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R W Tsien

Publications and source records attributed to R W Tsien.

At least 19 recordsLinked to original sources
PubMed1992

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1992

Persistent signalling and changes in presynaptic function in long-term potentiation.

Long-term potentiation (LTP) is an example of a persistent change in synaptic function in the mammalian brain, thought to be essential for learning and memory. At the synapse between hippocampal CA3 and CA1 neurons LTP is induced by a Ca2+ influx through glutamate receptors of the NMDA (N-methyl-D-aspartate) type (see Collingridge et al 1992, this volume). How does a rise in [Ca2+]i lead to enhancement of synaptic function? We have tested the popular hypothesis that Ca2+ acts via a Ca(2+)-dependent protein kinase. We found that long-lasting synaptic enhancement was prevented by prior intracellular injection of potent and selective inhibitory peptide blockers of either protein kinase C (PKC) or Ca2+/calmodulin-dependent protein kinase II (CaMKII), such as PKC(19-31) or CaMKII(273-302), but not by control peptides. Evidently, activity of both PKC and CaMKII is somehow necessary for the postsynaptic induction of LTP. To determine if these kinases are also involved in the expression of LTP, we impaled cells with microelectrodes containing protein kinase inhibitors after LTP had already been induced. Strikingly, established LTP was not suppressed by a combination of PKC and CaMKII blocking peptides, or by intracellular postsynaptic H-7. However, established LTP remained sensitive to bath application of H-7. Thus, the persistent signal may be a persistent kinase, but if so, the kinase cannot be accessed within the postsynaptic cell. Evidence for a presynaptic locus of expression comes from our studies of quantal synaptic transmission under whole-cell voltage clamp. We find changes in synaptic variability expected to result from enhanced presynaptic transmitter release, but little or no increase in quantal size. Furthermore, miniature synaptic currents in hippocampal cultures are increased in frequency but not amplitude as a result of a glutamate-driven postsynaptic induction. The combination of postsynaptic induction and presynaptic expression necessitates a retrograde signal from the postsynaptic cell to the presynaptic terminal.

Animals↗
PubMed1992

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.

Animals↗
PubMed1992

A caffeine- and ryanodine-sensitive Ca2+ store in bullfrog sympathetic neurones modulates effects of Ca2+ entry on [Ca2+]i.

1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]i) produced by voltage-dependent Ca2+ entry are influenced by a caffeine-sensitive Ca2+ store in bullfrog sympathetic neurones. Ca2+ influx was elicited by K+ depolarization and the store was manipulated with either caffeine or ryanodine. 2. For a time after discharging the store with caffeine and switching to a caffeine-free medium: (a) [Ca2+]i was depressed by up to 40-50 nM below the resting level, (b) caffeine responsiveness was diminished, and (c) brief K+ applications elicited [Ca2+]i responses with slower onset and faster recovery than controls. These effects were more pronounced as the conditioning caffeine concentration was increased over the range 1-30 mM. 3. [Ca2+]i, caffeine and K+ responsiveness recovered in parallel with a half-time of approximately 2 min. Recovery required external Ca2+ and was speeded by increasing the availability of cytosolic Ca2+, suggesting that it reflected replenishment of the store at the expense of cytosolic Ca2+. 4. During recovery, Ca2+ entry stimulated by depolarization had the least effect on [Ca2+]i when the store was filling most rapidly. This suggests that the effect of Ca2+ entry on [Ca2+]i is modified, at least in part, because some of the Ca2+ which enters the cytosol during stimulation is taken up by the store as it refills. 5. Further experiments were carried out to investigate whether the store can also release Ca2+ in response to stimulated Ca2+ entry. In the continued presence of caffeine at a low concentration (1 mM), high K+ elicited a faster and larger [Ca2+]i response compared to controls; at higher concentrations of caffeine (10 and 30 mM) responses were depressed. 6. Ryanodine (1 microM) reduced the rate at which [Ca2+]i increased with Ca2+ entry, but not to the degree observed after discharging the store. At this concentration, ryanodine completely blocked responses to caffeine but had no detectable effect on Ca2+ channel current or the steady [Ca2+]i level achieved during depolarization. 7. We propose that, depending on its Ca2+ content, the caffeine-sensitive store can either attenuate or potentiate responses to depolarization. When depleted and in the process of refilling, the store reduces the impact of Ca2+ entry as some of the Ca2+ entering the cytosol during stimulation is captured by the store.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

omega-Conotoxin GVIA receptors of Discopyge electric organ. Characterization of omega-conotoxin binding to the nicotinic acetylcholine receptor.

