Shoichi Minota

Long-term potentiation of transmitter release induced by repetitive presynaptic activities in bull-frog sympathetic ganglia.

Long‐lasting potentiation of transmitter release induced by repetitive presynaptic activities in bull‐frog sympathetic ganglia was studied by recording intracellularly fast excitatory post‐synaptic potentials (fast e.p.s.p.s.). Following a brief period of post‐tetanic potentiation or depression (less than 10 min), the amplitude of the fast e.p.s.p. was potentiated for a period between several tens of minutes and more than 2 h in response to tetanic stimulation of the preganglionic nerve in twenty‐one out of twenty‐eight cells. Quantal analysis revealed that this long‐term potentiation of the fast e.p.s.p. (l.t.p.) was accompanied by an increase in quantal content m (in nine out of twenty‐one cells), quantal size (four cells) or both (eight cells). The increased quantal content (presynaptic l.t.p.) declined exponentially (ten cells) or decayed gradually to a certain enhanced level which lasted several hours. In contrast, the increased quantal size grew with a relatively long latency (10‐25 min) and remained relatively constant for at least 2 h. The magnitude of presynaptic l.t.p. increased with increased duration of the presynaptic tetanus (33 Hz) from 2 to 5 s. No l.t.p. was elicited by a 1‐s tetanus, whereas the time course appears to be independent of the tetanus duration and the magnitude of l.t.p. There was a positive correlation between the magnitude of presynaptic l.t.p. and the pre‐tetanic quantal content up to m = 3, but the former deviated from linear regression when the value of the latter exceeded 3. No l.t.p. occurred when quantal content was less than 0.5. A tetanus (33 Hz, 10 s) applied in Ca2+‐free solution elicited no presynaptic l.t.p., while the same tetanus in normal Ringer solution produced a large presynaptic l.t.p. Presynaptic l.t.p. was enhanced in magnitude at low temperature (8‐10 degrees C). These results demonstrate the existence of a use‐dependent, long‐term potentiation of transmitter release in bull‐frog sympathetic ganglia. Several possible mechanisms are discussed in terms of Ca2+‐buffering mechanisms of the presynaptic nerve terminals.

Long-term potentiation induced by a sustained rise in the intraterminal Ca2+ in bull-frog sympathetic ganglia.

1. The mechanism of a long‐term potentiation of transmitter release (pre‐LTP) induced by a tetanic stimulation (33 Hz for 1‐30 s) applied to the preganglionic nerve was examined by intracellularly recording the fast excitatory postsynaptic potentials (fast EPSPs) in bull‐frog sympathetic ganglia. 2. Short‐term facilitation induced by paired pulses was decreased during the course of pre‐LTP; the extent of reduction paralleled with the magnitude of pre‐LTP. 3. The frequency of miniature EPSPs increased after tetanic stimulation that produced the pre‐LTP. 4. The Ca2+ ionophore, A23187, increased both the amplitude and quantal content of fast EPSPs and frequency of miniature EPSPs while it decreased short‐term facilitation. 5. A Ca2+ chelating agent, Quin‐2, loaded as acetoxymethyl ester, reduced the amplitude and quantal content of fast EPSPs and short‐term facilitation, and blocked the generation of pre‐LTP. 6. Activators of protein kinase C, phorbol 12,13‐dibutyrate and 1‐oleoyl‐2‐acetyl‐rac‐glycerol, and its inhibitors, H‐7 and staurosporine, did not block the generation of pre‐LTP, while the activators enhanced transmitter release. 7. Inhibitors of calmodulin, trifluoperazine and W‐7, blocked the generation of pre‐LTP, whereas the amplitude and quantal content of fast EPSPs were not influenced. 8. These results suggest that the pre‐LTP results from a sustained rise in the basal level of intraterminal Ca2+ and an activation of the Ca(2+)‐calmodulin‐dependent process in the preganglionic nerve terminals.

Differential effects of apamin on Ca2+-dependent K+ currents in bullfrog sympathetic ganglion cells

In B-type neurones of bullfrog sympathetic ganglia, apamin (10 nM) suppressed the Ca2+-dependent K+ current (IAH) involved in the afterhyperpolarization of an action potential, while it did not affect the Ca2+-dependent K+ current (Ic) underlying the spike repolarization. IAH was further separated into two exponential components which were differentially affected by apamin, voltage and alterations in Ca2+ influx, suggesting the existence of 3 different types of Ca2+-dependent K+ channel in bullfrog sympathetic neurones.

