Maintained depolarization of synaptic terminals facilitates nerve-evoked transmitter release at a crayfish neuromuscular junction

Presynaptic and postsynaptic potentials were examined by intracellular recording at a crayfish neuromuscular junction. During normal synaptic transmission, the action potentials were recorded in the terminal region of the excitatory axon and postsynaptic responses were obtained in the muscle fibers. We found that it was possible to modify the synaptic transmission by applying depolarizing or hyperpolarizing currents through the presynaptic intracellular electrode. Typically, a 7-15 mV depolarization lasting longer than 50 msec leads to a large (500%) enhancement of transmitter release, even though the preterminal action potential is reduced in amplitude. Hyperpolarization increases the amplitude of the action potential, but slightly reduces the transmitter release. These results are different from those reported for other neuromuscular synapses and the squid giant synapse, but are similar in many respects to the results reported for several invertebrate central synapses. We conclude, first, that different synapses may have markedly different responses to conditioning by membrane polarization and, secondly, that maintained low-level depolarization may induce a potentiated state in the nerve terminal, perhaps brought about by slow entry of calcium.     

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.     

Temperature dependence of spontaneous quantal and nonquantal transmitter release from motor nerve endings in the mouse

Spontaneous quantal and nonquantal acetylcholine release were investigated at an ambient temperature range of 10–35°C in a preparation of white mouse hemidiaphragm. Quantal transmitter release was assessed by the frequency of miniature endplate potentials and nonquantal secretion by the level of H-effect. Spontaneous quantal release rose exponentially in step with increasing temperature. Two relative maxima, one of 20°C and the other of 35°C were noted in the temperature dependence of nonquantal transmitter release. Nonquantal release of acetylcholine did not take place at a temperature of 10°C. The effective energy of activation of quantal release was calculated as 57.0 kJ/mole over the temperature range investigated; that of the nonquantal release process at intervals of 15–20°C and 25–35°C measured 45.5 and 38.2 kJ/mole respectively. It is suggested that an active transport system process rather than simple diffusion of acetylcholine molecules is responsible for nonquantal release of this neurotransmitter.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 3, pp. 361–367, May–June, 1986.     

Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode

Measurements of resting potential and action potential in presynaptic branches of the excitatory motor axon to the crayfish opener muscle were made with intracellular microelectrodes during application of serotonin (10(-9)-10(-3) M). A 5-min exposure to 10(-6) M serotonin produced enhancement of excitatory junction potentials (EJPs) lasting about 1 h. The membrane potential of the presynaptic terminal was depolarized by about 5 mV; the depolarization subsided within 1/2 h. Concomitant reduction in amplitude of the presynaptic action potential, not accompanied by spike broadening, was observed. The presynaptic depolarization, and the enhancement of EJPs, were dependent on the presence of extracellular sodium but not extracellular calcium. A possible mechanism for serotonin's effect involves initial entry of sodium into the nerve terminal, with consequent increased availability of intracellular calcium. The subsequent long-lasting phase of EJP enhancement may result from an additional effect on the metabolism of the nerve terminal.     

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.     

Identification of transmitter release sites in the motor nerve terminal

A model was produced of generation of postsynaptic current following release of a quantum of neurotransmitter from the nerve ending, whereby the law of current density attenuation is defined as j=I/r^b (A), where I is current density at the generation site and j stands at distance r from that site. Coefficient b was shown experimentally to be close to 1 using extracellular techniques of signal recording. Assuming that sites of signal generation and transmitter release are spatially identical, a new technique for determining the coordinates of the transmitter release site in the motor nerve terminal is suggested. This consists of measuring uniquantal signal amplitude by means of three extracellular microelectrodes spaced 5–10 µm apart. We were able to establish, by producing "spatial pictures" of transmitter release based on analysis of several hundred signals in the frog cutaneous pectoris muscle, that release sites are arranged in groups running diagonally to the nerve ending. These groups are thought to reflect transmitter release in active zones of the nerve ending. Advantages, disadvantages, and inaccuracies of the method are identified.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Moscow. V. I. Ulyanov-Lenin University, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 309–318, May–June, 1990.     

Does the motor nerve impulse evoke 'non-quantal' transmitter release?