A peptide toxin from a Conus marine snail, omega-conotoxin GVIA (omega-CgTx) has been used extensively as a probe for certain types of neuronal calcium channels. It is often assumed that omega-CgTx interacts with Ca2+ channels exclusively. We have tested this assumption in a study of omega-CgTx-binding sites in the electric organ of Discopyge ommata. Synaptosomal membranes from this tissue contain low affinity omega-CgTx receptor sites (Kd = 0.6 microM) in great abundance (280 pmol/mg of protein), as first reported by Ahmad and Miljanich (Ahmad, S. N., and Miljanich, G.P. (1988) Brain Res. 453, 247-256). However, we find that a large majority of these omega-CgTx-binding sites co-purify with the nicotinic acetylcholine receptor (nAChR) and can be immunoprecipitated by monoclonal antibodies generated against the nAChR of Torpedo. Cross-linking experiments with radiolabeled omega-CgTx show pronounced specific labeling of the alpha-subunit of the nAChR but not other subunits. Specific omega-CgTx binding to the nAChR is reduced by millimolar Ca2+ but not by alpha- or kappa-bungarotoxin, alpha-conotoxin, or carbamylcholine. Cross-linking experiments also reveal omega-CgTx-binding proteins of 170 and 60 kDa. The characteristics of the 170-kDa protein make it a likely candidate for the alpha 1-subunit of an N-type Ca2+ channel.

Affinity Labels↗
PubMed1991

Molecular diversity of voltage-dependent Ca2+ channels.

Voltage-dependent Ca2+ channels regulate Ca2+ entry and thereby contribute to Ca2+ signalling in many cells. Functional studies have uncovered several types of Ca2+ channel, distinguished by pharmacology, electrophysiology and tissue localization. More recently, molecular cloning has revealed an even greater diversity among Ca2+ channels, arising from multiple genes and alternative splicing. L-type, dihydropyridine-sensitive Ca2+ channels have been the most extensively characterized to date. Recently, Numa's group has reported the cloning and expression of a dihydropyridine-insensitive Ca2+ channel from brain that most closely resembles the P-type channel described by Llinas and colleagues. These results contribute to rapidly growing knowledge about molecular determinants of Ca2+ channel diversity.

Amino Acid Sequence↗
PubMed1991

Glutamate-induced increases in intracellular Ca2+ in cultured frog tectal cells mediated by direct activation of NMDA receptor channels.

Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels.

2-Amino-5-phosphonovalerate↗
PubMed1990

Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices.

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model system for understanding the cellular mechanisms of memory. In region CA1, LTP is triggered postsynaptically by Ca2(+)-dependent activation of protein kinases, but the locus of persistent modification remains controversial. Statistical analysis of synaptic variability has been proposed as a means of settling this debate, although a major obstacle has been the poor signal-to-noise ratio of conventional intracellular recordings. We have applied the whole-cell voltage clamp technique to study synaptic transmission in conventional hippocampal slices (compare refs 28-30). Here we report that robust LTP can be recorded with much improved signal resolution and biochemical access to the postsynaptic cell. Prolonged dialysis of the postsynaptic cell blocks the triggering of LTP, with no effect on expression of LTP. The improved signal resolution unmasks a large trial-to-trial variability, reflecting the probabilistic nature of transmitter release. Changes in the synaptic variability, and a decrease in the proportion of synaptic failures during LTP, suggest that transmitter release is significantly enhanced.

Animals↗
PubMed1990

Inhibition of Ca2+ and K+ channels in sympathetic neurons by neuropeptides and other ganglionic transmitters.

