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.     

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.     

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.     

Postsynaptic potentiation and desensitization at the myoneural synapse of the frog induced by rhythmic stimulation of a motor nerve

The contribution of postsynaptic potentiation (PSP) and desensitization (DS) to the changes in the amplitude and time course of miniature end-plate currents (mepc) recorded after 10 Hz repetitive stimulation of the motor nerve during 5 or 60 s was studied in the experiments on "cut" nerve-muscle preparation with inhibited acetylcholinesterase. After the short (5 s) train the mepc amplitude did not differ from the initial one while the decay time constant (tau mepc) increased by 32% (indication of PSP). The decrease of mepc amplitude by 23% was observed after the end of the long (60 s) train, while tau mepc did not differ from the original one (indication of DS). Similar but more marked two-phase changes occurred in the time course of end-plate currents. These effects were not observed when acetylcholinesterase was active. The PSP and DS manifestations were reproduced with acetylcholine addition into the bath. It was possible to change the ratio of PSP and DS under the action of aprodifen.     

The postsynaptic effects of substance P in the frog neuromuscular synapse]

The effect of substance P on the end-plate currents (EPC) and miniature EPC (MEPC) was studied in the "cut" sartorius muscle of the frog using voltage-clamp technique after acetylcholinesterase inhibition. Substance P in the concentration 5.10(-7)-1.10(-6) mol/l had no effect on the amplitude and time course of the single EPC and MEPC, but promoted significant prolongation of EPC decay during repetitive nerve stimulation (10/s), which indicated development of postsynaptic potentiation. Elevation of the substance P concentration to 5.10(-6) mol/l has led to the shortening of single EPS decay and more significant depression of the EPC amplitude in trains. This effect was connected with a decrease of the postsynaptic membrane sensitivity to acetylcholine, i. e. development of desensitization.     

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.     

Changes in the properties of synaptic channels opened by acetylcholine in denervated frog muscle

Acetylcholine (ACh)-activated channels in end-plates of frog sartorius muscle were studied at various times after denervation. Mean open times of the synaptic membrane channels were derived from the time constant of decay of miniature end-plate currents (tau MEPC) evoked by ACh quanta released from Schwann cells, which replace the motor nerve terminals after these degenerate. Membrane current noise, elicited by iontophoretic application of ACh to voltage-clamped end-plates, was also used to determine mean open time (tau noise) and conductance of the ion channels. About 1 week after denervation, soon after Schwann cell MEPCs appeared, they had a tau similar to that of the neural MEPC in innervated end-plates. However, 5-6 weeks after denervation tau MEPC was increased by a factor of about 5. Circa 4 weeks after denervation, cholinesterase activity of the denervated muscle decreased to 76% of that in the contralateral, innervated muscle, and even 4 months after the operation it was still 64%. Thus, it is unlikely that a change in acetylcholinesterase activity is the main factor responsible for the increase in tau of Schwann cell MEPC. About 1 week after denervation tau noise was close to that in innervated end-plates (about 2 ms). Twelve to 24 days after denervation the average channel open time was 4.5 +/- 1.0 ms, with some end-plates still showing normal 'fast' channels. However, in muscles denervated for 47-113 days the open time was 12.9 +/- 1.9 ms. In the early and intermediate periods, ACh-induced noise spectra with two components were obtained from many end-plates, indicating the simultaneous activation of two different types of channels. At some end-plates during the early and intermediate periods after denervation, but not after about 5 weeks, neostigmine caused the appearance of a component, which was as fast as that of normal end-plate channels. In other experiments small doses of alpha-bungarotoxin were applied in order to predominantly block extra-junctional receptors. In the early period of denervation, when two components were present in the noise spectra, alpha-bungarotoxin eliminated the slow component leaving channels as fast as in innervated end-plates. After prolonged denervation, a component with tau of about 5.5 ms was occasionally disclosed by application of alpha-bungarotoxin. tau noise and tau MEPC from the same end-plate closely agreed. Our results indicate that at frog end-plates the open time of the majority of the synaptic channels opened by ACh becomes longer with increasing time after denervation.     

