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W G Regehr

Publications and source records attributed to W G Regehr.

At least 37 records · Page 2Linked to original sources
PubMed1997

The mechanism of cAMP-mediated enhancement at a cerebellar synapse.

Increases in cAMP have been shown previously to enhance the strength of the granule cell to Purkinje cell synapse. We have examined the mechanisms underlying this enhancement in rat cerebellar brain slices. Elevation of cAMP levels by forskolin increased synaptic currents in a dose-dependent manner. Fluorometric calcium measurements revealed that forskolin did not affect presynaptic calcium influx or resting calcium levels. The waveform of the presynaptic volley was also unaltered, indicating that changes in the presynaptic action potential did not contribute to synaptic enhancement. However, forskolin enhanced the frequency but not the size of spontaneous miniature EPSCs. There was a one-to-one correspondence between increases of spontaneous and evoked neurotransmitter release. These results suggest that forskolin increases release at this synapse via presynaptic mechanisms that do not alter calcium influx. The effect of forskolin on paired-pulse facilitation was examined to assess the relative contributions of changes in the probability of release (p) and changes in the number of functional release sites (n) to this form of enhancement. These experiments suggest that although small changes in n cannot be excluded, most of the enhancement arises from increases in p.

Action Potentials↗
PubMed1997

Control of neurotransmitter release by presynaptic waveform at the granule cell to Purkinje cell synapse.

The effect of changes in the shape of the presynaptic action potential on neurotransmission was examined at synapses between granule and Purkinje cells in slices from the rat cerebellum. Low concentrations of tetraethylammonium were used to broaden the presynaptic action potential. The presynaptic waveform was monitored with voltage-sensitive dyes, the time course and amplitude of presynaptic calcium entry were determined with fluorescent calcium indicators, and EPSCs were measured with a whole-cell voltage clamp. Spike broadening increased calcium influx primarily by prolonging calcium entry without greatly affecting peak presynaptic calcium currents, indicating that the majority of calcium channels reach maximal probability of opening in response to a single action potential and that spike broadening increases the open time of these channels. EPSCs were exquisitely sensitive to elevations of calcium influx produced by spike broadening; there was a high power relationship between calcium influx and release such that a 23% increase in spike width led to a 25% increase in total calcium influx, which in turn doubled synaptic strength. The finding that even small changes in spike width influence neurotransmitter release suggests that altering the presynaptic waveform may be an important means of modifying the strength of this synapse. Waveform changes do not, however, contribute significantly to presynaptic modulation via activation of adenosine A1 or GABAB receptors. Furthermore, greatly reducing presynaptic calcium influx did not alter the presynaptic waveform, indicating that calcium channels and calcium-activated channels do not participate in shaping the presynaptic waveform.

Action Potentials↗
PubMed1997

Interplay between sodium and calcium dynamics in granule cell presynaptic terminals.

Fluorescent indicators were used to detect stimulus-evoked changes in presynaptic levels of intracellular sodium (Na(i)) and calcium (Ca(i)) in granule cell parallel fibers in brain slices from rat cerebellum. Ca(i) increased during stimulation, and three exponentials were needed to approximate its return to prestimulus levels. Ca(i) decayed to approximately 10% of peak levels with tau approximately 100 ms, to approximately 1% of peak values with tau approximately 6 s, and then returned to prestimulus levels with tau approximately 1-2 min. After stimulation, Na(i) accumulated in two phases; one rapid, the other continuing for several hundred milliseconds. The return of Na(i) to prestimulus levels was well approximated by a double exponential decay with time constants of 6-17 s and 2-3 min. Manipulations that prevented calcium entry eliminated both the slow component of sodium entry and the rapid component of Na(i) decay. Reductions of extracellular sodium slowed the rapid phase of Ca(i) decay. These Ca(i) and Na(i) transients were well described by a model in which the plasma membrane of presynaptic boutons contained both a sodium/calcium exchanger and a calcium ATPase (Ca-ATPase). According to this model, immediately after stimulation the sodium/calcium exchanger removes calcium from the terminal more rapidly than does the Ca-ATPase. Eventually, the large concomitant sodium influx brings the exchanger into steady-state, leaving only the Ca-ATPase to remove calcium. This perturbs the equilibrium of the sodium/calcium exchanger, which opposes the Ca-ATPase, leading to a slow return of Ca(i) and Na(i) to resting levels.

