Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

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, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), 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.     

Depolarization-activated potentiation of the T fiber synapse in the blue crab

The blue crab T fiber synapse, associated with the stretch receptor of the swimming leg, has a nonspiking presynaptic element that mediates tonic transmission. This synapse was isolated and a voltage clamp circuit was used to control the membrane potential at the release sites. The dependence of transmitter release on extracellular calcium, [Ca]o, was studied over a range of 2.5-40 mM. A power relationship of 2.7 was obtained between excitatory postsynaptic potential (EPSP) rate of rise and [Ca]o. Brief presynaptic depolarizing steps, 5-10 ms, presented at 0.5 Hz activated EPSP's of constant amplitude. Inserting a 300-ms pulse (conditioning pulse) between these test pulses potentiated the subsequent test EPSPs. This depolarization-activated potentiation (DAP) lasted for 10-20 s and decayed with a single exponential time course. The decay time course remained invariant with test pulse frequencies ranging from 0.11 to 1.1 Hz. The magnitude and decay time course of DAP were independent of the test pulse amplitudes. The magnitude of DAP was a function of conditioning pulse amplitudes. Large conditioning pulses activated large potentiations, whereas the decay time constants were not changed. The DAP is a Ca-dependent process. When the amplitude of conditioning pulses approached the Ca equilibrium potential, the magnitude of potentiation decreased. Repeated application of conditioning pulses, at 2-s intervals, did not produce additional potentiation beyond the level activated by the first conditioning pulse. Comparison of the conditioning EPSP waveforms activated repetitively indicated that potentiation lasted transiently, 100 ms, during a prolonged release. Possible mechanisms of the potentiation are discussed in light of these new findings.     

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

The calcium hypothesis and modulation of transmitter release by hyperpolarizing pulses.

Small presynaptic conditioning hyperpolarizing pulses reduce transmitter release to a depolarizing stimulus by a substantial amount, with little effect on release by a subsequent depolarization. This result, obtained at neuromuscular junctions and the squid giant synapse, has been offered as a disproof of the calcium hypothesis of transmitter release or the residual calcium hypothesis of synaptic facilitation. However, calculations based on several formulations of these hypotheses are shown to be consistent with the experimental results, and no fundamental modification of the hypotheses is necessary.     

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.     

Correlation of Transmitter Release with Membrane Properties of the Presynaptic Fiber of the Squid Giant Synapse

Depolarization of the presynaptic terminal by current produced a postsynaptic potential (PSP) which increased with increasing presynaptic polarization and then reached a plateau. Iontophoretic injection of tetraethylammonium ions (TEA) into the presynaptic axon near the terminal produced a prolonged presynaptic spike. The resulting PSP is increased in size and its time course closely followed that of the presynaptic spike. The presynaptic fiber no longer exhibited rectification and strong depolarizations revealed that the PSP reached a maximum with about 110 mv depolarization. Further depolarization produced a decrease in PSP amplitude and finally transmission was blocked. However, a PSP then always appeared on withdrawal of the depolarizing current. Under the conditions of these experiments, the PSP could be considered a direct measure of transmitter release. Bathing the TEA-injected synapse with concentrations of tetrodotoxin (TTX) sufficient to block spike activity in both pre- and postsynaptic axons did not greatly modify postsynaptic electrogenesis. However, doubling TTX concentration reversibly blocked PSP. Thus the permeability changes to Na and K accompanying the spike do not appear necessary for transmitter release. Some other processes related to the level of presynaptic polarization must be involved to explain the data. The inhibition of transmitter release by strong depolarizations appears to be related to Ca action. A membrane Ca current may also be necessary for normal transmitter release.     

Further Study of the Relationship between Pre- and Postsynaptic Potentials in the Squid Giant Synapse

The minimal presynaptic depolarization (MPD) for producing a detectable postsynaptic potential (PSP) was lower than 25 mv in normal or tetrodotoxin (TTX)-containing seawater. The MPD was about 10 mv when a small amount of tetraethylammonium ions (TEA) was injected into the presynaptic terminal. Application of linearly increasing depolarizing current to the normal presynaptic terminal at times produced a PSP before a presynaptic spike was evoked; the rate of rise of the resulting PSP was much slower than that of a PSP triggered by the normal presynaptic spike. A brief depolarizing pulse that preceded the presynaptic spike in normal seawater or the initial transient presynaptic depolarization in TTX decreased the PSP. It increased the PSP when it was applied during the spike or initial transient depolarization. Hyperpolarizing pulses had the reverse effect. The Off-PSP was also modified by inserting pulses at an initial part of the recovery phase of the strong presynaptic depolarization. These results indicate further that increases in Na⁺ and K⁺ conductance during presynaptic spike activity are not a requirement for transmitter release; the rate of release of transmitter can be controlled by electrical manipulation of the presynaptic terminal; there is a superficial correspondence between the time courses of presynaptic depolarization and the resulting PSP. Thus presynaptic depolarization appears to be only the first step in the series of events constituting excitation-transmitter release coupling. It may not be a necessary step for the release mechanism.     

