylcholine (ACh). The characteristic of the current-voltage relationship can be accounted for by the observed voltage and time dependency of the attenuation of the endplate current amplitude in the presence of histrionicotoxin during long conditioning steps. These effects of histrionicotoxin on the peak amplitude, and on the voltage and time dependence of the epc were concentration-dependent and slowly reversible upon washing out the toxin. Thus, the voltage-and time-dependent action of histrionicotoxin at the endplate is related to an increase in the affinity between the toxin and the ACh receptor-ionic channel complex. This increase in affinity is postulated to be due to a conformational change of the macromolecule in the presence of histrionicotoxin which is demonstrated to be relatively slow, i.e., on the order of tens of seconds.

A B S T R A C T The effect of tetraethylammonium (TEA) bromide on the neurally and iontophoretically evoked endplate current (EPC) of frog sartorius muscle was investigated using voltage-clamp and noise analysis techniques, and its binding to the acetylcholine (ACh) receptor ionic channel complex was determined on the electric organ of Torpedo oceUata. TEA (250-500 #M) produced an initial enhancement followed by a slow decline in the amplitude of the endplate potential and EPC, but caused only depression in the amplitude of the miniature endplate potential and current. In normal ringer's solution, the EPC current-voltage relationship was approximately linear, and the decay phase varied exponentially with membrane potential. Upon addition of 50-100 #.M TEA, the current-voltage relationship became markedly nonlinear at hyperpolarized command potentials, and with 250-2000 /~M TEA, there was an initial linear segment, an intermediate nonlinear segment, and a region of negative conductance. The onset of nonlinearity was dose-dependent, undergoing a 50 mV shift for a 10-fold increase in TEA concentration. The EPC decay phase was shortened by TEA at hyperpolarized but not depolarized potentials, and remained a single exponential function of time at all concentrations and membrane potentials examined. These actions of TEA were found to be independent of the sequence of polarizations, the length of the conditioning pulse, and the level of the initial holding potential. TEA shifted the power spectrum of ACh noise to higher frequencies and produced a significant depression of single channel conductance. The shortening in the mean channel lifetime agreed closely with the decrease in the EPC decay time constant. At the concentrations tested, TEA did not alter the EPC reversal potential, nor the resting membrane potential, and had little effect on the action potential duration. TEA inhibited the binding of both

Summary:Purpose: Status epilepticus (SE) has been considered an epileptogenic factor in humans. In the pilocarpine (PILO) model, after a brief period marked by SE, the rats exhibit recurrent spontaneous seizures, mimicking the clinical features of temporal lobe epilepsy. The aim of our study was to identify the molecular actions of PILO that could account for its ability to induce SE.Methods: Whole-cell mode of the patch-clamp technique was applied to cultured hippocampal neurons (2-3 weeks old) in the absence and in the presence of PILO (1-10 M), to study the spontaneous activity, the evoked, and the miniature postsynaptic currents. The postsynaptic currents were isolated pharmacologically.Results: PILO (1 and 10 M) caused an initial increase followed by a decrease in the frequency of spontaneous activity. The increase in the frequency of excitatory postsynaptic currents (EPSCs) and inhibitory PSCs (IPSCs) was blocked by atropine (1 M), indicating that this effect is mediated through muscarinic receptors. PILO also promoted a brief increase of the amplitude of IPSCs indirectly evoked by stimulation of a neuron synaptically connected to the neuron under study. Conversely, PILO promoted a sustained increase on the amplitude of electrically evoked EPSCs. In presence of tetrodotoxin (TTX; 300 nM), PILO (1 M) increased the frequency of miniature EPSCs and IPSCs without changing their amplitude during the first 3 min of application.Conclusions: These results indicate that PILO acting through muscarinic receptor causes an imbalance between excitatory and inhibitory transmission that can result in the generation of SE observed in animals acutely treated with PILO. Key Words: Pilocarpine-Whole cell-Cultured hippocampal neuron-IPSP-EPSP.Status epilepticus (SE) has been considered an epileptogenic factor in humans. Particularly the occurrence of SE or prolonged febrile seizures during childhood, as well trauma or meningitis, has been related to the late appearance of temporal lobe epilepsy (TLE) (1-3). This hypothesis suggests that TLE could be a consequence of initial precipitating injury resulting in a damage of selective hippocampal neuronal groups (4). Neuron loss would be followed by a reorganization of hippocampal excitatory and inhibitory circuitry and mossy fiber sprouting involved in the physiopathology of limbic seizures (2,5).Among the animal models, the pilocarpine (PILO) model (6) has been used to understand the mechanisms that underlie this pathology, because it displays several behavioral, electroencephalographic, and histopathologic similarities to TLE.Cavalheiro et al. (7) demonstrated that the application of pilocarpine, a muscarinic cholinergic agonist, induces a rapid onset of limbic seizures that result in the acute phase, marked by SE, which can last 12 h. After that, the rats have a symptom-free period for 4-44 days called the "silent phase," and then exhibit recurrent spontaneous seizures, the chronic phase. The key to the chronic phase seems to be the occurrence of SE, because those rats without S...

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Neuromuscular junctions from patients with early onset and chronic myasthenia gravis were examined by electrophysiological and ultrastructural techniques. Acetylcholine (AcCh) sensitivities were reduced by 34-63% in early onset myasthenia and 60-80% in chronic myasthenia. Ultrastructural analysis revealed that virtually all junctional folds of the early onset patients were intact but that the AcCh-receptor-rich crests of these folds were uniformly covered by an attached layer of 30 X 70 A particles arranged in small tufts or rosettes. In chronic myasthenic endplates, however, junctional fold crests were destroyed, apparently being replaced by vesicular membrane debris similarly labeled by tufts of 30 X 70 A particles. Thus, the initial reduction in junctional AcCh sensitivity observed in early onset myasthenia gravis may be attributed at least in part to in situ masking or inactivation of AcCh receptors, whereas the marked decrease in AcCh sensitivity observed in the chronic myasthenic patient may represent a combination of two factors: (a) in situ masking of AcCh receptors and (b) destruction of the receptor-containing crests of the junctional folds. These observations are compatible with an autoimmune etiology of myasthenia gravis initially involving an apparent antibody attachment to one or more components of the functional AcCh receptor complex, followed by systematic destruction and removal of junctional folds by both humoral and cell-mediated autoimmune responses.