Animals↗
PubMed1996

Neuronal firing: does function follow form?

Neurons generate diverse firing patterns to perform a range of specialized tasks. Experiments show that many features of these firing patterns arise from distinctive membrane properties, but theoretical work predicts that differences in neuronal morphology are also important.

Animals↗
PubMed1996

Timing of neurotransmission at fast synapses in the mammalian brain.

Understanding the factors controlling synaptic delays has broad implications. On a systems level, the speed of synaptic transmission limits the communication rate between neurons and strongly influences local circuit dynamics. On a molecular level, the delay from presynaptic calcium entry to postsynaptic responses constrains the molecular mechanism of vesicle fusion. Previously it has not been possible to elucidate the determinants of synaptic delays in the mammalian central nervous system, where presynaptic terminals are small and difficult to study. We have developed a new approach to study timing at rat cerebellar synapses: we used optical techniques to measure voltage and calcium current simultaneously from presynaptic boutons while monitoring postsynaptic currents electrically. Here we report that the classic view that vesicle release is driven by calcium entry during action-potential repolarization holds for these synapses at room temperature, but not at physiological temperatures, where postsynaptic responses commence just 150 micros after the start of the presynaptic action potential. This brisk communication is a consequence of rapid calcium-channel kinetics, which allow significant calcium entry during the upstroke of the presynaptic action potential, and extremely fast calcium-driven vesicle fusion, which lags behind calcium influx by 60 micros.

Action Potentials↗
PubMed1996

Determinants of the time course of facilitation at the granule cell to Purkinje cell synapse.

Short-term facilitation is a widely observed form of synaptic enhancement that is not well understood. Although presynaptic calcium has long been implicated in this process, its role is unclear, particularly at synapses in the mammalian brain. We tested the role of presynaptic residual free calcium ([Ca]res) in facilitation of synapses between granule cells and Purkinje cells in rat cerebellar slices. Paired-pulse facilitation of synaptic currents resulted in an approximately 2.5-fold enhancement that decayed with a time constant of approximately 200 msec, as assessed by voltage-clamp recordings. Measurements of [Ca]res using fluorescent calcium-sensitive indicators revealed that [Ca]res decayed more rapidly than did facilitation. Manipulation of [Ca]res dynamics by introducing EGTA into presynaptic terminals sped the decays of [Ca]res and facilitation in a dose-dependent manner. When [Ca]res was reduced to a brief impulse lasting several milliseconds, facilitation was still present, although reduced in amplitude and duration. Facilitation decayed with an intrinsic time constant of approximately 40 msec. These results suggest that facilitation at this synapse is produced by a calcium-driven process with a high affinity and a slow effective off-rate. A combination of [Ca]res dynamics and the properties of a calcium-driven reaction determine the time course and amplitude of facilitation.

Animals↗
PubMed1996

Contributions of calcium-dependent and calcium-independent mechanisms to presynaptic inhibition at a cerebellar synapse.

