Rami Rahamimoff

Pre- and Postfusion Regulation of Transmitter Release

We thank our colleagues, Alexander Butkevich, Brenda Farrell, Simona Ginsburg, Sir Bernard Katz, Jack McMahan, Piotr Marszalek, Alon Meir, Halina Meiri, and Naomi Melamed-Book for helpful comments.The work in Jerusalem was supported by the U.S.-Israel Binational Science Foundation, the German-Israel Foundation, and the Israeli Academy of Sciences. The work in Rochester was supported by NIH grants.The unfailing secretarial help of Ms. Debra Broide and Ms. Cynthia Camrud is greatly appreciated.

A study of tetanic and post‐tetanic potentiation of miniature end‐plate potentials at the frog neuromuscular junction.

1. The involvement of calcium sodium, potassium and magnesium in tetanic and post‐tetanic potentiation of miniature end‐plate potential frequency was examined at the frog neuromuscular junction using conventional electrophysiological techniques. 2. Tetanic potentiation is larger in calcium containing solutions, than in solutions which generate reversed electrochemical gradient for calcium during nerve activity. 3. Tetanic potentiation increases with stimulation frequency and duration, under both inward and reversed electrochemical gradient for calcium conditions. This indicates that factors, other than calcium entry, participate in tetanic potentiation. 4. Addition of the potassium conductance blocking agent, 3‐aminopyridine (5 mM), increases tetanic potentiation in calcium containing media, while depressing it under reversed calcium gradient. 5. Electronic depolarization of the nerve terminal in tetrodotoxin‐containing Ringer solution, produces tetanic potentiation under inward gradient, but fails to do so under reversed gradient. This indicates that the entry of sodium ions participates in the generation of tetanic potentiation. 6. Addition of magnesium ions suppresses tetanic potentiation in calcium containing solution, but increases tetanic potentiation under reversed gradient. 7. The results are explained by the hypothesis that calcium entry and intracellular calcium translocation participate in the generation of tetanic potentiation. 8. Both the fast and the slow components (augmentation and potentiation respectively) of post‐tetanic potentiation increase in duration, with increase in the tetanic stimulation rate. 9. The decay of post‐tetanic potentiation increases: when [Ca]o is elevated by ionophoretic application during the decay phase only, when ouabain is present in the medium or when [Mg]o is elevated. These finding suggest that calcium, sodium and possibly magnesium take part in post‐tetanic potentiation.

Presynaptic action of the neurosteroid pregnenolone sulfate on inhibitory transmitter release in cultured hippocampal neurons

The effects of the neurosteroid pregnenolone sulfate (PS) were studied in 3- to 9-week-old hippocampal cultures from neonatal rats. Cells were voltage clamped using CsCl filled electrodes, while action potentials and excitatory glutamatergic currents were abolished by superfusing with a combination of tetrodotoxin, 6-cyano-7-nitroquinoxaline (CNQX) and 2-amino-5-phosphonopentanoic acid (AP-5). Under these conditions spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were seen as inward currents at a holding potential of -70 mV. Their amplitude distributions were skewed without clearly detectable peaks. PS at 1-50 microM concentrations decreased the frequency of sIPSCs, with 1 microM being the most effective concentration. The effect appeared after 10-15 min of steroid application and the magnitude of the reduction increased during the early wash period. No recovery of sIPSC frequency was found after 30 min of washing with steroid-free medium. sIPSC amplitudes were not significantly changed at the time the effect of PS on sIPSC frequency was observed. The slow onset of this effect and its duration suggest a novel presynaptic action of the neurosteroid PS on GABAergic inhibition in the mammalian brain.

Ionic basis of tetanic and post-tetanic potentiation at a mammalian neuromuscular junction.