The effect of "nonquantal" acetylcholine on miniature end-plate currents in the mouse

The development of postsynaptic potentiation (PSP) and desensitization (DS) under the nonquantal secretion (NS) of acetylcholine (ACh) was studied using characteristics of miniature end-plate currents (MEPCs) in mice diaphragm muscle. The H-effect (the measure of the NS level) fell to zero during 3 hours after acetylcholinesterase (AChE) inhibition by armine at 20 degrees C. The MEPCs decay time constant (tau) decreased in parallel with the H-effect, though there was no reduction of the MEPCs amplitude in this case; tau did not change when NS was absent (early denervation). The maximal prolongation of tau after AChE inhibition was the same both in innervated (NS was normal) and denervated (NS was absent) muscle. The reduction of tau accelerated with the temperature rise and occurred after AChE inhibition by neostigmine. There was no changes in tau when AChE was active. It is suggested that nonquantal ACh decreases tau due to the DS development. NS induces no significant PSP after the AChE inhibition, but can do it later according to DS development, so that signs of DS may partially be masked by PSP.     

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.     

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.     

Postsynaptic structure may account for variations in miniature endplate current shapes along frog neuromuscular junctions

Intracellular recordings were made in the presence of 6-9 microM neostigmine with an electrode placed halfway between the distal end and the proximal region of a long simple branch of a frog neuromuscular junction. During the intracellular recording period, an extracellular electrode was placed alternatively at a distal and at a proximal site. The temporal correspondence between the extracellular miniature endplate current and intracellular miniature endplate potential permitted us to identify two miniature endplate potential (MEPP) populations originating, respectively, from the distal and proximal regions. The frequency and intracellular amplitude of MEPPs were higher in the proximal than in the distal region. The rise time and the decay time of the extracellularly recorded MEPC were longer in the proximal region. A significant correlation was observed between the amplitude (in mV) and surface (in mV.ms) of MEPCs. The slope of this regression line was steeper for the proximal region. Furthermore, the correlation coefficient was lower for that region owing to variations of MEPC shape. Our observations suggest that in the presence of anticholinesterase, ACh rebinds more often to acetylcholine receptors in the proximal region because of the presence of longer postjunctional folds.     

l-Carnitine: therapeutic strategy for metabolic encephalopathy

The effects of 4-aminopyridine (4-AP) on membrane electrical properties and synaptic transmission in neurons of the isolated rabbit superior cervical ganglion were investigated. 4-AP (0.03-0.1 mM) increased the amplitude of the fast excitatory postsynaptic potential (f-EPSP) without affecting appreciably either the acetylcholine (ACh) depolarization induced by iontophoresis of ACh or the passive and active membrane properties of the neurons. At concentrations of 1-5 mM, 4-AP reversibly depressed the amplitude of the f-EPSP as well as the ACh depolarization; a slight to moderate prolongation of the action potential duration was observed. In addition to the effects on evoked synaptic potentials, 4-AP induced spontaneous discharges which were abolished reversibly by curare, low Ca solution or Co. The results indicate that 4-AP at low concentrations facilitated evoked as well as spontaneous release of ACh by a presynaptic mechanism, whereas at higher concentrations it exerted a curare-like effect on the postsynaptic membrane.     

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.     

Physostigmine prolongs the elementary event underlying decay of inhibitory postsynaptic currents in Aplysia

Inhibitory postsynaptic currents (IPSCs) recorded at cholinergic synapses between identified neurons of the buccal ganglia of Aplysia decay exponentially. IPSC decay has been ascribed to a receptor-channel-dependent kinetic process, rather than to changes in subsynaptic acetylcholine (ACh) concentration, but is prolonged by the anticholinesterase eserine. Our data are consistent with a direct effect of 1.6 X 10(-4) M eserine on an elementary channel process. IPSC decay remains single exponential during eserine prolongation, suggesting that eserine slows the kinetic process responsible for decay, rather than substituting an additional kinetic process. In either control sea water or eserine, noise spectra computed from currents induced by pressure ejection of ACh are fitted by the sum of two Lorentzians. The slow corner frequency f1 is reduced from 6.3 to 3.5 Hz by eserine, consistent with a direct eserine action on receptor-channel kinetics, whereas apparent single-channel conductance was unchanged. Both IPSCs and ACh noise recorded in the same cells were comparably slowed by eserine. In eserine, decay time constant tau agreed with noise f1; however, tau and f1 in control sea water differed by 31%. The discrepancy may be accounted for by invoking an additional component to the recorded noise spectra, perhaps produced by synaptically active choline. In addition to the direct effect on kinetics, prolonged exposure to eserine produces a slow extra component to the IPSC decay tail.     