Activation of either adenosine A1 receptors or GABAB receptors inhibits many excitatory synapses in the mammalian brain. However, the extent to which different mechanisms contribute to such synaptic modulation is unclear. We examined the manner in which activation of adenosine A1 receptors and GABAB receptors modulates synaptic strength at the granule cell to Purkinje cell synapse in rat cerebellar slices. Optical determination of presynaptic calcium influx revealed that presynaptic calcium channels were modulated by 2-chloroadenosine (2CA) and baclofen, agonists of the adenosine A1 receptor and the GABAB receptor, respectively. 2CA and baclofen differentially affected three classes of calcium channels without altering the shape of the presynaptic volley, suggesting that changes in presynaptic waveform do not contribute significantly to synaptic modulation. 2CA affected neither the amplitude nor the frequency of spontaneous miniature postsynaptic currents, whereas baclofen reduced the frequency by approximately 40% without affecting the amplitude. In addition, 2CA and baclofen do not change either fiber excitability or presynaptic residual calcium. Taken together, our data indicate that activation of the adenosine A1 receptor reduces synaptic strength by modulating presynaptic calcium channels. Baclofen modulates presynaptic calcium channels as well but also affects release processes downstream from calcium entry.

2-Chloroadenosine↗
PubMed1995

Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice.

The control of neurotransmitter release by modulation of presynaptic calcium influx was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory post-synaptic currents were measured using whole cell voltage clamp, and changes in presynaptic Ca influx were determined with the Ca-sensitive dye mag-fura-5. Single stimuli of the parallel fibers evoked rapid changes in mag-fura-5 fluorescence which increased from 10 to 90% in 1.4 msec, and then decayed within hundreds of milliseconds to prestimulus levels. These fluorescence changes were unaffected by disruption of internal stores with ryanodine or thapsigargin, and were reduced by 79% by the calcium channel toxin omega-conotoxin-MVIIC. We conclude that these signals result from calcium entry into presynaptic terminals through voltage gated calcium channels opened by action potentials. These fluorescence signals allow us to quantitate changes in calcium influx. We used this approach to study the enhancement of stimulus-evoked synaptic currents by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and antagonist of adenosine receptors. Both enhancement of calcium influx into presynaptic terminals, and reduction in the firing threshold of the parallel fibers, were found to contribute to IBMX-mediated synaptic enhancement. Changes in presynaptic calcium influx were also quantified with a novel method, which is unaffected by changes in fiber threshold. These studies illustrate some of the difficulties encountered when determining the factors responsible for synaptic enhancement and demonstrate how measurements of presynaptic calcium influx can contribute to our understanding of synaptic modulation. The approach described here promises to be widely useful in elucidating the role of calcium influx in the modulation of synapses in brain slice.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Calcium control of transmitter release at a cerebellar synapse.

The manner in which presynaptic Ca2+ influx controls the release of neurotransmitter was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory postsynaptic currents were measured using whole-cell voltage clamp, and changes in presynaptic Ca2+ influx were determined with the Ca(2+)-sensitive dye furaptra. We manipulated presynaptic Ca2+ entry by altering external Ca2+ levels and by blocking Ca2+ channels with Cd2+ or with the toxins omega-conotoxin GVIA and omega-Aga-IVA. For all of the manipulations, other than the application of omega-Aga-IVA, the relationship between Ca2+ influx and release was well approximated by a power law, n approximately 2.5. When omega-Aga-IVA was applied, release appeared to be more steeply dependent on Ca2+ (n approximately 4), suggesting that omega-Aga-IVA-sensitive channels are more effective at triggering release. Based on interactive effects of toxins on synaptic currents, we conclude that multiple types of Ca2+ channels synergistically control individual release sites.

Animals↗
PubMed1995

Calcium transients in cerebellar granule cell presynaptic terminals.

Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons.

Animals↗
PubMed1995

A quantitative analysis of presynaptic calcium dynamics that contribute to short-term enhancement.