Presynaptic potentials and facilitation of transmitter release in the squid giant synapse

Presynaptic potentials were studied during facilitation of transmitter release in the squid giant synapse. Changes in action potentials were found to cause some, but not all, of the facilitation during twin-pulse stimulation. During trains of action potentials, there were no progressive changes in presynaptic action potentials which could account for the growth of facilitation. Facilitation could still be detected in terminals which had undergone conditioning depolarization or hyperpolarization. Facilitation could be produced by small action potentials in low [Ca++]o and by small depolarizations in the presence of tetrodotoxin. Although the production of facilitation varied somewhat with presynaptic depolarization, nevertheless, approximately equal amounts of facilitation could be produced by depolarizations which caused the release of very different amounts of transmitter.     

Mechanism of long-term potentiation of transmitter release induced by adrenaline in bullfrog sympathetic ganglia

A mechanism of the long-term potentiation of transmitter release induced by adrenaline (ALTP) was studied by recording intracellularly the fast excitatory postsynaptic potentials (fast EPSPs). The ALTP was produced during the blockade of K+ channels at the presynaptic terminals by tetraethylammonium (TEA). The synaptic delay, possibly reflecting a relative change in the duration of an action potential at the presynaptic terminal, was not changed during the course of the ALTP. By contrast, it was significantly lengthened by TEA and other K+ channel inhibitors (4-aminopyridine and Cs+) that markedly enhanced the evoked release of transmitter. The magnitude of facilitation of the fast EPSP, induced by a conditional stimulus to the preganglionic nerve, was decreased during the generation of the ALTP, but was unchanged during the potentiation of transmitter release caused by TEA. These results, together with theoretical considerations applying the residual Ca2+ hypothesis to the facilitation, suggest that the enhancement of transmitter release during the ALTP is not caused by an increased Ca2+ influx during a presynaptic impulse owing to the blockade of K+ channel or the modulation of Ca2+ channel, but presumably is induced by a rise in the basal level of free Ca2+ in the presynaptic terminal.     

Post-tetanic decay of evoked and spontaneous transmitter release and a residual-calcium model of synaptic facilitation at crayfish neuromuscular junctions

The post-tetanic decay in miniature excitatory junction potential (MEJP) frequency and in facilitation of excitatory junction potentials (EJPs) was measured at crayfish neuromuscular junctions. A 2-s tetanus at 20 Hz caused the MEJP frequency to increase an average of 40 times and the EJP amplitude to increase an average of 13 times. Both MEJP frequency and EJP facilitation decayed with two time constants. The fast component of MEJP frequency decay was 47 ms, and that of EJP facilitation was 130 ms. The slow component of MEJP frequency decay was 0.57 s, and that of EJP facilitation was approximately 1 s. These results were consistent with the predictions of a residual calcium model, with a nonlinear relationship between presynaptic calcium concentration and transmitter release.     

Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltage-dependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating models of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.     

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Calcium and facilitation at two classes of crustacean neuromuscular synapses

The closer muscle of the crab, Chionoecetes, has at least two classes of excitatory neuromuscular synapses. In one class of synapses an action potential depolarizing the synaptic region releases much more transmitter if it has been preceded recently by another action potential. The other class of synapses shows this property, called facilitation, to a far lesser extent. Immediately after one conditioning stimulus the level of facilitation is similar in both classes. The rate of the ensuing decay of the facilitation is the critical factor differentiating the two classes of synapses. The relationship between external Ca⁺⁺ concentration and transmitter release is similar for both classes of synapses. The slope of a double logarithmic plot of this relationship varies from 3.1 between 5 and 10 mM Ca⁺⁺ to 0.9 between 30 and 40 mM Ca⁺⁺. Facilitation does not significantly change when tested in external Ca⁺⁺ concentrations ranging from 7 to 30 mM. The extracellularly recorded nerve terminal action potential does not increase in amplitude during facilitation. The results suggest that the mechanism of synaptic facilitation is similar for both classes of synapses and occurs after the stage in transmitter release involving Ca⁺⁺.     

Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release

Evoked transmitter release depends upon calcium influx into synaptic boutons, but mechanisms regulating bouton calcium levels and spontaneous transmitter release are obscure. To understand these processes better, we monitored calcium transients in axons and presynaptic terminals of pyramidal neurons in hippocampal slice cultures. Action potentials reliably evoke calcium transients in axons and boutons. Calcium-induced calcium release (CICR) from internal stores contributes to the transients in boutons and to paired-pulse facilitation of EPSPs. Store depletion activates store-operated calcium channels, influencing the frequency of spontaneous transmitter release. Boutons display spontaneous Ca2+ transients; blocking CICR reduces the frequency of these transients and of spontaneous miniature synaptic events. Thus, spontaneous transmitter release is largely calcium mediated, driven by Ca2+ release from internal stores. Bouton store release is important for short-term synaptic plasticity and may also contribute to long-term plasticity.     

Cyclic AMP, 5-HT, and the modulation of transmitter release at the crayfish neuromuscular junction

In this study it was found that several agents which elevate cAMP levels in cells also increase dramatically the quantity of transmitter released from crayfish excitatory nerve terminals in response to a stimulus. With respect to time course and magnitude, the increase produced by one of these agents, the cyclic nucleotide phosphodiesterase inhibitor Squibb 20,009 (SQ 20,009), is unlike any reported for such a drug at a synapse. Additionally, SQ 20,009 potentiated the facilitation of transmitter release produced by serotonin (5-HT) at this synapse. These results establish a possible role for cAMP in the control and modulation of transmitter release at the crayfish neuromuscular junction (NMJ). They further suggest that 5-HT functions here by activation of a presynaptically located adenylate cyclase.     

Effects of membrane polarization on sarcoplasmic calcium release in skeletal muscle

Calcium release from the sarcoplasmic reticulum was investigated in voltage-clamped, tetrodotoxin-treated frog skeletal muscle fibres injected with arsenazo III. Short (5 ms) depolarizing pulses (test pulses) produced a transient change in arsenazo III absorption, signalling an increase in intracellular calcium in concentration (calcium transient). Conditioning subthreshold depolarizations, which preceded the test pulse, potentiated the calcium transient triggered by the test pulse. Conditioning hyperpolarizations, applied either before or after the test pulse, inhibited the calcium transient. These effects of conditioning polarizations on the calcium transient may explain similar effects of subthreshold polarizations on muscle contraction that have previously been reported. The potentiating effect of subthreshold depolarizations was observed only when the test pulse was short (5 ms). The potentiating effect develops at -48 mV with a time constant of about 7 ms at 6.5 degrees C; this seems to be slower than that predicted by the potential spread from the surface along the tubular system. Thus, part of the effect could arise from the coupling process between tubular depolarization and calcium release.     

Modulating action of barium and other ions on the sensory activity of frog labyrinth

The effect of Ba2+, TEA, 4-AP and CoCl2 on the EPSP and spike discharges recorded from single fibres of the posterior nerve in the isolated frog labyrinth has been investigated. In Ca-free solution Ba2+ preserved, at low concentration (0.3 mM), the resting activity and at higher levels (up to 6 mM) it resulted in a pronounced facilitation of the EPSP and spike discharges. Facilitation increased on increasing Ba2+ concentration up to 4-5 mM and it was more evident in those units exhibiting a low resting spike firing. The effect of Ba2+ (1 mM) was completely antagonized by 10 mM Ca2+ X CoCl2 (3 mM) suppressed the resting rate at the normal external Ca2+ concentration; the Co2+ block was partially relieved by 1.8 mM Ba2+ X TEA (20 mM) evoked a clear-cut increase in the EPSP and spike discharges which, however, was less consistent than that produced by Ba2+. By comparing the effect of TEA on the spike frequency with that obtained at different Ba2+ levels, the Ba2+ capacity to carry the Ca2+ current was dissected. Such an effect is dose-dependent and it is more evident in low-frequency units. Conversely, 4-AP did not affect the resting discharge frequency. These results indicate that either the Ca2+ or the Ba2+ current sustain the transmitter release at the cyto-neural junction. The effect of TEA suggests that the Ca2+-dependent K+ current may play an important role in supporting the neurosecretory process by controlling the membrane potential of the hair cells.