Role of desensitized acetylcholine receptors in the development of post-tetanic potentiation

A hypothesis that desensitized acetylcholine (ACh) receptors are responsible for postsynaptic potentiation (PSP) was verified in experiments performed on the sartorial muscle of the frog using a voltage-clamp recording technique. The PSP was estimated, at active and inhibited acetylcholinesterase, with the use of paired stimulation of the motor nerve at various interpulse intervals, according to an increase in the time constant () of the second end-plate current (EPC) relative to that of the first EPC. The appearance of desensitized receptors on the postsynaptic membrane, caused by the application of a promoter of desensitization proadiphen or by application of exogenous ACh, was followed by acceleration of the decay of the first EPC and by a decrease in its amplitude. However, this did not intensify PSP, which even decreased in the presence of proadiphen under the influence of ACh and membrane hyperpolarization. A sharp increase in calcium concentration after certain period of ACh influence, which increased quantum ACh release, did not enhance PSP, which allows us to suggest the role of the presynaptic factor to be insignificant. The results provide arguments against the direct involvement of desensitized acetylcholine receptors in the PSP development.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 361–367, September–December, 1995.     

Pre-synaptic and post-synaptic factors influencing the synchronism in the transmitter secretion and the amplitude and temporal parameters of a postsynaptic response in the neuromuscular junction: a model study

Using a model of the frog neuromuscular junction, we studied the influence of pre-synaptic and post-synaptic factors on the amplitude and temporal parameters of end-plate currents (EPC). A nerve terminal (NT) was supposed to include linearly distributed active zones (AZ) that are able to release a transmitter quantum with a definite temporal distribution of the release probability (AZ DRP) after successive activation of these zones by a spreading action potential (AP). An increase in the length of a terminal, distance between AZ, and time constant of the DRP decline, or a decrease in the AP conduction velocity along the NT determines a decrease in the EPS amplitude and prolongation of its rising phase. These effects result from an increased asynchronism in the transmitter release. An expansion of the temporal parameters of minature EPC leads to an increase in the EPC amplitude, i.e., provides minimization of its loss. Various EPC models are compared, and contributions of the examined pre-synaptic and post-synaptic factors in modifications of the amplitude and temporal EPC parameters are evaluated.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 163–179, May–June, 1995.     

Effects of quantal content,membrane potential,and cholinoceptor density on the time course of end-plate currents in the rat under during acetylcholinesterase inhibition

The time-course of multiquantal end-plate currents (EPCs) was compared with monoquantal synaptic responses, i.e., miniature end-plate currents (MEPCs), in voltage-clamped rat diaphragm muscle fibers. In the presence of active acetylcholinesterase (AChE), the time constant of the decay of EPCs, that were composed of 25–140 quanta, was 1.2 times greater than that of MEPCs. After inhibition of AChE with armine or proserine the decay of the EPC was longer than the decay of the MEPC by 10–100 times, and unlike the MEPC, in the majority of synapses it could be described by the sum of two (n=34) or three (n=9) exponentials: monoexponential EPCs were noted in only three cases. The nature and duration of the EPC decay depended on its quantal content. After a reduction in the quantal content a three-exponential EPC decay could be successively reduced to a two- and a mono-exponential decay. A ,slow, component of the EPC decay, unlike the MEPC decay, was extremely sensitive to changes in the membrane potential, and extracellular magnesium ion concentration. When the cholinoceptors were irreversibly blocked by -bungarotoxin the MEPC decay accelerated, and the monoexponential EPC decay initially slowed down before accelerating, but even during a profound blockade the open-times of the ion channels were not affected. It therefore appears that during the generation of multiquantal EPCs when AChE is inhibited, not only does the synchronicity of the ion channel opening change, but so do their kinetics, possibly because of ion channel blockade by endogenous acetylcholine.S. V. Kurashov Institute of Medicine, Russian Ministry of Public Health, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 269–279, May–June, 1992.     

