Estimation of the Temporal Course of Evoked Secretion of Transmitter Quanta in the Frog Neuromuscular Junction

In experiments on the cutaneothoracic muscle of the frog, we recorded, using the technique of two-electrode voltage clamp at a normal Ca²⁺ concentration (1.8 mM), multiquantum end-plate currents (EPC) and miniature uniquantum EPC (mEPC). Multiquantum signals, when compared with uniquantum currents, were characterized by longer leading and trailing edges. The quantum composition of multiquantum signals estimated according to the ratios of EPC and mEPC amplitudes was, on average, 27% lower than that calculated according to the ratios of their integral values (areas). These data demonstrate that stimulus-evoked transmitter secretion from the motor nerve endings is noticeably asynchronous. Based on the parameters of the experimental EPC and mEPC, we estimated the temporal course of evoked secretion using various techniques: spectral analysis, a system of linear equations, and Van der Kloot's method. Using convolution with uniquantum signals, we found that spectral analysis is the best technique for such estimation. Calculated parameters of the temporal course of secretion were the following: risetime 0.20 msec and decay time constant 0.33 msec. The respective distribution significantly differed from that of the synaptic delays of extracellularly recorded uniquantum EPC by longer durations (150-200%) of the leading and trailing edges. We hypothesize that these differences are related to the geometry of the junction and the temporal sequence of switching on of the active zones in the nerve ending upon their activation by spreading action potentials. Factors influencing the temporal course of evoked secretion of the transmitter in the junction under study (its asynchronicity, in particular) are discussed.     

Mediator secretion in the nerve-muscle synapse in the frog following long-term effect of calcium-free solutions

The changes of spontaneous and evoked transmitter release in condition of long time (1-4 hours) incubation in Ca-free solution with EGTA adding, were investigated with extracellular recordings in experiments on the nerve-muscular junction of the frog cutaneous-pectoris muscle. Using the method of three extracellular microelectrodes recordings of the monoquantal postsynaptic signals, it was shown that during action of Ca-free solutions the topography of transmitter release changed, the specific spatial organization of points of transmitter release was disrupted. These changes remained after returning to the initial solution. The obtained data suggest that the Ca2+ free solution leads to disruption of active zones of nerve ending. In condition of low initial extracellular Ca2+ concentrations (0.15-0.4 mmol/l), the active zones disorganization led to decreasing of average amplitude of the end-plate currents (EPC) by decreasing their quantal content, increasing their time-course and decreasing the frequency of the miniature end-plate currents (MEPC). The sharp displacement of dependence of quantal contents of EPC in extracellular Ca2+ concentration to a higher Ca2+ concentration without significant changes of slope was revealed. In condition of high (1.8 mmol/l) concentration of Ca2+, the long action of Ca-free solutions leads to decreasing of amplitude of EPC too, but it was less obvious than in condition of initial low Ca2+ concentration. It is supposed that intra- and extracellular Ca concentration provides the support of the typical morpho-functional organization of the mechanisms of transmitter release at the nerve ending of the frog. The disorganization of active zones leads to separation of the elements, which take part at the transmitter release process and reduces the efficiency of secretion.     

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.     

Transmitter release at the active zone of motor nerve endings

Transmitter release sites were located in the motor nerve ending of the frog cutaneous-pectoris muscle using three extracellular electrodes. Transmitter release sites were found to be grouped in a direction cutting across the nerve ending and reflecting transmitter release and active release zones (AZ). Measurements from these groups showed that most transmitter release takes place at the center of the AZ, declining towards the periphery and to either side of this zone. All AZ were found to take place in spontaneous release with a low extracellular concentration of calcium ions present, compared with only a proportion in evoked release. Advantages of the triple as opposed to the dual micro-electrode technique are analyzed. It was found that transmitter release in spatially isolated AZ at the nerve ending leads to a polymodal distribution pattern of the amplitude of uniquantal signals during extracellular recording. The part played by AZ in transmitter release is discussed.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Moscow. V. I. Ul'yanov-Lenin State University, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 318–327, May–June, 1990.     

Desensitisation of postsynaptic membrane in the neuro-muscular junction due to an increase in spontaneous quantal release of a mediator

Dependence of the amplitude of miniature end-plate currents on frequency of spontaneous quantal release modulated by the elevation of K+ concentration was studied in the frog voltage clamped neuromuscular junctions. A sharp increase of mEPC frequency (not less than approximately 50 per sec) was followed by an obvious fall in both their amplitude and acceleration of decay only in the presence of 3 microM prostigmine (acetylcholinesterase inhibitor) and 5 microM proadiphene, these agents promoting a desensitization of cholinergic postsynaptic membrane. Probable depletion of transmitter store is not involved in the phenomenon observed which is mainly due to the repetitive activation of the postsynaptic zones and the increase of the desensitized cholinoreceptor number.     