Previous experiments have indicated that there is a continuous leakage of acetylcholine (ACh) from resting motor nerve terminals which can produce a small depolarization in anti-esterase treated endplates (Katz & Miledi 1977; Vyskocil & Illés 1978). This leakage might be expected to be intensified during the presynaptic action potential and also lead to a very small non-quantal endplate response. This hypothesis was examined, in frog and mammalian endplates, by stimulating the motor nerve in a calcium-deprived medium and recording the summated average response to several hundred stimuli. The result was completely negative; no trace of a non-quantal endplate potential was ever observed, with the limit of detection being always less than 10 microV, and sometimes as low as 2 microV. These experiments suggest that the leakage of ACh either does not originate predominantly from the synaptic region of the axon terminal, or that it occurs by a mechanism that is not directly influenced by the membrane potential.     

Adenosine depresses spontaneous transmitter release from frog motor nerve terminals by acting at an A1-like receptor

Adenosine (1 microM to 1 mM) depressed spontaneous transmitter release from frog motor nerve terminals without producing any observable postsynaptic effects. Since this action of adenosine was blocked by 20 microM theophylline and 1 microM 8-phenyltheophylline, adenosine probably acts at a specific receptor on motor nerve terminals to reduce spontaneous transmitter output. The effects of the adenosine analogs, L-N6-phenylisopropyladenosine (L-PIA, 100 pM to 1 microM), D-PIA (100 nM to 100 microM), and 5'-N-ethylcarboxamidoadenosine (NECA, 10nM to 100 microM), were tested on spontaneous transmitter release at the frog neuromuscular junction. L-PIA depressed mepp frequency at a threshold concentration of about 1 nM, was thirteen times more potent than NECA, and was 294 times more effective than D-PIA. The rank-order potency of these analogs indicates that adenosine acts at an A1-like receptor to depress spontaneous transmitter release. Inhibitory actions of maximally effective concentrations of adenosine and L-PIA were also blocked by the A1-specific antagonist, 1-3-dipropyl-8-cyclopentylxanthine (DPCPX) at a concentration of 100 nM. Micromolar concentrations of NECA, an agonist with approximately equal affinity for the A1 and A2 receptors, produced biphasic effects on mepp frequency. Thus, a second adenosine receptor, perhaps of the A2 subtype, may be present on motor nerve terminals and may mediate an increase in spontaneous transmitter release.     

The formation and function of single transmitter release sites at mature amphibian motor—nerve terminals

The extent of quantal transmitter release from single sites of synaptic vesicle accumulations along the length of motor-nerve terminal branches at the amphibian neuromuscular junction has been investigated. Such a determination involves development of a model for the generation of quantal potential fields at single styryl-dye stained sites along the length of a branch. Successful testing and application of this model indicates that the extent of quantal release at a dye-stained site is proportional to the total length of active zone at the site. The stability of these sites and of their ensheathing terminal Schwann cell processes was also investigated. Following simultaneous injection of the terminal Schwann cell and nerve terminal with different fluorescent dyes, terminal branches were observed to show dynamic changes in their length, with these occurring in relatively short periods of hours or less. Redistribution of styryl dye stained sites at the ends of branches also occurred in such short periods of time. These were accompanied by changes in the configuration of terminal Schwann cells, which generally occurred prior to changes in the length of nerve terminal branches.     

Dopamine release from nerve endings induced by polysialogangliosides

Polysialogangliosides but not monosialoganlioside or a neutral glycosphingolipid induce release of [³H] -dopamine from synaptosomes in presence of Ca⁺⁺, presumably by exocytosis. This effect is discussed in relation to the ability of polysialogangliosides to induce membrane fusion in chicken erythrocytes and to their behaviour in lipid monolayers. It is suggested that characteristic interactions with phosphatidylcholine involving decreases of surface potential are participating in the polysialoganglioside-induced neurotransmitter release.     