Effect of carbachol on spontaneous transmitter release from rat motor nerve endings depending on extracellular potassium concentration

The effect of carbachol (10 µM) on the frequency of miniature end-plate potentials (MEPP) was studied in experiments on the Wistar rat soleus muscle during a change in extracellular potassium concentration from 2 to 15 mM. Between the range of potassium concentrations from 2 to 7.5 mM the cholinomimetic had no effect on spontaneous transmitter release. In higher potassium concentrations carbachol caused an increase in the frequency of MEPP. This facilitatory effect increased in strength with an increase in potassium concentration; at 15 mM the frequency of MEPP was increased up to 160%. The results confirmed the previous hypothesis that the action of the mimetic on spontaneous transmitter release, relaized through presynaptic acetylcholine receptors, depends on the initial level of polarization of nerve endings.S. V. Kurashov Kazan' State Medical Institute. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 470–475, July–August, 1984.     

Effect of carbachol on miniature end-plate potentials and currents of rat skeletal muscle

Experiments on rats showed that carbachol (10 µM) appreciably quickens miniature end-plate potentials in the soleus muscle kept in medium with potassium chloride concentration increased to 13 mM. Potentiation of spontaneous mediator release also took place when the membrane potential of the muscle fibers was clamped at the level of the potassium equilibrium potential. It is concluded that the presynaptic action of carbachol on synaptic liberation of acetylcholine from motor nerve endings is unconnected with depolarization of the postsynaptic membrane but takes place as a result of the direct effect of the mimetic on motor nerve endings.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 185–189, March–April, 1982.     

Effect of axoplasmic transport blockade on end-plate currents in frog muscle fibers

Evoked and spontaneous end-plate currents (EPC) were studied in normal voltage-clamped frog sartorius muscle fibers and 2 weeks after application of colchicine to the nerve innervating the muscle to block axoplasmic transport in its fibers. Application of colchicine was found to reduce the rate of rise and to prolong decay of EPC without affecting the amplitude of the EPC and miniature EPC, the quantum composition of EPC, and the frequency of miniature EPC. The histogram of distribution of the time constant () of EPC decay under normal conditions follows the normal law, but after application of colchicine to the nerve it is shifted to the right, with separation of two modes (₁ and ₂). Three types of synapses can be distinguished from the character of EPC decay: monoexponential decay with ₁ (44%), biexponential decay with ₁ and ₂ (39%), and monoexponential decay with ₂ (19%). An increase in of EPC decay is accompanied by strengthening of the dependence of this process on the clamping voltage. The current-voltage characteristic and reversal potential of EPC are unchanged. It is suggested that the change in character of EPC decay after application of colchicine to the motor nerve is due to the appearance of acetylcholine-activated ionic channels in the muscle membrane with a longer duration of the open state and with potential-dependence of the open state similar to that taking place after muscle denervation.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 204–211, March–April, 1985.     

Postsynaptic effects of substance P in frog neuromuscular junction

We have studied the effect of substance P on the end-plate currents (EPC) and the miniature EPC (MEPC) after acetylcholine esterase (ACE) inhibition in the cut neuromuscular preparation of the frog sartorius muscle using the voltage-clamp technique. At concentrations of 5·10^–7–1·10^–6 moles/liter substance P had no effect on the amplitude and the time characteristics of single EPC and MEPC but promoted prolongation of EPC decay on repetitive stimulation of the nerve with a frequency of 10/sec, indicating intensification of postsynaptic potentiation. Elevation of the concentration of the given peptide to 5·10^–6 moles/liter led to the shortening of the decay of single EPC and a more marked depression of the EPC amplitude in the trains as compared to the control, reflecting a decrease in the sensitivity of the postsynaptic membrane to the mediator, i.e., development of desensitization.S. V. Kurashov State Medical Institute, Kazan. I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 436–441, July–August, 1991.     