Changes in the topography of transmitter release in a neuromuscular synapse reflect plastic alterations occurring in active zones

The topography of transmitter release along the motor nerve terminals (NT) was studied on the frogcutaneous pectoris muscle under normal conditions and following denervation. Coordinates of release sites (RS) of transmitter quanta were determined by extracellular recording of postsynaptic signals using three microelectrodes. It was shown that RS form groupings that reflect transmitter release in individual active zones (AZ). The topography of transmitter release in the distal parts of the NT under normal conditions was shown to differ from that observed in the proximal parts. The difference consists in a lower probability of transmitter release in AZ and a higher probability of this process between AZ, as well as in a change of release profile in individual AZ. Similar differences were found following denervation. It is suggested that these properties may reflect plastic reorganization occurring in AZ in the course of remodelling of neuromuscular synapse and its degeneration.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 253–260, July–August, 1995.     

Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 μM OA. At lower doses (0.5–5 μM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, Which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

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.     

Location and functioning of transmitter release sites in frog neuromuscular junction

End-plate potentials (EPP) and miniature EPP (MEPP) were recorded in a single neuromuscular synapse of the frog sartorius muscle by means of two microelectrodes with a resistance of 0.5–2.0 M. Groups of signals (fields), reflecting transmitter secretion in spatially distinct release sites were identified by extracellular recording on MEPP amplitude scatter diagrams. Release sites in the nerve ending were found to be unevenly distributed, to be grouped in certain areas, and to differ in their probability of secretion of a quantum of transmitter. Comparison of fields on MEPP and uniquantal EPP amplitude scatter diagrams in solution with low Ca⁺⁺ concentration (0.2–0.4 mM) showed that ability to induce evoked and spontaneous transmitter release at the release site differs, and that sometimes a release site does not participate in evoked secretion. The results of simultaneous recording of synaptic potentials using extra- and intracellular electrodes indicate that transmitter secretion in spatially distinct groups of release sites leads to the appearance of polymodality in the distribution of amplitudes of intracellularly recorded MEPP and uniquantal EPP.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 152–160, March–April, 1985.     

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.     

Presynaptic effects of arachidonic acid and prostaglandin E2 in the frog neuromuscular synapse

Arachidonic acid and prostaglandin E2 decreased the frequency of miniature endplate currents without changing their amplitude-temporary parameters. They also reduced the evoked transmitter release and the amplitude of the 3rd phase of nerve ending response corresponding to the voltage-dependent K(+)-current. Using perineural recording, It was shown that arachidonic acid and prostaglandin E2 decreased the Ca2+ currents of nerve endings. Indometacin: inhibitor of cyclooxygenase, enhanced the evoked transmitter release and decreased the 3rd phase of nerve ending response. Indometacin prevented the effects of arachidonic acid on evoked transmitter release, whereas the effects of arachidonic acid on the 3rd phase was preserved. Prostaglandin E2 seems to mediate the effects of arachidonic acid on spontaneous and evoked transmitter release, Ca(2+)- and Ca(2+)-activated K(+)-currents. Moreover, the arachidonic acid and prostaglandin E2 exerted their own effects upon voltage-dependent potassium current of motor nerve ending.     

Effect of noradrenaline on the amplitude and temporal parameters of multiquantal currents and on the kinetics of evoked quantal secretion of a transmitter]

Noradrenaline caused a significant increase of the multiquantal endplate currents (EPC) amplitude, the EPC rising phase/the rising phase of miniature EPC ratio diminishing, at that. Noradrenaline seems to synchronise the process of quanta secretion in the EPC generation. Thus when a single stimulation of motor nerve results in release of several tenths of a transmitter, noradrenaline may increase the multiquantal EPC amplitude through synchronising of the transmitter release involved in the generation.     

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.     

Electrophysiological study of mediator secretion in the frog neuromuscular synapse under a colchicine block of axoplasmic transport

Two weeks after colchicine nerve treatment the evoked transmitter release was blocked in part of the frog sartorius synapses, with spontaneous activity being absent from some of them. In the synapses with evoked and spontaneous transmitter release preserved within this period of time, the magnitudes of the absolute refractory phase of nerve terminals were significantly higher than the control ones, while in part of synapses, the frequency of miniature end plate potentials (MEPP) was considerably increased. Nerve stimulation (5 imp.s-1) led to a rise of the amplitude of evoked potentials and of MEPP frequency followed by irreversible blockade of synaptic activity. It is concluded that substances transported by rapid axonal flow control the level of membrane potential of nerve terminals and are fairly important for presynaptic membrane integrity.     

