End plate currents with a physiological level of quantal secretion and after potentiation of the mediator release by 4-aminopyridine

The role of postsynaptic potentiation (PSP) and asynchronous secretion of acetylcholine (ACh) in generation of multiquantal and plate currents (EPC) was studied under voltage clamp conditions in the cut sartorius muscle of frog before and after 4-aminopyridine (4-AP) treatment. If compared with miniature EPC (MEPC), multiquantal EPC had larger amplitude and more prolonged both rise-time and decay. In the presence of 4-AP the difference between the decay time constant of EPC (tau epc) and MEPC (tau mepc) became much more pronounced. Mean quantal content of EPC was 249 in control 373 and 423 in the presence of 1 X 10(-5) and 1 X 10(-4) mol/l of 4-AP, respectively. Magnesium ions (6-10 mmol/l) reduced the amplitude and tau epc of EPC without affecting its rise-time. Repetitive activity (10 Hz, 60s) resulted in the reduced amplitude and tau epc but in increased rise-time of EPC. D-tubocurarine (5 X 10(-7) mol/l) and alpha-bungarotoxin (1 X 10(-6) g/ml) diminished the difference between tau epc and tau mepc. In the presence of 4-AP all these effects were much more pronounced. It was suggested that asynchronous secretion of ACh from motor nerve terminals caused prolongation of the rise-time, reduction in the amplitude of EPC, but had little or no effect on the decay rate of EPC. The slow decay of multiquantal EPC both in the absence and in the presence of 4-AP was almost entirely due to postsynaptic interaction of ACh quanta, i. e. PSP.     

Reactivating and cholinolytic action of dipyroxime in the myoneural synapse of warm-blooded animals

Reactivating (RA) and cholinolytic (Chl) effects of dipyroxime in soleus and diaphragm muscles of the rat were estimated by amplitudes and durations of miniature end-plate potentials (MEPP) and currents (MEPC). After armin-induced inhibition of acetylcholinesterase (AchE), the action of dipyroxime in concentrations of 5.10(-6)-5.10(-4) mol/l on the amplitude and duration of MEPPs and MEPCs represents the combination of RA and Chl effects. Separate study of the RA effect (after washing out of the reactivator) has shown that this effect increased within the whole range of concentrations used. Complete reactivation of phosphorylated AchE was achieved at dipyroxime concentrations of 2-5.10(-4) mol/l. Separate analysis of Chl effect (in voltage-clamp experiments with intact AchE) has shown that dipyroxime suppressed acetylcholine-induced responses due to a block of cholinoreceptors in the open conformation. This block was characterized by prolongation of MEPCs decay without affecting its exponential nature. It is concluded that dipyroxime is a "very fast blocker".     

Miniature currents of the endplates of the muscle fibers of the diaphragm of the rat after inhibition of acetylcholinesterase with galanthamine

Miniature end-plate currents (MEPC) in rat diaphragm were studied with voltage-clamp technique when synaptic acetylcholinesterase (AChE) was inhibited with different concentrations of galanthamine. The MEPC amplitude and time course were increased progressively with galanthamine concentrations in the range of 3.16 X 10(-8) - 10(-6) g/ml. The decay of MEPC was always exponential. The input resistance of muscle fibres increased. Galanthamine (10(-5) g/ml) produced a curare-like action: the amplitude and duration of MEPC were less as compared with those at galanthamine concentration 10(-6) g/ml, the decay of MEPC became biphasic. During washing out of the drug, the duration of MEPC began to increase and then to diminish, returning to the initial value 3 hours later. The decay of MEPC became exponential. A positive correlation was found between half-decay time and amplitude of MEPC both in the presence and in the absence of anticholinesterase. It is supposed that the functional role of synaptic AChE in limiting the postsynaptic effect of acetylcholine is not so significant as it is usually considered, therefore it is possible to use the parameters of MEPC for the estimation of functional AChE activity.     