Augmentation and posttetanic potentiation--two forms of short-term synaptic enhancement produced by repetitive presynaptic action potentials--are dependent on the buildup and decay of nerve terminal residual calcium that occurs on the seconds to minutes time scale. With the goal of providing a quantitative understanding of these kinetics, we measured the buildup and decay of calcium ions in nerve terminals at the crayfish neuromuscular junction under a variety of intracellular buffer conditions and stimulation paradigms. The calcium extrusion process in the terminals was characterized by analysis of calcium levels reached during long stimulus trains as a function of action potential frequency. The extrusion was linearly dependent on the free calcium ion concentration. Using this result, we developed a mathematical model and computer simulation of the residual calcium kinetics. The model demonstrates the experimentally observed dependence of decay rate on exogenous calcium buffer concentration, and can be explicitly solved to provide an expression for the limiting exponential time course of calcium decay following trains in terms of calcium buffer and extrusion characteristics. Methods to determine the calcium influx per action potential, characteristics of endogenous buffer, and the rate of calcium extrusion are suggested by our analysis and demonstrated experimentally.

Animals↗
PubMed1994

Participation of multiple calcium channel types in transmission at single climbing fiber to Purkinje cell synapses.

The sensitivity of synaptic transmission to antagonists of different calcium channels was examined at the powerful climbing fiber synapse between neurons from the inferior olive and cerebellar Purkinje cells. In rat brain slices, climbing fibers were activated with extracellular electrodes, and synaptic currents were recorded with whole-cell patch clamp. Dihydropyridines did not discernibly affect synaptic strength. omega-Conotoxin GVIA, a potent antagonist of N-type calcium channels, reduced synaptic currents by an average of 29%. omega-Agatoxin-IVA, a high affinity blocker of P-type calcium channels, reduced synaptic strength by an average of 77%. Together, the two toxins virtually eliminated synaptic transmission (91% inhibition). These results indicate that omega-agatoxin-IVA-sensitive calcium channels play an important role in transmission at the climbing fiber synapse. They also suggest that in single climbing fibers, release is evoked by at least two pharmacologically distinct calcium currents, one sensitive to omega-agatoxin-IVA, the other to omega-conotoxin GVIA.

Animals↗
PubMed1994

Dendritic calcium dynamics.

Further characterization of the biochemical components that contribute to calcium handling, together with advances in optical imaging of ion concentration, are providing quantitative information on the dynamics of calcium in the dendrites of neurons in tissue culture, brain slices and in vivo. It has recently been demonstrated that strong spatial gradients and transient calcium elevations result from the geometry and membrane properties of dendrites. These studies are adding to our understanding of calcium's role in synaptic plasticity and in shaping the electrophysiological properties of neurons.

Animals↗
PubMed1994

The role of presynaptic calcium in short-term enhancement at the hippocampal mossy fiber synapse.

The mossy fiber synapse between dentate granule cells and CA3 pyramidal cells in the guinea pig hippocampus shows a robust short-term synaptic enhancement. We have simultaneously measured presynaptic residual free calcium ([Ca2+]i) and postsynaptic field potentials at this synapse to examine the role of [Ca2+]i in this enhancement. Single action potentials produced an increase in [Ca2+]i of 10-50 nM that decayed to resting levels with a time constant of about 1 sec. Trains of action potentials produced larger [Ca2+]i increases that returned more slowly to resting levels. Following the onset of moderate frequency stimulus trains (0.1-5 Hz), synaptic transmission and [Ca2+]i both increased and eventually plateaued. During the steady-state phase a linear relationship between [Ca2+]i and synaptic enhancement was observed. During the initial buildup, however, [Ca2+]i rose more rapidly than synaptic enhancement. Similarly, during the decay phase immediately following termination of a stimulus train, [Ca2+]i returned to prestimulus levels faster than synaptic enhancement. High concentrations of the calcium buffer EGTA in the presynaptic terminal slowed the buildup and decay of both [Ca2+]i and synaptic enhancement produced by stimulus trains. Under these conditions, the time course of [Ca2+]i and synaptic enhancement were well matched. This suggests that, despite the differences in kinetic rates observed for normal buffering conditions, increases in [Ca2+]i play a causal role in short-term enhancement. An increase in [Ca2+]i of 10-30 nM produced a twofold enhancement. We propose a simple kinetic model to explain these results. The model assumes that synaptic enhancement is controlled by a Ca-dependent first-order reaction. According to this scheme, a change in [Ca2+]i alters neurotransmitter release, but the slow kinetics of the underlying reaction introduces a temporal filter, producing a delay in the change in synaptic enhancement.