Evidence for a presynaptic action of chlordimeform at the insect neuromuscular junction

The mechanism of action of chlordimeform on the mealworm nerve-muscle preparation was studied with microelectrodes. Chlordimeform affected neither the mean amplitude nor the frequency of spontaneous miniature excitatory postsynaptic potentials. Extracellular focal recordings show that in the presence of 0.8 mM chlordimeform the presynaptic spike is almost unchanged, but the quantal content for evoked transmitter release is reduced. It is suggested that chlordimeform decreases the influx of calcium at the presynaptic terminal during the active phase of the nerve terminal action potential, thereby inhibiting evoked transmitter release.     

Effects of pyrocatechol on neuromuscular transmission

Effects of pyrocatechol on neuromuscular transmission were studied both in the frog pectoral-cutaneous muscle and in the mouse phrenic-diaphragmatic preparation by means of extracellular microelectrode recording of synaptic signals. Pyrocatechol applied in a concentration of 0.05 mM increased the frequency of miniature end-plate currents (MEPC) and the amplitude of end-plate current (EPC) by increasing its quantum content. Pyrocatechol also increased the duration of presynaptic response. When voltage-dependent potassium channels had been blocked, pyrocatechol affected neither the EPC quantum content nor the duration of presynaptic response. It is suggested that the pyrocatechol-induced enhancement of transmitter release results from modulatory effects of pyrocatechol on voltage-dependent potassium current in the membrane of a nerve terminal.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 405–408, November–December, 1993.     

Potassium channel blocker-induced presynaptic modulation of inhibitory synaptic transmission in hippocampal neurons of rats

The effects of blockers of voltage-gated potassium channels, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), on inhibitory postsynaptic currents (IPSC) evoked by local electrical stimulation of zones of unitary synaptic terminals on hippocampal neurons were studied using a voltage-clamp technique under conditions of low density cell culture. At activation of the transmitter release in the absence of action potentials (when the terminals are in a tetrodotoxin-containing medium), external application of 5 mM 4-AP reversibly increased the averaged IPSC amplitude by 90±30%, while a similar effect of 10 mM TEA reached only 20±7%. The amplitudes of individual evoked IPSC varied between 10 and more than 150 pA. Amplitude histograms of IPSC in all studied neurons (n=14) were of a polymodal nature and could not described by a Gaussian law. An increase in the averaged IPSC amplitude under the influence of potassium channel blockers cannot be described as resulting only from modification of the number of trials without transmitter release (blank events). The mechanism of potassium channel blocker-induced facilitation of IPSC evoked by single synaptic terminals is discussed.     

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

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.     

Calcium Channels in the GABAergic Presynaptic Nerve Terminals Projecting to Meynert Neurons of the Rat

Abstract : Effects of selective Ca²⁺ channel blockers on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in the acutely dissociated rat nucleus basalis of Meynert (nBM) neurons attached with nerve endings, namely, the “synaptic bouton” preparation, and in the thin slices of nBM, using nystatin perforated and conventional whole-cell patch recording modes, respectively. In the synaptic bouton preparation, nicardipine (3 × 10^-6M) and ω-conotoxin-MVIIC (3 × 10^-6M) reduced the frequency of spontaneous postsynaptic currents by 37 and 22%, respectively, whereas ω-conotoxin-GVIA had no effect. After blockade of L- and P/Q-type Ca²⁺ channels, successive removal of Ca²⁺ from external solution had no significant effect on the residual spontaneous activities, indicating that N-, R-, and T-type Ca²⁺ channels are not involved in the spontaneous GABA release. Thapsigargin, but not ryanodine, increased the frequency of spontaneous IPSCs in both the synaptic bouton and slice preparations, suggesting the partial contribution of the intracellular Ca²⁺ storage site to the spontaneous GABA release. In contrast, ω-conotoxin-GVIA (3 × 10^-6M) and ω-conotoxin-MVIIC (3 × 10^-6M) suppressed the evoked IPSCs by 31 and 37%, respectively, but nicardipine produced no significant effect. The residual evoked currents were abolished in Ca²⁺-free external solution but not in the external solution containing 10^-5M Ni²⁺, suggesting the involvement of N-, P/Q-, and R-type Ca²⁺ channels but not L- and T-type ones in the evoked IPSCs. Neither thapsigargin nor ryanodine had any significant effects on the evoked IPSCs. It was concluded that Ca²⁺ channel subtypes responsible for spontaneous transmitter release are different from those mediating the transmitter release evoked by nerve stimulation.