Regulation of two ion channels by a common muscarinic receptor-transduction system in a vertebrate neuron

In bullfrog sympathetic ganglion cells, muscarine produced an inward current (Imus) through the activation of a subtype (M1) of muscarinic acetylcholine receptor (mAChR) by suppressing an outward M-current (IM), and/or activating cation-selective current (ID; see below). The former was induced with a potency (Kd = 0.5 microM) higher than the latter (Kd = 5 microM) before and after blocking a fraction of the receptor with an irreversible blocker. Activators of protein kinase C mimicked muscarines actions. Blocking IM by Ba2+ increased ID. These results suggest that activation of M1-mAChR both closes M-channel and opens cation-selective D-channel through phosphoinositide breakdown and the subsequent activation of protein kinase C and that a difference in potency at the last step of the cascade determines the order in which channels are regulated.

Patch clamp experiments on nicotinic acetylcholine receptor-ion channels in bullforg sympathetic ganglion cells

Nicotinic acetylcholine-receptor ion channels (AChR channels) were studied in bullfrog sympathetic ganglion cells cultured for 1 day to 3 weeks, using a patch clamp technique. Microsuperfusion of ACh (2–10 μM) to the ganglion cell under the whole cell clamp produced an inward current at membrane potentials negative to −60 mV, which had a fast onset and decay. This rapid ACh-induced current was accompanied by a large current fluctuation, decreased and increased in amplitude by membrane depolarization and hyperpolarization, respectively, and blocked by d-tubocurarine. Thus, this current must be induced by the nicotinic action of ACh, but not by a muscarinic effect to activate a slow cation-selective current. At depolarized levels more than −50 mV, Ach induced an additional inward current which was slow in time course, accompanied by no or decreased current fluctuation and increased in amplitude by membrane depolarization. Accordingly, this slow ACh-induced current could result from the suppression of a voltage-dependent K+ current (M-current: Brown and Adams 1980) by the muscarinic action of ACh. Fluctuation analysis of the rapid ACh-induced current at potentials negative to −50 mV revealed the elementary conductance of 14 pS and a power spectral density distribution of the double Lorentzian function which yielded the time constants of 5.4 and 62.5 ms at −60 to −80 mV. The variance of either component was independent of the mean current.Under both the cell-attached and outside-out modes, ACh (1–10 μM: applied by microsuperfusion or bath application for the latter mode) caused single channel currents which reversed at a membrane potential close to 0 mV and had a conductance of 18–28 pS. In some patches, single channel currents of a smaller conductance (12 pS) were also observed in the presence of ACh. The open time distribution of the main AChR channel population followed a single or double exponential function depending on patches. The time constant of the single exponential distribution and that of the fast component of the double exponential distribution were similar and approximately 0.9 ms, while that of the slow component of the latter was 6.4 ms. Furthermore, the magnitude of each component in double exponential distributions varied largely among patches. These results suggest that there are at least two or three types of AChR channels in cultured bullfrog sympathetic ganglion cells: fast and slow AChR channels of 18–28 pS and possibly a fast AChR channel of smaller conductance of 12 pS.

Nicotinic acetylcholine receptor-ion channels involved in synaptic currents in bullfrog sympathetic ganglion cells and effects of atropine.

The nicotinic acetylcholine receptor-ion channels (AChR channels) of the bullfrog sympathetic ganglion cells were studied with a two-electrode voltage clamp technique. The decay phase of the fast excitatory postsynaptic current (fast e.p.s.c.) in B-type neurones followed a double exponential function whose time constants were 3.2 and 8.0 ms at −60 mV and increased with membrane hyperpolarization. Likewise, the decay phase of the fast e.p.s.c. in C-type neurones was double-exponential with time constants of 4.4 and 12.3 ms (at −60 mV). The miniature e.p.s.c. in B-type neurones also decayed with a double exponential function (2.7 and 15.4 ms at −100 mV). Analysis of acetylcholine-induced current fluctuations revealed the power spectral density distribution of a double Lorentzian function which yielded the time constants of elementary events [τnoise(f) and τnoise(s): 1.7 and 29.7 ms, respectively, at −100 mV] and the averaged elementary conductance (γ: 7.8 pS).The amplitude of fast e.p.s.c. and the time constant of the fast component of its decay phase decreased during the initial (“acute”) phase (within 15 min) of the action of atropine (3 μM), but recovered during the later (“chronic”) phase (more than 30 min after application) of the action. The slow component was affected by atropine in a manner similar to the fast component during the “acute” phase.During the “chronic phase”, however, the slow time constant recovered and exceeded the control value. Furthermore, this prolongation remained for at least 1 h after the removal of atropine. τnoise(s) during the “chronic” phase of the action was also prolonged to 50.3 ms, while [τnoise(f) (2.1 ms) was similar to the control value. The amplitude and quantal size of the fast excitatory postsynaptic potential were also decreased during the “acute” phase of atropine action, and recovered during the “chronic” phase. Interestingly, they were reduced transiently during the course of removal of atropine from the bath.These results revealed that the decay phases of the fast e.p.s.c. and miniature e.p.s.c. have two components which are explained either by the existence of two types of AChR channels having different open times or by a single type having three states with rate constants of certain relationships in the bullfrog sympathetic ganglion cells and suggested that their open forms are blocked by atropine only transiently and later desensitized to the blocking action.