The use of the driving function in the analysis of endplate current kinetics

The time course of transmitter release at the neuromuscular junction is non-instantaneous and is more prolonged for nerve-evoked endplate currents (EPCs) than for miniature endplate currents (MEPCs). This creates difficulties in the interpretation of the effects of ion channel blocking drugs which greatly increase the rate of EPC decay, resulting in a substantial number of channels being blocked during the EPC rising phase. Apparent changes in peak current and in the EPC/MEPC peak current ratio can be erroneously interpreted as receptor block or effects on quantal content. A method is described for a more precise assessment of receptor block or quantal content in the presence of ion channel block, by calculation of the EPC driving function. The driving function is a measure of the rate of opening of the ion channels in response to transmitter release, and is independent of ion channel block effects. Simulated EPCs with the same decay rate can be created from the driving functions of EPCs in control and in the presence of the drug. The peak current of such EPCs can be compared to reveal any additional receptor block or quantal content effects independent of a drug's ion channel blocking effects.     

Progressive supranuclear palsy--parkinsonian disorder with tau pathology

Progressive supranuclear palsy (PSP), also known as Steele-Richardson-Olszewski syndrome, is a rare form of parkinsonism characterised by abundant tau pathology. Only a few familial cases have been reported, therefore PSP can be considered as a sporadic tauopathy. Recent case-control studies of patients with sporadic PSP suggest that PSP has a recessive pattern of inheritance. Strong genetic evidences for the involvement of the tau gene variability in the pathogenesis of PSP have been demonstrated in several Caucasian populations. We review the most important DNA polymorphisms (e.g.: A0 polymorphism and H1 haplotype) contributing to the risk of PSP. Moreover, we discuss how these DNA polymorphisms may influence the exon 10 splicing, and thus the proportion of 4R/3R tau isoforms, leading to a class II tau pathology in PSP patients.     

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.     

Analysis of postsynaptic currents in the phasic and tonic muscle fibers of the extraocular muscles of the rat

The miniature end-plate currents (MEPCs), miniature postjunctional currents (MPJCs) and mean values of life-time of open ionic channels (tau chan) were compared in experiments on fast and tonic muscle fibres. The rise-time and decay-time constants of MPJCs in tonic fibres were 2.5 and 4-5 times (respectively) longer than those of MEPCs in fast fibres; tau chan in tonic synapses was 2.2 times higher than in fast ones. Inhibition of AChE induced more pronounced prolongation of MEPCs (4.4 times) than MPJCs (1.8 times). As a result, the difference between the time decay of MPJCs and MEPCs reduced to 1.6 times. Thus, a more prolonged decay of MPJCs in tonic fibres in comparison with MEPCs in fast fibres was due to a larger value of tau chan and lower activity of AChE, that favours repetitive interaction between ACh-molecules and choline receptors causing prolongation of the decay of the synaptic response. The time course of MEPCs and tau chan in skeletal slow-twitch muscle fibres was significantly shorter than those in extraocular tonic muscle fibres.     

Nicotinic receptors of frog ganglia resemble pharmacologically those of skeletal muscle

The actions of ACh antagonists were studied on synaptic currents of autonomic ganglia of the frog. Fast excitatory synaptic currents (ESCs) were recorded from cardiac and paravertebral neurons with the use of the 2-microelectrode voltage-clamp method. The actions of 4 ACh antagonists, tubocurarine, hexamethonium, trimetaphan, and decamethonium were studied. Tubocurarine was effective at reducing the peak amplitude of ESCs (50% inhibition at 3 microM). In contrast, tubocurarine (1-30 microM) reduced the time constant of ESC decay by only 9% compared with controls. Both of these effects of tubocurarine were independent of membrane potential. Hexamethonium was a weak inhibitor of ESCs; at 600 microM peak amplitude was reduced only to about 60% of controls and decay time constants were unaffected at concentrations between 10 and 600 microM. These effects of tubocurarine and hexamethonium are consistent with these drugs being receptor antagonists with no evidence of ion channel block. Trimetaphan (3-100 microM) and decamethonium (100 microM) reduced the peak amplitude of ESCs. In the presence of 100 microM trimetaphan or 10 microM decamethonium, ESC decays were biexponential. The 2 exponential components induced by the presence of these drugs were faster and slower, respectively, than the single-exponential component of control ESC decays. The effects of these 2 drugs were more pronounced at hyperpolarized potentials and are consistent with a channel-blocking action. The actions of the 4 ACh antagonists on frog autonomic ganglia are similar to their effects at the neuromuscular junction but dissimilar to their effects on the rat submandibular ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)