Effects of "non-quantal" acetylcholine on the sensitivity of the postsynaptic membrane: action of ouabain and imitation of these effects by exogenous acetylcholine]

Development of postsynaptic potentiation (PSP) and desensitization (DS) caused by "non-quantal" acetylcholine after acetylcholinesterase inhibition was studied by means of ouabain, an agent known to modulate (initially increase and then decrease) the level of non-quantal secretion of ACh. Ouabain had no effect on the MEPC parameters when AChE was active. After AChE inhibition ouabain initially increased the decay time constant of MEPC (tau), i.e. caused postsynaptic potentiation (PSP). This effect of ouabain grew with time between inhibition of AChE and application of ouabain. The PSP stage was followed by shortening of MEPCs decay, due to the development of desensitization (DS), and that process was more pronounced than in control. Applied before AChE inhibition, ouabain had no effect on tau. Thus neither PSP nor DS developed under those conditions. Exogenous ACh (20 nmol/l) applied simultaneously with inhibitor of AChE partially prevented the shortening of MEPCs decay, but decreased the amplitude of MEPC. Applied after MEPCs shortening, exogenous ACh (50 nmol/l) tended to return the initial value of tau. It is concluded that nonquantal ACh produces PSP and DS on the postsynaptic membrane after inhibition of ACh and that the DS persists after cessation of nonquantal secretion for a long time.     

The characteristics of the action of calcium ions on miniature end-plate currents after the disruption of mediator hydrolysis

Effect of calcium on the miniature end-plate currents (MEPC) at the frog neuromuscular junction was studied by the voltage-clamp technique. Rise of the calcium concentration in the Ringer solution up to 9 mmol/l caused a decrease of the MEPC amplitude which was related to the reduction of the end-plate channel conductance. Calcium had no effect on the time course of MEPCs at the active acetylcholinesterase (AChE) but accelerated MEPC decay by 26% after AChE inhibition by neostigmin or armin. It is supposed that the shortening effect of calcium on the MEPC decay phase is based on the ability of calcium to modulate the block of ionic channels by acetylcholine or to accelerate the process of desensitization of the postsynaptic membrane.     

Effect of the quantal composition, membrane potential and cholinoreceptor density on the temporal flow of the end plate current in the rat under conditions of acetylcholinesterase inhibition]

The factors determining the decay of multiquantal end plate currents (EPC) were studied in the diaphragm muscle of rat by the comparison of EPC and miniature EPC (MEPC) amplitude--temporal characteristics. The decay of EPC (quantal content 25-100) was 1.2 times slower than the decay of MEPC when AChE was active. The AChE inhibition by armine or neostigmine made this difference 10-100 times higher. In most synapses the decay of multiquantal EPC can be approximated by a sum of two or three exponents. It depended on the quantal content and 3-exponential EPC could be transformed in 2-exponential and later to monoexponential ones if increasing concentration of magnesium ions. A slow component of EPCs (but not of MEPC) decay was highly sensitive to concentration of magnesium ions and had 3 times higher dependence of the membrane potential value than that one of MEPC. The irreversible blocking of receptors by alpha-bungarotoxin (alpha-BuTX) accelerated the decay of MEPC but the decay of multiquantal EPC changed in two phases: it was prolonged at the beginning of alpha-BuTX action followed by its acceleration, but never the time of the decay of EPC had achieved the apparent open time of ACh-activated ionic channels. It is suggested that during the multiquantal EPC generation not only the synchronization of opening but the kinetic of ACh-activated channels is changed, probably by blocking of this channels by high concentrations of endogenous ACh.     

The depressing effect of GABA on synaptic excitation in isolated rat hippocampal slices

The action of various gamma-aminobutyric (GABA) concentrations on the amplitude of population EPSPs (PEPSPs) recorded from dendrites was examined in hippocampal slices. GABA produced reversible dose-dependent diminishing of the amplitude of PEPSPs without features of desensitization. CA1 hippocampal PEPSPs were much more sensitive to GABA (threshold concentrations--3.10(-5)-2.10(-4)mol/l; IC50--5.10(-4)-1.10(-3) mol/l) the action of GABA on PEPSPs was not depressed by bicuculline, picrotoxin or penicillin. The inhibitory action of GABA on dendritic antidromic population spike (DAPS) (post-synaptic inhibitory effect) was greatly reduced by these drugs. Baclofen was more potent inhibitor of CA1 PEPSPs than GABA (threshold concentration--1.10(-6)mol/l; IC50--3.10(-6)mol/l), but even in high concentration it induced only slight reduction of DAPS amplitude. It is concluded that GABA inhibitory effects on CA1 hippocampal PEPSPs are caused in the main by its presynaptic action mediated by GABA(B)-receptors located probably on axon terminals of the Schaffer collaterals.     