Restoration of the nicotinic receptor-channel activity from the blockade by atropine in bullfrog sympathetic ganglia

Atropine (3 microM) reduced both the nicotinic and muscarinic acetylcholine (ACh) potentials of the bullfrog sympathetic ganglion cell. However, the former was completely restored within 1 h during a sustained exposure to atropine, while the latter remained blocked. Similar restorations were observed for the depressant effects on both the amplitude and decay phase of a nerve-induced postsynaptic current. The results suggest that the sustained or repetitive binding of atropine to this site results in a new conformational state capable of passing ions almost normally, but resistant to the blockade by atropine.

General Characteristics and Mechanisms of Nicotinic Transmission in Sympathetic Ganglia

The quantal release of acetylcholine (ACh) by an impulse at preganglionic terminals generates an excitatory postsynaptic potential (EPSP) at the subsynaptic membrane of a postganglionic neuron. This EPSP exhibits a rapid rise and a relatively slow decay with a time constant somewhat longer than the membrane time constant. In view of its temporal characteristics, it has been termed the fast EPSP (cf. Nishi, 1974; Kuba and Koketsu, 1978] to distinguish it from other EPSPs such as the slow EPSP and the late slow EPSP. The fast EPSP is unequivocally induced by the nicotinic action of ACh, since it is depressed by D-tubocurarine or β-erythroidine (Eccles, 1963; Blackman et al., 1963) and less by atropine and is augmented by anticholinesterases.

Delayed onset and slow time course of the non-M-type muscarinic current in bullfrog sympathetic neurons

The onset and time course of the muscarinic currents induced by brief applications of acetylcholine (ACh) were examined in voltage-clamped neurons of bullfrog sympathetic ganglia bathed in a solution containing d-tubocurarine. At a potential of −40 mV, the ACh-induced current (IACh) appeared within 1.2 s and rapidly increased to its peak with a half-activation time of 2.2 s. This initial current was termed the fastIACh and was blocked by 4 mM Ba2+. At a potential more negative than −60 mV, the fastIACh disappeared and the remainingIACh activated with a delay of 3.9 s and slowly increased to its peak with a half-activation time of 8.2 s. This delayed current was termed the slowIACh and is thought to be associated with inhibition of a K+ current, orIM, as well as activation of an inward current through non-M-type muscarinic cation channels. The slowIACh was not inhibited by Ba2+, but its amplitude was reduced with depolarization (the extra-polated reversal potential was +3 mV). In Na+-free solution, the amplitude of the slowIACh reduced, but its polarity did not reverse in the voltage region examined (-30 to −100 mV). The slow excitatory postsynaptic current was also recorded, and was shown to have a similar delay in onset and slow time course. The results demonstrate that ACh activates the non-M-type muscarinic current three times more slowly than it inhibitsIM.

An Increased Basal Calcium Hypothesis for Long-Term Potentiation of Transmitter Release in Bullfrog Sympathetic Ganglia

Long-term potentiation (LTP) of synaptic transmission, a basis for learning and memory (cf. Tsukahara, 1981), occurs in response to conditioning stimuli in various neuronal elements at both central and peripheral synapses. In bullfrog sympathetic ganglia, there are two types of long-term potentiation of transmitter release, one induced by conditional tetanic stimulation of the preganglionic nerve through a Ca2+ -dependent mechanism (presynaptic LTP, pre-LTP; Koyano et al., 1985), and the other generated by the action of epinephrine through a cAMP-dependent mechanism (epinephrine-induced LTP, adrLTP; Kuba et al., 1981; Kuba and Kumamoto, 1986). We describe here novel mechanisms of these LTPs in which a rise in the basal level of the intracellular free Ca2+ ([Ca2+]i) in the presynaptic terminal plays an important role.