1. The ionic basis of tetanic and post‐tetanic potentiation (TP and PTP) was studied at the rat soleus neuromuscular junction (NMJ), using the miniature endplate potential (MEPP) frequency as an index for transmitter release. Conventional intracellular recording and computer‐assisted data analysis were employed. 2. The experimental results in this study indicate that contrary to previous suggestions, there is a substantial similarity in the ionic basis of TP and PTP at the mammalian and amphibian motor nerve terminals which can be subdivided into [Ca2+]o‐dependent and [Ca2+]o‐independent parts. 3. Tetanic and post‐tetanic increase in MEPP frequency at the rat soleus NMJ is similar to that at the frog NMJ in the following aspects: (i) Tetanic potentiation is substantially larger in calcium‐containing solutions than in calcium‐deficient solutions. About 90% of tetanic potentiation is contributed by extracellular calcium. (ii) Increase in [Mg2+]o reduces tetanic potentiation in calcium‐containing solutions and enhances TP in calcium‐defient solutions. Elevated [Mg2+]o prolongs the post‐tetanic potentiation both in calcium‐containing and in calcium‐deficient solutions. (iii) A post‐tetanic jump in MEPP frequency was observed in 44% of the experiments performed in calcium‐deficient solutions. (iv) The augmentation phase of post‐tetanic potentiation, evident in calcium‐containing solutions, is completely abolished by removal of [Ca2+]o. (v) Tetanic and post‐tetanic potentiations are enhanced by increasing the rate and duration of tetanic stimulation in calcium‐containing solutions. 4. The [Ca2+]o‐independent part of tetanic potentiation is presumably due to entry of sodium ions and their accumulation in the nerve terminal, since it is increased by measures known to inhibit the sodium pump: reduction in [K+]o and partial substitution of sodium by lithium. 5. Sodium ions contribute substantially to the [Ca2+]o‐independent part of posttetanic potentiation, since its duration is markedly prolonged by ouabain, reduction in [K+]o and partial substitution of sodium by lithium. 6. Tetanic potentiation is manifested earlier in calcium‐containing media than in calcium‐deficient media. This difference may indicate that sodium entry into the terminal during tetanic stimulation is at locations remote from the releasing sites. Alternatively, this time difference may be due to the delay between intracellular sodium accumulation and the increase in transmitter release.

Quelling of spontaneous transmitter release by nerve impulses in low extracellular calcium solutions.

1. The effect of nerve stimulation on spontaneous transmitter release was studied at the frog neuromuscular synapse which was bathed in a solution containing very low extracellular calcium concentration. Conventional methods for intracellular and extracellular recording were used and the pattern of quantal liberation following the nerve stimulus was determined. 2. Stimulation of the motor nerve (at rates between 0.09 and 2Hz) caused a reduction in the frequency of the miniature e.p.p.s in comparison to the prestimulation values. 3. The mean distribution of the time of occurrence of the miniature e.p.p.s during the interstimulus period showed periodic oscillations. 4. The quelling effect of nerve stimulation on transmitter release is explained by the hypothesis that a low [Ca]o a reversed electrochemical gradient for calcium occurs and nerve stimulation causes an increased calcium conductance leading to calcium efflux which in turn temporarily reduces [Ca]i and transmitter release.

The revival of the role of the mitochondrion in regulation of transmitter release