Effect of magnesium ions on the functional state of postsynaptic membrane by modulation of the level of non-quantal secretion of the mediator]

The role of non-quantal secretion (NS) of acetylcholine (ACh) in shortening of miniature end-plate currents (MEPC) after initial prolongation of MEPC due to acetylcholinesterase (AChE) inhibition was studied in the mouse diaphragm muscle under the voltage clamp conditions. Elevation of NS level by removal of magnesium ions from solution accelerated the shortening effect. After elimination of NS due to an increase of magnesium concentration up to 3 mmol/l the shortening of MEPC was absent. It was suggested that after AChE inhibition the magnesium ions in physiological range of concentrations can modulate the desensitization development on postsynaptic membranes by NS level regulation.     

Characteristics of the time course of miniature endplate currents in different parts of the neuromuscular junction in the frog

Extracellular glass microelectrodes were used for recording miniature endplate currents (MEPC) from proximal and distal parts of frog sartorius synapse. The amplitude and half-time of decay (t1/2) of MEPC in proximal parts exceeded those in distal parts. These differences were eliminated by tubocurarine and increased under the influence of armin. In 80% of experiments the positive correlation between the amplitude and t1/2 of MEPC was revealed, which reflects repetitive binding of acetylcholine (ACh) molecules with cholinoreceptors (ChR). Positive correlation was observed in all investigated proximal parts of terminal, and only in half of distal ones. The electron microscopic study showed that postsynaptic folds in proximal parts occur more frequently, are poorly branching and rather shallow. A conclusion is made that the more prolonged time course of MEPC in proximal synapse parts is due to the more pronounced repetitive binding of ACh molecules with ChR of the postsynaptic membrane, which may be explained by high density of ChR and special, features of the synaptic cleft geometry in proximal parts.     

Effect of vinblastine and colchicine on the postsynaptic membrane of the myoneural junction in the frog

Postsynaptic effects of vinblastine (VB) and colchicine (CCH) were studied in the frog neuromuscular junction under voltage-clamp conditions. The same concentrations of VB and CCM, which did not affect the amplitude and duration of miniature end plate currents (MEPC), the current-voltage relationship of peak end plate currents (EPC) and voltage sensitivity of the decay phase of EPC, markedly reduced the responses to iontophoretic application of acetylcholine. Both VB and CCH accelerated the EPC decay when acetylcholinesterase was inhibited; they also caused a pronounced depression in the trains of EPC (10 Hz) and trains of responses to acetylcholine application (5-10 Hz). It was concluded that use-dependent effects of VB and CCH are not connected with their influence on the cytoskeleton of the muscle fibre, but can be a result of accelerated desensitization of cholinoreceptors.     

Concentration-dependent effects of neostigmine on the endplate acetylcholine receptor channel complex

The concentration-dependent actions of neostigmine, a carbamate anticholinesterase agent, were studied on the acetylcholine receptor channel complex in voltage-clamped twitch fibers of costocutaneous muscles of garter snakes. Low concentrations of neostigmine (10(-6) or 10(-5) M) increased miniature endplate current (MEPC) amplitude and the time constant of MEPC decay without changing the relationship between the MEPC decay time constant and membrane potential. Acetylcholine- or carbachol-induced endplate current fluctuation spectra were well fitted by a single Lorentzian curve with a characteristic frequency and single-channel conductance unaltered by low concentrations of neostigmine. Concentrations of neostigmine greater than 5 X 10(-5) M decreased MEPC amplitude and split the decay of MEPCs into two components, one faster and one slower than the control rate. These effects were both voltage and concentration dependent. Spectra of current fluctuations recorded in concentrations greater than or equal to 5 X 10(-5) M neostigmine required two time constants, one faster and one slower than the control. Two component spectra were also obtained with carbachol-induced current fluctuation spectra, indicating that these effects of neostigmine were direct and not a consequence of acetylcholinesterase inhibition. Similar results were also obtained in muscles pretreated with collagenase to remove junctional acetylcholinesterase. The fast and slow time constants obtained from current fluctuation spectra decreased and increased, respectively, with either increases in the concentration of neostigmine or membrane hyperpolarization when analyzed in the same fiber. The effects of neostigmine on channel lifetime were reversible with washing. These results indicate that the effects of neostigmine are concentration dependent. Concentrations greater than 2.5 X 10(-5) M exhibit direct effects on the endplate receptor channel complex which are unrelated to acetylcholinesterase inhibition. These actions include: a prolongation of the gating kinetics of the endplate receptor channel complex, the production of an altered state of the receptor channel complex evidenced by a high frequency component to current fluctuation spectra, and a direct action to block the acetylcholine receptor.     

