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R S Zucker

Publications and source records attributed to R S Zucker.

13 recordsLinked to original sources
PubMed1992

Monensin can transport calcium across cell membranes in a sodium independent fashion in the crayfish Procambarus clarkii.

Monensin, a Na(+)-selective ionophore, enhances transmitter release when applied to crustacean and frog neuromuscular junctions. Monensin is believed to raise intracellular sodium ([Na+]i) which in turn elevates intracellular calcium ([Ca2+]i). Using the fluorescent indicator fura-2, we measured [Ca2+]i in crayfish Procambarus clarkii presynaptic terminals during monensin application in normal Ringer, zero-calcium Ringer and zero-sodium Ringer to determine if [Ca2+]i increases with monensin application and if so by what mechanism. In normal Ringer, monensin, 10 microM and 100 microM, elevated [Ca2+]i by 440 nM and 7 microM respectively. This rise in [Ca2+]i was dependent on external calcium, as [Ca2+]i did not increase in zero-calcium Ringer. However, in a zero-sodium Ringer, monensin (10 microM) elevated [Ca2+]i by 370 nM. It is important to recognize that monensin, thought to be a sodium-selective ionophore, can transport calcium across the cytoplasmic membrane in a sodium-independent manner.

Animals↗
PubMed1992

Effects of photolabile calcium chelators on fluorescent calcium indicators.

The fluorescence properties of the calcium indicators Fura-2 and Fluo-3 have been investigated in the presence of the 'caged calcium' photolabile chelators Nitr-5 and DM-nitrophen. The excitation spectra of dilute solutions of these indicators was distorted by the presence of photolabile chelators, owing to differential absorbance of excitation light by the chelators, as well as calcium-dependent fluorescence of the chelators themselves. This distortion was altered on partial photolysis of the chelators, due to changes in their absorbance and fluorescence. At high concentrations of indicators (100 microM) and photolabile chelators (10 mM), similar to those used experimentally, DM-nitrophen quenched the fluorescence of Fluo-3 at low calcium concentrations. The results suggest that Fura-2 may be used with either chelator, and Fluo-3 with Nitr-5, to measure calcium released on photolysis of the caged compounds, but that careful calibration of the chelator-indicator mixture after the appropriate degree of photolysis is necessary.

Acetates↗
PubMed1992

Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation.

The induction of long-term potentiation (LTP) in hippocampal CA1 pyramidal cells requires a rise in postsynaptic intracellular Ca2+ concentration ([Ca2+]i). To determine the time for which Ca2+ must remain elevated to induce LTP, the photolabile Ca2+ buffer diazo-4 was used to limit the duration of the rise in postsynaptic [Ca2+]i following a tetanus. The affinity of diazo-4 for Ca2+ increases approximately 1600-fold upon flash photolysis, permitting almost instantaneous buffering of [Ca2+]i without disturbing resting [Ca2+]i prior to the flash. Photolysis of diazo-4 1 s following the start of the tetanus blocked LTP, while delaying photolysis for more than 2 s had no discernible effect on LTP. Photolyzing diazo-4 at intermediate delays (1.5-2 s) or reducing photolysis of diazo-4 often resulted in short-term potentiation (STP). These results indicate that a tetanus-induced rise in postsynaptic [Ca2+]i lasting at most 2-2.5 s is sufficient to generate LTP. Smaller increases or shorter duration rises in [Ca2+]i may result in STP.

Action Potentials↗
PubMed1992

Time course of transmitter release calculated from simulations of a calcium diffusion model.

A three-dimensional presynaptic calcium diffusion model developed to account for characteristics of transmitter release was modified to provide for binding of calcium to a receptor and subsequent triggering of exocytosis. When low affinity (20 microM) and rapid kinetics were assumed for the calcium receptor triggering exocytosis, and stimulus parameters were selected to match those of experiments, the simulations predicted a virtual invariance of the time course of transmitter release to paired stimulation, stimulation with pulses of different amplitude, and stimulation in different calcium solutions. The large temperature sensitivity of experimental release time course was explained by a temperature sensitivity of the model's final rate limiting exocytotic process. Inclusion of calcium tail currents and a saturable buffer with finite binding kinetics resulted in high peak calcium transients near release sites, exceeding 100 microM. Models with a single class of calcium binding site to the secretory trigger molecule failed to produce sufficient synaptic facilitation under this condition. When at least one calcium ion binds to a different site having higher affinity and slow kinetics, facilitation again reaches levels similar to those seen experimentally. It is possible that the neurosecretory trigger molecule reacts with calcium at more than one class of binding site.