Effect of imidazole,guanidine, and theophylline on transmitter release in the frog neuromuscular synapse in relation to temperature and calcium ion concentration in the medium

The effects of imidazole, guanidine, and theophylline on spontaneous (frequency of miniature end-plate potentials) and evoked (quantum composition of end-plate potentials) transmitter release were compared in isolated sartorius muscles ofRana temporaria at different temperatures and during changes in the calcium concentration in the external solution. All three substances increased the quantum composition of the end-plate potentials and the frequency of the miniature end-plate potentials at 20°C and in 0.5 mM calcium. As regards their effect on the quantum composition the substances could be arranged in the following order: imidazole guanidine theophylline; as regards their effect on frequency: theophylline imidazole guanidine. Theophylline increased spontaneous release, whereas imidazole and guanidine increased evoked transmitter release more than the rest. Comparison of the effect of these substances at 20 and 7°C showed that only the action of theophylline on spontaneous release depends on temperature. The effect of imidazole and theophylline on frequency was independent of the calcium concentration in the medium. Differences in the mechanism of action of these compounds on spontaneous and evoked acetylcholine release are discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 430–436, July–August, 1977.     

Role of Potassium Channels in Facilitation of Transmitter Release from Frog Motor Nerve Ending (Electrophysiology and Mathematical Simulation)

In experiments on neuromuscular junctions in the frog m. thoraco-cutaneous, we studied changes in the transmitter release and shape of the nerve ending (NE) response related to high-frequency (10 or 50 sec^-1) rhythmic stimulation of the motor nerve; an extracellular recording technique was used. At a low extracellular Ca²⁺ concentration, rhythmic stimulation resulted in a gradual increase in the quantum content of end-plate currents, i.e., in facilitation. Simultaneously, the third (positive) phase of the NE response became smaller, the amplitude of the second (negative) phase of this response also decreased, while the duration of this phase increased. Modifications of the NE response upon stimulation with a 10 sec^-1 frequency were more clearly expressed than those at 50 sec^-1 stimulation. In Ca²⁺-free solutions, rhythmic stimulation was accompanied by analogous modifications of the shape of NE responses, and the dynamics of these changes were the same at both the above-mentioned stimulation frequencies. When 0.5-1.0 mM tetraethylammonium was applied, 10 sec^-1 stimulation was accompanied by no facilitation of transmitter release; at 50 sec^-1 stimulation, this phenomenon was observed but was weaker than in the control, and the shape of NE responses underwent only mild changes. Simulation of electrogenesis in the studied structure showed that modifications of the NE response shape related to rhythmic 10 sec^-1 stimulation can develop in the case of a gradual decrease in the voltage-dependent potassium membrane conductivity, which results in prolongation of the de- and repolarization phases of action potentials and increases in the amplitude and duration of the inward calcium current. At higher stimulation frequencies (50 sec^-1), this mechanism is accompanied by a gradual increase in the Ca²⁺-dependent potassium conductivity, due to an increase in the intracellular Ca²⁺ concentration. These data allow us to conclude that the intensity of facilitation of transmitter release from the frog motor NE is related not only to accumulation of residual calcium, but also to changes of presynaptic calcium current due to modification of the kinetics of functioning of the potassium channels.     

Comparison of Pre- and postsynaptic process kinetics in neuro-muscular synapse using the end-plate current model

The effect of correlation between the kinetics of acetylcholine (ACh) quanta release and the kinetics of the postsynaptic membrane channel activity on end-plate currents (EPCs) was studied in model experiments. The presynaptic process was described by a transmitter secretion probability distribution (SPD), and the postsynaptic process by characteristicsof miniature EPC (MEPC) with standard amplitude and variable decay duration (_MEPC). The SPD was represented by a curve with an S-like rise and exponential decay. The main portion of the data was analyzed after being adjusted to 20°C. The effect of the EPC amplitude loss (the difference between the EPC amplitudes in cases of synchronous and non-synchronous release of the ACh quanta) due to temporal dispersion of the transmitter secretion process was 43% at _MEPC=1.0 msec and 20% at _MEPC=4–5 msec under conditions when acetylcholinesterase (AChE) was inhibited.When _MEPC varied over a wide range, the effect of the EPC amplitude loss could be described by a curve with steeply and gently sloping portions; the boundary between these two portions corresponded to approximately 1.0 msec _MEPC, which is nearly the channel lifetime at resting membrane potential. The rise time of the EPC only slightly related to changes in _MEPC. The rate of EPC decay exceeded that in MEPC only when the lifetimes of the ACh-activated ionic channels were lower than their physiological values. In this case, the kinetics of transmitter release became a factor determining the EPC decay time course.Three variants of an increase in temporal dispersion of the ACh quanta secretion were modeled for constant _MEPC: one resulting from an increase in the SPD rising phase; a second resulting from equal increases in the SPD rising and declining phases, and a third resulting from an increase in the SPD declining phase. The results of the first variant corresponded most closely to experimental data on changes in EPC during a short-term period of rhythmical activity. This was also characterized by minimal effect of EPC amplitude loss.It has been suggested that the interrelation between the parameters of SPD and _MEPC is an important factor regulating the amplitude and duration of the postsynaptic signal. The maintenance of this interrelation at a constant level ensures reliability of synaptic transmission.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 126–132, March–April, 1993.     