The article by David, 1998et al. in this issue of The Journal of Physiology helps to solve a long-standing dilemma regarding the possible role of the mitochondrion in the regulation of transmitter release. Transmitter is released from presynaptic nerve terminals as preformed multimolecular packages or quanta, most probably represented by synaptic vesicles (see Katz, 1969). One of the main determinants of the number of quanta released by the nerve impulse is the free intracellular calcium ion concentration ([Ca2+]i). There are many molecular mechanisms that control the [Ca2+]i. They include ion channels and transporters at the surface membrane and intracellular organelles (Pozzan et al. 1994). More than 20 years ago it was proposed that the mitochondrion is one of the intracellular organelles taking part in intracellular calcium homeostasis in the nerve terminal and is part of the repertoire of cellular processes regulating [Ca2+]i and thus transmitter release (Alnaes & Rahamimoff, 1975). This proposal was based on the abundance of the mitochondria in the nerve terminal (6.59 % of the cross-section of the frog terminal in electron microscopy), on their ability to take up calcium and on the increase in transmitter release when the mitochondria were inhibited; it encountered, however, substantial opposition, based on two indirect observations. First, it was found, using electron-probe microanalysis, that the amount of calcium in synaptosomal and in other mitochondria is low (Blaustein et al. 1980). The second finding was that the apparent KD for calcium uptake into isolated mitochondria is high compared with the presumed levels of [Ca2+]i. Hence it was considered highly unlikely that the mitochondria take a significant part in the physiological regulation of [Ca2+]i and thus of transmitter release. Experiments done in a number of laboratories during the past 5 years indicate that a re- evaluation is probably necessary of the notion that mitochondria play only a minor role in the regulation of [Ca2+]i and thus of transmitter release in physiological conditions. The first set of experiments was done by Pozzan and co-workers (Rizzuto et al. 1995). Using techniques derived from molecular biology, they were able to add a targeting sequence to the calcium indicator aequorin and to direct this molecule into a number of intracellular organelles, including the mitochondria. Thus they were able to measure the mitochondrial [Ca2+] and found that there is a very substantial amount of calcium in the mitochondria in living tissue. Thus the first obstacle to the acceptance of the mitochondrial involvement in the regulation of transmitter release was removed. It now seems that the mitochondria are important members of an intracellular calcium network, which co-ordinates calcium controlling organelles (Babcock et al. 1997). The excitability of the mitochondria makes them highly suitable for signalling (Ichas et al. 1997). The elegant article by David, 1998et al in this issue of The Journal of Physiology provides direct proof of the role of mitochondria in the regulation of [Ca2+]i. They used the very powerful technique of laser scanning confocal microscopy of the presynaptic nerve terminals of the lizard neuromuscular junction, and different calcium indicators that report separately the changes in [Ca2+] in the cytosol and in the mitochondria. They made three very important findings. First, they found that repetitive nerve stimulation causes an increase in [Ca2+]i in the cytosol and, after some delay, in the mitochondria. It was sufficient to give only twenty-five or fifty action potentials to detect a significant increase in mitochondrial [Ca2+]. This was achieved after a very small increase in the cytosolic [Ca2+]i of about 200 nm. The second finding was that, after the end of the stimulation, there was a gradual decline in [Ca2+]i. The decay in the mitochondrial [Ca2+] was much slower than that of the cytosolic [Ca2+]i, making it a possible source of calcium in frequency modulation of transmitter release. The third finding was that inhibition of the calcium uptake by the mitochondria caused a much larger increase in the cytosolic [Ca2+]i after stimulation, which may correspond to the observed increase in transmitter release. Taken together, these findings show in a very convincing way that the mitochondrion is one of the [Ca2+]i regulators in the nerve terminals in vivo and it may take part in the control of neurosecretion. It may also provide an important link between cellular metabolism and synaptic transmission. It will be of interest to see whether the reported periodic oscillations in the nerve terminal [Ca2+]i (Melamed et al. 1993) and in transmitter release (Meiri & Rahamimoff, 1978) are related to the periodic oscillations found in mitochondrial [Ca2+] (Hajnoczky, Robb-Gaspers, Seitz & Thomas, 1995). One of the main features of the nervous system is its tremendous plasticity, achieved mainly by modulation of synaptic transmission. There is a growing body of evidence that the mitochondria make an important contribution to this plasticity by altering calcium signals and transmitter release. They seem to participate in short term frequency modulation such as post-tetanic potentiation (Tang & Zucker, 1997) and perhaps also in longer term phenomena due to the ability of NO to release calcium from the mitochondria (Richter et al. 1997).

The non-specific ion channel in Torpedo ocellata fused synaptic vesicles.