Postsynaptic potentiation of miniature currents of muscle fiber endplates in the rat diaphragm. The effect of an acetylcholinesterase inhibitor, temperature and curare

Miniature end-plate currents (MEPC) were recorded in voltage-clamped end-plates of the rat diaphragm. A positive correlation between the amplitude and half-decay time of individual MEPC was found in control, after acetylcholinesterase (AChE) inhibition the correlation increased: correlation coefficients--0.29 and 0.49, respectively (28 degrees C). Addition of curare after the AChE inhibition caused a decrease in the amplitude and duration of MEPC, but had no effect on the correlation between these parameters. Cooling down to 18 degrees C led to the essential reduction of this correlation in control as well as after the AChE inhibition. Effect of the prolongation of the half-decay time due to the AChE inhibition was more significant at 28 degrees C than at 18 degrees C. The Q10 value for duration of the rising MEPC phase (about 2) was less than that for the half-decay time (about 3). The role of factors determining the MEPC duration is discussed. The results are explained by the postsynaptic potentiation in terms of the cooperative action of agonists on cholinoreceptors and of peculiarities of the acetylcholine diffusion from the synaptic cleft.     

The effect of intracellular calcium on an increase in the cAMP-mediated calcium current]

Intracellularly perfused neurons of Helix pomatia in which serotonin-induced increase of calcium current (Ica) is mediated by cAMP were studied by the voltage clamp technique. It was established that an increase of the calcium intracellular concentration ([Ca2+]i) caused inhibition of the serotonin (5-HT) effect. The 5-HT effect value at 10(-6) and 10(-7) mol/l [Ca2+]i was 60 and 32% of the control one, respectively. Addition of the calmodulin antagonist (5.10(-5) M trifluoperasine) and phosphodiesterase blockers (5 mM theophylline or 10(-4) mol/l IBMX) sharply reduced the Ca2+ ability to inhibit the 5-HT effect. It is concluded that the Ca(2+)-calmodulin-dependent phosphodiesterase is a key link in the interaction between two signal transduction pathways--Ca2+ and cAMP in modulation of the calcium channel activity.     

Effect of changes in membrane potential and temperature on the post-synaptic potential on the neuromuscular junction of the frog

The decay time-constants of the 2nd and 1st nerve-evoked paired end-plate currents (epc) were recorded in transversely cut muscle preparations of frog. After the inhibition of synaptic acetylcholinesterase by prostigmine (3 X 10(-6) mol/l) the decay of the 2nd epc was 39 +/- 8% slower than the decay of the 1st epc (the interstimulus interval being 100 ms) due to postsynaptic potentiation (PSP). It was found that PSP does not depend on the membrane potential level in the range of-30-120 mV. A drop in temperature from 22 degrees to 12 degrees resulted in several effects: an increase in the decay time constant of epc and meps; a slight decrease in mepc amplitude; a fall of epc quantal content. The comparison of paired epc of equal quantal content showed that PSP was more pronounced at lower temperature. The temperature coefficient (Q10) for the ratio of decay time constants of the 2nd and the 1st epc was 2.0 +/- 0.2. Evidently, the trace of preceding activity of the transmitter does not depend on the membrane potential level but becomes stronger with a fall of the temperature.     

Pharmacologic study of the inhibition of the glomerular input to the olfactory tubercle of the frog induced by a puff of air on the olfactory mucosa

The effects of 4-aminopyridine (10(-2) mol/l), aminooxyacetic acid (AOAA, 10(-4)-10(-3) mol/l), beta-alanine (10(-3)-10(-2) mol/l) and bicuculline (10(-5), 10(-4) mol/l) applications on the frog olfactory bulb (OB) were studied in vivo. The suppression of the orthodromic potential postsynaptic components evoked by a puff on the olfactory mucosa (OB input inhibition), or by single electrostimulation of the olfactory nerve (postsynaptic inhibition) was evaluated. 4-aminopyridine greatly reduced the OB input inhibition and strongly increased the postsynaptic inhibition. AOAA and bicuculline increased and beta-alanine slightly reduced the OB input inhibition. Clear-cut, simple alterations of the postsynaptic inhibition under the influence of the same drugs were not observed. The results confirm the hypothesis, that the OB input inhibition evoked by a puff on the olfactory mucosa may appear due to the release of GABA from glial cells and its binding with presynaptic GABAB-receptors in glomeruli.     