Animals↗
PubMed1992

Posttetanic potentiation at the crayfish neuromuscular junction is dependent on both intracellular calcium and sodium ion accumulation.

The fluorescent indicator fura-2 was used to measure cytoplasmic calcium in presynaptic terminals in the crayfish Procambarus clarkii under conditions that raise intracellular sodium to examine whether sodium can elevate intracellular calcium concentration ([Ca2+]i) or prolong its efflux and thus influence the magnitude and duration of posttetanic potentiation (PTP). Sodium was elevated in presynaptic terminals at rest by either (1) injection of sodium into the excitatory axon, (2) application of veratridine to open sodium channels, or (3) addition of ouabain to block Na/K exchange, with [Ca2+]i increasing by either 430, 400, or 180 nM, respectively. Intracellular calcium concentration increased only when external calcium was present, indicating that calcium influx occurred through Na/Ca exchange. Additionally, ouabain enhanced excitatory junctional potentials (EJPs) eightfold. Elevation of sodium using a high-frequency stimulation in zero-calcium Ringer's did not elevate [Ca2+]i during the train or immediately afterward when calcium-containing Ringer's was re-introduced. This indicates that a physiological sodium load does not release calcium from internal stores or reverse Na/Ca exchange to levels where [Ca2+]i accumulation is detectable. We examined the ability of sodium to interfere with calcium efflux from presynaptic terminals by loading boutons with both sodium and calcium or calcium alone using high-potassium depolarization. Elevation of internal sodium slowed calcium efflux from the terminal (12.3 min) compared to calcium removal without a sodium load (4.0 min). When sodium loading was increased during a tetanus by application of ouabain, the time constants for decay of EJP potentiation, 17.3 min, and for [Ca2+]i, 35 min, were longer than control values, 4.4 min and 5.8 min, respectively. In addition, using lithium to inhibit the efflux of calcium by Na/Ca exchange following a PTP-inducing train also lengthened the decay of [Ca2+]i to 15.7 min. Intracellular sodium accumulation in presynaptic terminals slows the efflux of calcium through Na/Ca exchange, and may therefore augment and prolong PTP.

Animals↗
PubMed1991

Action potentials must admit calcium to evoke transmitter release.

There are two hypotheses to explain how neurons release transmitter. The calcium hypothesis proposes that membrane depolarization is necessary only for opening calcium channels and increasing internal calcium concentration ([Ca2+]i) near membrane transmitter-release sites. These calcium ions trigger a transient release of neurotransmitter. The calcium-voltage hypothesis postulates that voltage induces a conformational change in a membrane protein rendering it sensitive to calcium such that, in the presence of high [Ca2+]i, depolarization directly triggers transmitter release. Here we report that when calcium influx is blocked by cobalt or manganese ions in a calcium-free Ringer, as measured with Fura-2, and [Ca2+]i is elevated by liberation from a caged calcium compound, transmitter release at the crayfish neuromuscular junction is unaffected by presynaptic action potentials. These results support the calcium hypothesis.

Action Potentials↗
PubMed1991

Presynaptic calcium in transmitter release and posttetanic potentiation.

This review gives some indication of the progress that has been made in understanding synaptic transmission by use of new methods for measuring and controlling presynaptic [Ca2+]i. Many unsolved problems remain. We still do not have a clear idea of the exact relationship between [Ca2+]i and transmitter release and whether this relationship is the same under all circumstances. The apparently different [Ca2+]i-dependence of evoked transmitter release and of PTP suggest multiple molecular sites of calcium action that remain to be identified. A complete and comprehensive model of transmitter release has yet to be devised, and questions raised by our experiments may indicate that it is still too early to try to construct a precise model. We also do not know just how serotonin acts to modulate transmitter release, only that it does not appear to alter either resting or entering calcium. Some of these questions may be approachable with the techniques described here; others are not and require different methods for their resolution. The work continues.

Action Potentials↗
PubMed1991

Presynaptic calcium and serotonin-mediated enhancement of transmitter release at crayfish neuromuscular junction.