1. Synaptic vesicles were isolated and fused into large structures with a diameter of more than 20 microns to characterize their ionic channels. The ‘cell’‐attached and inside‐out configurations of the patch clamp technique were used. 2. Two types of ion channels were most frequently observed: a low conductance chloride channel and a high conductance non‐specific channel. 3. The non‐specific channel has a main conducting state and a substate. The main conducting state has a slope conductance of 246 +/‐ 15 pS (+/‐ S.E.M., n = 15), in the presence of different combinations of KCl and potassium glutamate. 4. From the reversal potentials of the current‐voltage (I‐V) relation, it was concluded that this channel conducts both Cl‐ and K+. 5. The non‐specific channel is highly voltage dependent: under steady‐state voltages it has a high open probability near 0 mV and does not inactivate; when the membrane is hyperpolarized (pipette side more positive), the open probability decreases dramatically. 6. Voltage pulses showed that upon hyperpolarization (from holding potentials between ‐20 and + 20 mV), the channels deactivated; when the membrane was stepped back to the holding potential, the channels reactivated rapidly. 7. In a number of experiments, when the pipette side was made more negative than the bath, the open probability also decreased. 8. Frequently, a substate with a conductance of about 44 +/‐ 4% (+/‐ S.E.M., n = 3) of the main state was detected. 9. We speculate that this non‐specific ion channel may have different roles at the various stages of the life cycle of the synaptic vesicle. When the synaptic vesicle is an intracellular structure, it might help its transmitter‐concentrating capacity by dissipating the polarization. After fusion with the surface membrane, it might constitute an additional conductance pathway, taking part in frequency modulation of synaptic transmission.

Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non‐specific ion channel in the synaptic vesicle membrane, in the context of the post‐fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non‐specific ion channel was examined using the fused vesicle‐attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non‐specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

A bursting potassium channel in isolated cholinergic synaptosomes of Torpedo electric organ.

1. Pinched‐off cholinergic nerve terminals (synaptosomes) prepared from the electric organ of Torpedo ocelata were fused into large structures (greater than 20 microns) using dimethyl sulphoxide and polyethylene glycol 1500, as previously described for synaptic vesicles from the same organ. 2. The giant fused synaptosomes were easily amenable to the patch clamp technique and 293 seals with a resistance greater than 4 G omega were obtained in the ‘cell‐attached’ configuration. In a large fraction of the experiments, an ‘inside‐out’ patch configuration was achieved. 3. Several types of unitary ionic currents were observed. This study describes the most frequently observed single‐channel activity which was found in 247 out of the 293 membrane patches (84.3%). 4. The single‐channel current‐voltage relation was linear between ‐60 and 20 mV and showed a slope conductance of 23.8 +/‐ 1.3 pS when the pipette contained 350‐390 mM‐Na+ and the bath facing the inside of the synaptosomal membrane contained 390 mM‐K+. 5. From extrapolated reversal potential measurements, it was concluded that this channel has a large selectivity for K+ over Na+ (70.4 +/‐ 11.5, mean +/‐ S.E.M.). Chloride ions are not transported significantly through this potassium channel. 6. This potassium channel has a low probability of opening. The probability of being in the open state increases upon depolarization and reaches about 1% when the inside of the patch is 20 mV positive compared to the pipette side. 7. The mean channel open time increases with depolarization; thus the product current x time (= charge) also increases upon depolarization, showing properties of an outward rectifier. 8. The potassium channel in the giant synaptosome membrane has a bursting behaviour. Open‐time distribution, closed‐time distribution and a Poisson analysis indicate that the minimal kinetic scheme requires one open state and three closed states.

Regulation of Acetylcholine Liberation from Presynaptic Nerve Terminals1

Acetylcholine is liberated from motor nerve terminals either as a molecular leakage or as quantal packages; the latter form of release is responsible for signaling across the neuromuscular synapse. Three main factors determine the number of quanta liberated by the nerve impulse: the degree of presynaptic depolarisation, the frequency of activation of the nerve terminal, and calcium ion concentration in the extracellular medium. These factors seem to act yb changing the free calcium ion concentration [Ca]in in the presynaptic nerve terminal. Thus, processes that change [Ca]in will determine efficiency of synaptic transmission. These processes include fluxes of calcium ions across the presynaptic membrane and reversible translocation by intracellular organelles such as mitochondria, vesicles and soluble molecules. The level of intracellular [Ca] can be changed by ion-containing liposomes. One of the main physiological determinants of the level of transmitter release is potentiation, where the increase in transmitter release is caused by transmembranal processes and intracellular translocation.