Maximum likelihood estimation of non-uniform transmitter release probabilities at the crayfish neuromuscular junction

The classical model of quantal release of neurotransmitter assumes that a fixed number of quantal units are available for release in the presynaptic terminal, and that each unit has the same probability of being released. This model also assumes that different units are released independently of one another. We consider two variations of the classical model. In the first case we assume that release is independent, but with potentially different release probabilities at different sites. In the second case we allow for dependence among the release units. A maximum likelihood procedure for the estimation of model parameters is developed, and an estimator of the number of quantal units is proposed. The performance of the method is assessed through a simulation study, and the procedures are applied to the analysis of a sequence of post-synaptic potentials recorded intracellularly at the crayfish neuromuscular junction. Goodness of fit and hypothesis test procedures reject the classical model in favor of an independent release mechanism with differing release probabilities. A more general release mechanism, allowing for dependence in the release process, also provides a good fit to the data analyzed.     

Effect of substances blocking calcium channels (verapamil,D-600, manganese ions) on transmitter release from motor nerve endings in frog muscle

Experiments on isolated frog nerve-muscle preparations showed that manganese ions (0.4–5.0 mM) inhibit evoked transmitter release by reducing the quantum composition of the end-plate potentials, and they intensify spontaneous transmitter release to a certain extent by increasing the frequency of miniature potentials. Verapamil (1 · 10^–6–5·10^–5 g/ml) and D-600 (2.5·10^–5 g/ml), by contrast with manganese ions, do not inhibit evoked release, but also intensify spontaneous release of the transmitter. All the agents tested prevent the potentiating effect of imidazole (3 mM). During repetitive stimulation, verapamil disturbs action potential generation in the motor nerve. Manganese ions had no such action. It is concluded that between the calcium channels of motor nerve endings and the calcium channels of heart muscle or the neuron soma there are molecular differences, expressed as sensitivity to the blocking action of verapamil and D-600.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 415–422, July–August, 1977.     

Calcium currents and calcium-activated potassium currents at the frog motor nerve endings

Intracellular recordings were made of evoked electrical response of the nerve endings during experiments on the frog cutaneous pectoral muscle. A delayed inward current was discovered when superfusing the neuromuscular preparation with a calcium-free solution containing tubocurarine in the response evoked at the nerve endings, using CaCl₂-filled electrodes. This was replaced by the opposite (outward) type of current when 4-aminopyridine was added to the external solution. The outward current was dependent on the calcium concentration at the electrode, decreased after local increase on potassium concentration at the electrode, and disappeared under the effects of cobalt. Local iontophoretic application of tetraethylammonium led to the disappearance of the outward current and the appearance of a powerful and protracted inward current. Similar readings of inward and outward currents were obtained when recording electrical signals using electrodes filled with SrCl₂, BaCl₂, and MgCl₂. It was deduced that the late inward current is carried through voltage-dependent calcium channels and outward delayed current through calcium-activated potassium channels at the nerve terminal. The part played by these currents in transmitter secretion from the motor nerve ending is discussed, together with the relationship between them.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR. V. I. Ul'yanov-Lenin State University, Kazan'. Translated from Neirofiziologiya, Vol. 19, No. 4, 1987, pp. 467–473, July–August, 1987.     

Effects of stimulus timing on transmitter release and postsynaptic membrane potential at crayfish neuromuscular junctions

Summary Different synaptic terminals of the single excitor axon to the opener muscle of crayfish (Procambarus clarkii) often release transmitter in a very different manner when stimulated with the same equal-interval, doublet, or triplet patterns. Compared to synapses that show little facilitation (low F_e synapses), highly facilitating (high F_e) synapses show greater percentage increases in several measures of synaptic efficacy when stimulated with any of these patterns. Low F_e synapses usually show the greater absolute changes in these measures of synaptic efficacy. Changes in the span and pattern of doublets and triplets can independently affect both pre- and postsynaptic measures of synaptic efficacy at either low F_e or high F_e synapses.Abbreviations EJP excitatory junctional potential - MJP spontaneous miniature EJP - F _e ratio of EJP at 1 Hz to EJP amplitude at 10 Hz - F ₁ zero-time facilitation - A ₂,B ₂,C ₂ doubly corrected EJP amplitude of a particular pulse - average amplitude of doubly corrected EJPs in a train of equal-interval, doublets, and triplets, respectively - A_m, B_m, C_m maximum depolarization reached by a particular EJP - time constant of decay