Fura-2 microfluorometric measurements of calcium concentrations in excitor motor nerve terminals of the first walking leg of crayfish Procambrus clarkii were made during application of serotonin (5-HT) to examine whether changes in residual calcium concentration or calcium influx are responsible for the 5-HT-induced increase in transmitter release at this synapse. Calcium concentrations and EPSPs were monitored simultaneously during 0.5-Hz and moderate-frequency (3-16 Hz) stimulation before, during, and after a 5-min application of 5-HT at concentrations between 5 and 100 microM. We did not observe a significant increase in resting calcium concentration [Ca2+]i in presynaptic terminals during or after application of 5-HT despite simultaneously recorded excitatory junction potential (EJP) enhancement. Injection of the calcium buffer EGTA to concentrations sufficient to reduce significantly the buildup of frequency-dependent facilitation during trains of action potentials did not reduce 5-HT-mediated EJP enhancement. We therefore conclude that 5-HT does not release calcium from intracellular stores to an extent sufficient to overcome calcium buffering and removal systems. Spontaneous release of transmitter quanta was increased by 5-HT to the same extent in normal and increased-osmolarity saline in the absence of measured changes in [Ca2+]i. Thus, changes in residual calcium cannot explain 5-HT-induced synaptic enhancement. Increasing the action-potential-mediated calcium influx by broadening the spike or by increasing extracellular calcium increased evoked transmitter release and increased the accumulation of calcium during trains of action potentials. However, though 5-HT produced a comparable synaptic enhancement, we found no evidence for increased calcium accumulation during trains of action potentials, suggesting that net calcium influx, buffering, and removal are not affected by 5-HT. Therefore, we conclude that 5-HT's action does not result from effects on calcium metabolism and suggest that it acts on the neurosecretory apparatus to increase the effectiveness of calcium for stimulating release. Furthermore, based on its relatively constant effects on facilitated and unfacilitated action-potential-evoked release and spontaneous transmitter release, we conclude that 5-HT actions are independent of the concentration of calcium that is driving release.

Action Potentials↗
PubMed1979

Effect of TEA on light emission from aequorin-injected aplysia central neurons.

Aplysia central neurons were injected with the calcium-sensitive photoprotein aequorin and stimulated with trains of identical depolarizing voltage-clamp pulses. The light emissions grew and the outward currents declined in successive pulses. Tetraethylammonium (TEA) enhanced the light emissions to single depolarizing pulses and suppressed the outward current. The remaining net inward current is carried primarily by calcium ions and does not facilitate. The aequorin emissions were larger at all amplitudes of depolarizing pulses that elicited emissions, and the facilitation of emissions in a train of pulses was reduced. The effect of TEA on outward current was nearly maximal when sodium ions were partially replaced with 0.1 M TEA, while the aequorin emissions were further enhanced by increasing the TEA concentration to 0.459 M. TEA enhanced the aequorin emissions at all voltages. These observations suggest that the action of TEA on aequorin emissions is not strictly a consequence of its better known outward current blocking action. The effects of TEA could be partly due to the lowered sodium concentration of these solutions. Replacement of sodium by Tris, sucrose or mannose, however, all produced no enhancement of emissions. Tetramethylammonium (TMA) replacement of sodium had effects similar to those of TEA. Thus TEA and TMA appear to have a specific effect. Part of the enhancement of light emissions by TEA is due to the removal of a series resistance error in the voltage clamp, and this may also account partly for the reduced facilitation of aequorin emissions in TEA. The remainder of the action of TEA on aequorin emissions evidently reflects a specific but previously unrecognized action on the cellular metabolism of calcium ions or on the voltage-dependent calcium channels.

Aequorin↗
PubMed1978

Prevention of the cortical reaction in fertilized sea urchin eggs by injection of calcium-chelating ligands.

Eggs from the sea urchin, Lytechinus pictus, were injected with either EGTA or EDTA, and were subsequently fertilized. EGTA prevented cortical vesicle discharge and formation of the fertilization membrane. EDTA had either no effect, or sometimes retarded the elevation of the fertilization membrane, or reduced the percentage of eggs with elevated membranes. Theoretical considerations lead to estimates of the probable effects of EGTA and EDTA on the internally released calcium which triggers the cortical reaction. Whether or not cytoplasmic calcium buffers are considered, it is concluded: (1) that normally several times the threshold calcium concentration for the cortical reaction is released into a subsurface space; (2) that if a rapidly-equilibrating high-affinity buffer is present, it is locally saturated by the calcium released internally; (3) the injected EDTA reduces the subsurface free calcium concentration normally reached to approximately threshold for the cortical reaction, while injected EGTA reduces the calcium concentration to below this threshold; and (4) a rise in the internal ionic calcium concentration is a necessary step in the activation of the cortical reaction at fertilization.

Animals↗