Synaptic effects of nicotinic and muscarinic agonists in the sympathetic ganglia of the frog

Superfusion of isolated frog sympathetic ganglia with nicotinic agonists (suberyldicholine, tetramethylammonium, DMPP), as well as with acetylcholine in the presence of atropine, caused short-term depolarization of a single ganglion cell and blockade of synaptic transmission. Muscarinic agonists (methylfurmethide, methyl dilvasen, acetylcholine) caused sustained depolarization which was not accompanied by transmission failure. Oxotremorine did not change membrane potential at concentrations up to 1.10(-5) mol/l, but at a lower concentration (1.10(-6) mol/l) it produced about a two-fold decrease of EPSP quantum content. This allows the presynaptic muscarinic receptors to be related to M2 type. Inhibition of acetylcholinesterase markedly potentiated postsynaptic effect of methylfurmethide; as a result, there was a shift of the dose-response curve to the lower concentrations of agonist. The postsynaptic muscarinic receptors were found to have high stereoselectivity that was seen in the case of enantiomers of methyl dilvasen (F-2268). The obtained results elucidate changes in the ganglionic transmission, the transmitter being present in the synaptic cleft.     

Effect of thiamine on various types of synaptic junctions

Thiamine (1.10(-14) -- 1.10(-4) mol/l) reversibly increased the frequency of miniature excitatory postsynaptic potentials, amplitude and quantal content of excitatory postsynaptic potentials in crayfish glutaminergic synapse. Thiamine also increased spontaneous electrical activity and amplitude of synaptic potentials in guinea-pig taenia coli. In synaptosomes from the rat brain thiamine produced depolarization of nerve endings. The role of thiamine in the regulation of synaptic transmission and mechanism of its action are discussed.     

Repetitive firing in motor nerve endings: modulation by divalent cations, rhythmic activation and cholinergic agents]

The phenomenon of repetitive firing (RG) in motor nerve endings induced by 4-aminopyridine (4-AP) was studied in cut sartorius frog muscle under the voltage clamp conditions. In the presence of 4-AP (1.10(-4) mol/l) one stimulus applied to the nerve induced two end plate currents (EPC) in half of cells studied (n = 35). The elevation of calcium ion concentration up to 5.4 mmol/l or magnesium to 5-9 mmol/l or rhythmic activity (0.05 Hz and above) abolished RF. Substitution of calcium by strontium or barium enhanced RF (the number of EPC during the burst of RF). EPC with very slow time course were observed in some cells in the presence of barium, that disrupted to the burst of RF by repetitive nerve stimulation. Neostigmine, an anticholinesterase agent, increased the number of EPC in the burst of RF, but alpha-bungarotoxin exerted no effect on the RF. The role of calcium and calcium-activated potassium currents in generation and termination of RF in motor nerve endings is discussed.     

Thiamine blockade of neuromuscular transmission

The effects of thiamine on neuromuscular transmission were studied. Thiamine (0.2-2 mM) decreased the quantal height (q) of the evoked end-plate current (EPC). In the presence of 2 mM thiamine, the shape of the decay phase of the EPC remained exponential but the decay time constant increased dramatically. Thiamine changed the EPC height/holding potential and log (EPC decay time constant)/holding potential relationships from linear to non-linear. The reversal potential and the rate of rise of the EPC were unaffected. Analysis of the ACh-induced noise revealed that thiamine increased the mean channel lifetime and decreased the single channel conductance. Under normal conditions, the ACh-induced single channel conductance had no membrane voltage dependency, while thiamine caused the single channel conductance to decrease with hyperpolarization. The effects of thiamine could be explained in computer simulation of the peak EPC/membrane potential relationship by the assumption that thiamine prolonged the channel lifetime and decreased the single channel conductance with hyperpolarization. It is concluded that thiamine modifies the kinetics of ACh-receptor ion channel complex and that this effect is dependent on the membrane voltage.     

An attempt to estimate various characteristics of neuro-muscular transmission from the rising phase of miniature end-plate currents

A simple expression describing the relation between the duration of rising phase and amplitude of miniature end-plate currents (MEPC) is suggested. Parameters of MEPC of the rat diaphragm with different degrees of acetylcholinesterase inhibition by galanthamine were used to estimate the lower levels of acetylcholine diffussion coefficient (0.86.10-6 cm2/s) and channel opening rate constant (21000 s-1).