Neuropeptides are known to modulate the excitability of frog sympathetic neurons by inhibiting the M-current and increasing the leak current, but their effects on Ca2+ channels are poorly understood. We compared effects of LHRH, substance P, epinephrine, and muscarine on Ca2+, K+, and leak currents in dissociated frog sympathetic neurons. At concentrations that inhibit M-current, LHRH and substance P strongly reduced N-type Ca2+ current and induced a leak conductance that may contribute to slow EPSPs. In contrast, muscarine produced little reduction of Ca2+ current, even in cells in which it strongly suppressed the M-current. We find that peptidergic inhibition of Ca2+ channels involves G proteins, but does not require protein kinases. In addition, it leads to reductions in Ca2(+)-activated K+ current and catecholamine release.

Animals↗
PubMed1989

Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP.

Long-term potentiation (LTP) of synaptic transmission is a widely studied cellular example of synaptic plasticity. However, the identity, localization, and interplay among the biochemical signals underlying LTP remain unclear. Intracellular microelectrodes have been used to record synaptic potentials and deliver protein kinase inhibitors to postsynaptic CA1 pyramidal cells. Induction of LTP is blocked by intracellular delivery of H-7, a general protein kinase inhibitor, or PKC(19-31), a selective protein kinase C (PKC) inhibitor, or CaMKII(273-302), a selective inhibitor of the multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII). After its establishment, LTP appears unresponsive to postsynaptic H-7, although it remains sensitive to externally applied H-7. Thus both postsynaptic PKC and CaMKII are required for the induction of LTP and a presynaptic protein kinase appears to be necessary for the expression of LTP.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Alpha-adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating.

In sympathetic neurons, catecholamines interact with prejunctional alpha-adrenergic receptors to reduce delivery of transmitter to postjunctional target organs. This autoinhibitory feedback is a general phenomenon seen in diverse neurons containing a variety of transmitters. The underlying mechanisms of alpha-adrenergic inhibition are not clear, although decreases in cyclic AMP and cAMP-mediated phosphorylation have been implicated. We have studied depolarization-induced catecholamine release and calcium-channel currents in frog sympathetic neurons. Here we show that alpha-adrenergic inhibition of transmitter release can be explained by inhibition of Ca2+-channel currents and not by modulation of intracellular proteins. Noradrenaline strongly reduces the activity of N-type Ca2+ channels, the dominant calcium entry pathway triggering sympathetic transmitter release, whereas L-type Ca2+ channels are not significantly inhibited. The down-modulation of N-type channels involves changes in rapid gating kinetics but not in unitary flux. This is the first detailed description of inhibition of a high-voltage activated neuronal Ca2+ channel at the single-channel level. The coupling between alpha-adrenergic receptors and N-type channels involves a G protein, but not a readily diffusible cytoplasmic messenger or protein kinase C, and may be well suited for rapid and spatially localized feedback-control of transmitter release.

Animals↗
PubMed1989

Voltage-gated calcium channels: direct observation of the anomalous mole fraction effect at the single-channel level.

Voltage-gated Ca channels are very efficient pores: even while exhibiting strong ionic selectivity, they are highly permeant to divalent cations. Studies of the mechanism of selectivity and ion permeation have demonstrated that whole-cell Ca channel current in mixtures of Ca and Ba ions can be smaller than with equimolar concentrations of either ion alone. This anomalous mole fraction effect (AMFE) has provided an important impetus for proposed mechanisms of ion selectivity and permeation that invoke multiple ion binding sites. However, recordings of unitary L-type Ca currents did not demonstrate the AMFE [Marban, E. & Yue, D.T. (1988) Biophys. J. 55, 594a (abstr.)], raising doubts about whether it is an expression of ion permeation through open Ca channels. We have made patch-clamp recordings from single L-type Ca channels in PC-12 pheochromocytoma cells. Our results demonstrate a significant AMFE at the single-channel level but also indicate that the AMFE can only be found under restrictive conditions of permeant ion concentration and membrane potential. While the AMFE is clear at 0 mV when permeant ions are present at 10 mM, it is not evident when the divalent cation concentration is increased to 110 mM or the membrane potential is hyperpolarized to -40 mV. We compared our experimental observations with predictions of a single-file, two-binding-site model of the Ca channel. The model accounts for our experimental results. It predicts an AMFE under conditions that favor ion-ion interactions, as long as the outer binding site is not saturated due to high permeant ion concentration or negative membrane potential.

Adrenal Gland Neoplasms↗