Repetitive firing in motor nerve endings: Divalent cation,rhythmic activity,and cholinergic agent modulation

Repetitive firing (RF) as two or more end plate currents (EPC) induced by a single stimulus of the motor nerve was studied in sartorius frog muscle under voltage clamp conditions. In the presence of 4-aminopyridine (1.10^–4 M), RF as two EPC with 3–8 msec intervals was found in half of the cells (n=35). When calcium ion concentration was increased from 1.8 to 5.4 mM, magnesium to 5–9 mM, and rhythmic activity beginning with 0.05 Hz and above, RF stopped, but when strontium or barium was substituted for calcium, RF intensified. In the presence of barium, the repeated EPC quantity reached six-eight per one nerve impulse. Slow currents arose in some of the cells when disruption of the EPC series was stimulated. Proserine, an anticholinesterase agent, enhanced RF, whereas -bungarotoxin, a cholinoreceptor blocker, had no effect. The role that calcium and calcium-activated potassium currents play in generating and terminating mechanisms of RF, is discussed.S. V. Kurashov Medical Institute, Russian Federation Ministry of Public Health, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 387–395, July–August, 1992.     

Rhythmic Activity of Murine Neuromuscular Junctions upon Activation of Release of Stored Calcium in the Presence of a Calcium Buffer

Using a two-electrode voltage-clamp technique, we recorded end-plate currents (EPCs) in neuromuscular synaptic junctions of the murine diaphragm upon rhythmic stimulation of the n. phrenicus with frequencies of 7, 20, 50, 70, and 100 sec⁻¹. Parameters of EPC series were analyzed against the background of the action of a mobilizer of intracellular calcium, ryanodine (0.5 μM), after the loading of terminals by 1.2 mM BAPTA (calcium buffer with rapid dynamics of binding of calcium), and upon the action of ryanodine in the presence of BAPTA. Under the action of ryanodine, the amplitude and quantum content of EPC within the plateau phase increased by 100 to 150% (P < 0.05). Loading with BAPTA evoked sharp decreases in the quantum content of unitary EPCs, the intensity of the initial facilitation, and the level of the EPC plateau in series within the entire range of stimulation frequencies used. Against the background of the action of BAPTA, the facilitatory effect of ryanodine increased; inhibitory effects of BAPTA with respect to the amplitude of unitary EPC and the level of the initial facilitation were completely compensated, whereas the level of EPC at the plateau stage increased to levels exceeding the control values by 50 to 70%. The ability of ryanodine to facilitate the transmitter (acetylcholine) release, which was enhanced in the presence of BAPTA, was completely neutralized by a blocker of L-type calcium channels, verapamil (5 μM). In the absence of BAPTA, verapamil did not influence the effects of ryanodine. We hypothesize that in the presence of BAPTA calcium channels of L type whose activity is resistive to the buffer action of BAPTA are disinhibited. The calcium current through L-type channels, perhaps, is capable of stimulating calcium release from the stores of nerve terminals and, as a consequence, of intensifying the facilitatory effect of ryanodine on the release of acetylcholine. After verapamil-induced blockade of this current, BAPTA demonstrates the ability to prevent the facilitatory effect of ryanodine on the transmitter release. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 330–338, July–August, 2005.     

