Effects of hypertonic solutions on quantal transmitter release at the crayfish neuromuscular junction (original) (raw)
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The Journal of General Physiology
Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK~c~, on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK~c~) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with g~c~ not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK~c~ by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltagedependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gKtc~ by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK<ca~ was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (> 50 ms) suggest either that residual Zucker, R. S., and L. O. Lara-Estrella. 1983. Post°tetanic decay of evoked and spontaneous transmitter release and a residual calcium model of synaptic facilitation at crayfish neuromuscular junctions.
The Journal of physiology, 1982
1. The effects of the anion-transport blocking reagents Probenecid, 4,4'diisothiocyanostilbene-2,2'-disulphonate (DIDS), and pyridoxal phosphate on the number of quanta released (m) and the time course of the probability of quantal release, alpha(t), following an action potential were studied at single synaptic sites on the opener muscle in the crayfish leg.2. Low concentrations (10-50 muM) of DIDS or Probenecid produced decreases in m to about 30-50% of the initial value; at higher concentrations (100 muM or greater), evoked release failed. Much greater concentrations (500 muM) of pyridoxal phosphate were necessary to produce decreases in m.3. Synaptic depression in 50 muM-Probenecid was reversible by removing the agent; after treatment with 50 muM-DIDS, however, m partially regained the initial value upon removal of the drug.4. The time course of alpha(t) after an action potential was unaffected by any of the reagents.5. It is concluded that the reagents decrease the numbe...
Neuroscience Letters, 1988
Recordings of synaptic currents from the crayfish opener muscle were made with a macro-patch recording technique, permitting clear detection of neurotransmitter quanta at individual nerve terminals before and after induction of long-term facilitation (LTF). Depolarization of the terminal by propagated action potentials or by local intracellular pulses induced LTF. The quantal content was increased on average by 93%. Binomial analysis indicated increased probability of release and also increased number of available quantal units. The increase occurred regardless of a blockade of sodium, calcium or potassium channels by appropriate pharmacological agents. Presynaptic recording with an intracellutar microelectrode showed no change in presynaptic electrical properties. Also, there were no changes in the synaptic delay. It is concluded that LTF results from a depolarization-dependent alteration of synaptic release sites.
The Journal of General Physiology
Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, g~c~), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK~c,), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K + on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals. Protocol
Increased presynaptic ATP levels coupled to synaptic activity at the crayfish neuromuscular junction
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
Levels of ATP and related adenylates were measured in the terminal region of efferent nerves in the crayfish opener muscle using the luciferin-luciferase method. Following 1 min of stimulation at 50 Hz, the average (+/- SE) ATP content rose from 13.4 (+/- 1.5) to 19.0 (+/- 2.1) nmol/mg dry weight. The amounts of ADP, AMP, and the phosphagen phosphoarginine did not change significantly. Thus, the increased ATP was not derived from any of these potential sources. The increase was found to depend on synaptic activation, however, for its magnitude was directly related to the concentration of extracellular Ca2+, and it was blocked when CoCl2, verapamil, ruthenium red, or gamma-methylglutamate and picrotoxin were added to the bath. Addition of ATP to the bath solution also increased nerve ATP levels. Based upon measurements of sucrose distribution, only 50% of this increase was in the extracellular water space. The remainder of the ATP had either entered the nerve, become adsorbed extrace...
2003
The quantum-vesicular hypothesis holds that release of transmitter from nerve endings occurs in discrete portionsquanta. A transmitter quantum is located in the synaptic vesicle and is released into the synaptic cleft by exocytosis in response to increases in the intracellular Ca ion concentration . Electron microscopic studies of neuromuscular synapses in frogs have demonstrated that vesicles are located in the immediate vicinity of the presynaptic membrane in electron-dense formations, which have been termed active zones . Contemporary electrophysiological studies have demonstrated convincingly that the active zone is the site of transmitter secretion processes . Freeze-thaw investigations have shown that the active zone region of the presynaptic membrane contains large intramembrane particles (diameter 10 nm), running across the nerve ending in the form of two parallel rows; synaptic vesicles are located in two rows near these . Numerous data have been obtained indicating that these particles are both calcium channels, responsible for temporary increases in calcium ion concentrations close to synaptic vesicles, and calcium-activated potassium channels, which control the parameters of local depolarization in the area of the active zones . It is currently believed that secretion of transmitter into the active zone area requires formation of morphofunctional transmitter Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5-6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings. At initially low extracellular Ca ion concentrations (0.15-0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra-and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.