Effects of vinblastine and colchicine on the postsynaptic membrane at the frog neuromuscular junction

The possible effects of the alkaloids vinblastine and colchicine on the postsynaptic membrane of the frog neuromuscular junction were investigated using voltage-clamp techniques. Concentrations of vinblastine and colchicine which had been shown to exert no effect on the amplitude and duration of miniature endplate currents (MEPC) and the current-voltage relationship of low-quantal endplate currents (EPC) together with the coefficient of voltage-dependent EPC decay did produce a considerable rise in the amplitude of response to iontophoretically applied acetylcholine (ACh). In addition, vinblastine and colchicine accelerate MEPC and EPC during acetylcholine esterase inhibition while further depressing the amplitude of multi-quantal EPC succeeding at the rate of 10 Hz as well as response to regular (5–10 Hz) application of ACh from a micropipet. The dosage-frequency effects of vinblastine and colchicine on the postsynaptic membrane (as described) are presumed to be unconnected with the action of these agents on muscle fiber cytoskeleton but the results of accelerated desensitization of cholinoreceptors.S. V. Kurashov Medical Institute, Kazan. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 75–81, January–February, 1988.     

End plate currents at the physiological level of quantal secretion and after potentiation of transmitter release by 4-aminopyridine

The role of postysynaptic potentiation (PSP) and asynchronous secretion of acetylcholine (ACh) in the generation of multiquantal currents and end plate currents (EPC) was investigated under voltage clamp conditions in transected sartorius muscle of the frog before and after 4-aminopyridine (4-AP) treatment. Compared with miniature EPC (MEPC), showing an average quantum content of 249, multiquantal EPC has a larger amplitude, longer rise-time, and longer decay-time (_epc). Magnesium ions (6–10 mM) reduce the amplitude and _spec of EPC without affecting its rise-time. Rhythmical stimulation (10 Hz for 60 sec) results in reduced amplitude and but increased rise-time of EPC. D-turbocurarine (5×10^–7 M) and -bungarotoxin (1×10^–5 gm/ml) diminishes the difference between _epc and _mepc. In the presence of 4-AP, all these effects are much more pronounced. It is proposed that asynchronous secretion of ACh from motor nerve teminals causes prolongation of the rise-time and reduction of the amplitude of EPC but has little or no effect upon the decay rate of EPC. The slow decay of multiquantal EPC, both in the absence and in the presence of 4-AP, is almost entirely due to postynaptic interaction of ACh quanta, i.e., PSP.Kurashov Meidcal Institute, Kazhan. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 48–56, January–February, 1991.     

Biochemical characteristics of a muscle perfusate and its effects on mediator release in the neuromuscular synapse

Histidine-containing compounds (HCC) were found, using diazoreaction and reaction with diethylpyrocarbonate, a specific histidine reagent, in the perfusate of the frog hindlimb vasculature. After gel filtration on sephadex G-25 about 80% of HCC were eluted with a fraction with molecular mass less than 5000 kD. The studies of the perfusate influence on the characteristics of quantum secretion of transmitter in the preparation of frog cutaneopectoral muscle have shown that the perfusate increased the quantum content of end-plate potentials (EPP) due to increasing binominal parameter and decreasing frequency of miniature EPP. The characteristics of presynaptic action of the perfusate were similar to those of exogenous histidine.     

Effects of Noradrenaline on End-Plate Currents and the Kinetics of Transmitter Release under Long-Lasting Repetitive Stimulation

Noradrenaline causes a significant increase in the amplitude of multiquantum end-plate currents (EPC) and also diminishes the EPC rising phase vs the rising phase of the miniature EPC ratio in the frog neuromuscular junction under conditions of low-frequency long-lasting stimulation of the motor nerve. Noradrenaline changes the kinetics of transmitter release due to synchronization of the quantum transmitter secretion. The synchronizing action of noradrenaline can underlie its de-fatiguing effect in the neuromuscular junction.     

Facilitation and Depression of Neuromuscular Transmission under Conditions of Blockade of Ryanodine Receptors

In our experiments on mice, end-plate currents (EPC) evoked by stimulation of the phrenic nerve were intracellularly recorded in neuromuscular synaptic junctions of the phrenic muscle. We studied the effects of a specific blocker of ryanodine receptors, ryanodine (10 to 20 M), on the amplitude and time parameters of EPC under conditions of tetanic facilitation and depression of synaptic transmission at frequencies of stimulation of 4 to 200 sec^-1. Ryanodine inhibited facilitation at stimulation frequencies of 7 to 70 sec^-1 (with maximum effect at 20 sec^-1) and accelerated depression. In the presence of ryanodine, an initial rundown of the EPC amplitude in the course of depression of transmission increased at high frequencies of stimulation (50 to 100 sec^-1), whereas the EPC amplitude at the plateau level decreased already at low frequencies (4 to 7 sec^-1). We concluded that the changes in facilitation and depression resulted from blocking of the presynaptic ryanodine receptors by ryanodine. It seems probable that calcium release from the calcium stores in murine motor terminals is a factor involved in the control of processes of transmitter secretion during short-term rhythmic activation of the junction.     

Mechanisms of neurotransmitter release facilitation in strontium solutions

Mechanisms of neurotransmitter release facilitation were studied using electrophysiological recording of end-plate currents (EPC) and nerve ending (NE) responses after substitution of extracellular Ca ions with Sr ions at the frog neuromuscular junction. The solutions with 0.5 mM concentration of Ca ions (calcium solution) or 1 mM concentration of Sr ions (strontium solution) were used where baseline neurotransmitter release (at low-frequency stimulation) is equal. Decay of paired-pulse facilitation of EPC at calcium solutions with increase of interpulse interval from 5 to 500 ms was well described by three-exponential function consisting of early, first and second components. Facilitation at strontium solutions was significantly diminished due mainly to decrease of early and first components. At the same time, EPC facilitation with rhythmic stimulation (10 or 50 imp/s) at strontium solutions was significantly increased. Also more pronounced decrease of NE response 3rd phase, reflecting potassium currents was detected under rhythmic stimulation of 50 imp/s at strontium solutions comparing to calcium solutions. It was concluded that facilitation sites underlying first and early components had lower affinity to Sr ions than to Ca ions. The enhancement of frequency facilitation at strontium solutions is mediated by two mechanisms: more pronounced broadening of NE action potential and increase of bivalent cation influx due to feebly marked activation of Ca(2+)-dependent potassium current by Sr ions, and slower dynamics of Sr(2+) removal from NE axoplasm comparing to Ca(2+).     

Some effects of prolonged polarization on membrane currents in bullfrog atrial muscle

Summary Bullfrog atrial trabecula were voltage-clamped using a double-sucrose-gap method. Step depolarization produced a slowly changing outward current which was studied by analyzing the current tail produced by repolarization. The initial phase of the current tail (time constant 0.1 to 0.7 sec at –60 mV) had a reversal potential which depended upon the duration and magnitude of the preceding depolarization. Calculations based on trabecular geometry and the behavior of the currents in high external potassium suggest that part of the current tail reflects a restoration to a lower steadystate concentration of external potassium which had accumulated in narrow clefts between cells during the preceding depolarization. Step hyperpolarization produced a declining inward current (time constant 0.3 sec at –100 mV) which can be explained on the basis of a depletion of potassium from these intercellular clefts (about 0.5% of the trabecular volume).     

Effect of High-Frequency Stimulation on the Time Course of End-Plate Currents

We investigated the effects of repetitive high-frequency (10 sec^-1) nerve stimulation on the time course of evoked and miniature end-plate currents (EPC and mEPC, respectively) in the frog neuromuscular junction. The data obtained indicate that at a physiological Ca²⁺ level in the bath medium, 10 sec^-1 stimulation results in prolongation of the growth phase of multiquantum EPC without any effect on the mEPC time course. It is concluded that timing of acetylcholine quantum secretion may be affected by high-frequency stimulation.