Animals↗
PubMed1992

Sodium action potentials in the dendrites of cerebellar Purkinje cells.

We report here that in cerebellar Purkinje cells from which the axon has been removed, positive voltage steps applied to the voltage-clamped soma produce spikes of active current. The spikes are inward, are all-or-none, have a duration of approximately 1 ms, and are reversibly eliminated by tetrodotoxin, a Na channel poison. From cell to cell, the amplitude of the spikes ranges from 4 to 20 nA. Spike latency decreases as the depolarizing step is made larger. These spikes clearly arise at a site where the voltage is not controlled, remote from the soma. From these facts we conclude that Purkinje cell dendrites contain a sufficient density of Na channels to generate action potentials. Activation by either parallel fiber or climbing fiber synapses produces similar spikes, suggesting that normal input elicits Na action potentials in the dendrites. These findings greatly alter current views of how dendrites in these cells respond to synaptic input.

Action Potentials↗
PubMed1992

Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells.

The spatial and temporal dynamics of many electrophysiological and biochemical processes in nerve cells are in turn dependent on the concentration dynamics of the second messenger calcium. We have used microfluorimetry of the calcium indicator fura-2 (Grynkiewicz et al., 1985) to measure and characterize synaptically activated calcium changes in individual CA1 pyramidal cells contained within guinea pig hippocampal slices. One component of the calcium changes was largely produced by influx through voltage-dependent Ca2+ channels (VDCCs). It consisted of large transient accumulations in the proximal-apical and basal dendrites; the amplitude was smaller in the distal-apical dendrites and the soma. This spatial profile was insensitive to the method of cell activation: stimulation of inputs located at different positions on the dendritic tree as well as antidromic stimulation produced only slight modifications. This component was not blocked by the NMDA antagonist 5-amino-4-phosphonovalerate (AP5) (Collingridge et al., 1983), was greatly reduced by Cd2+, partially reduced by nifedipine, and was increased by Bay-K 8644, providing the evidence that it was largely produced by influx through VDCCs. Blocking postsynaptic Na+ channels with QX-314 greatly reduced the accumulation amplitude, and spatial differences between proximal-dendritic and distal-dendritic regions were less pronounced, suggesting that active sodium conductances contribute significantly to the spatial activation of calcium conductances. Residual spatial differences that persist in QX-314 experiments are consistent with the idea that VDCCs have decreased density on distal-apical dendrites. A second component of accumulation was induced by ionic currents through NMDA receptor channels. It was blocked by AP5, unaffected by QX-314, attenuated and slowed down by elevated calcium buffering, and spatially localized to regions receiving activated synaptic inputs. The magnitude of this component was strongly dependent on the frequency and amplitude of synaptic activation. At high frequency, it was generally very large, often saturating the fura-2 (> 2 microM). Measurements made with the indicator furaptra also showed large localized AP5-sensitive fluorescence changes. Our results suggest that in dendritic regions near activated input fibers calcium levels may reach 2-10 microM. In general, our measurements of calcium dynamics provide an experimental basis for evaluating the spatial distribution of calcium conductances, the spatial distribution of calcium-activated electrophysiological and biochemical processes, and the spatial uniformity of calcium buffering and removal systems in CA1 hippocampal pyramidal cells. The time course and amplitude of Ca2+ transients we measured suggest that activation of Ca(2+)-dependent conductances [e.g., IK(Ca)] will be markedly different for different cellular regions.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1992

Electrical activity increases growth cone calcium but fails to inhibit neurite outgrowth from rat sympathetic neurons.

Previous studies have shown that the growth of axons from both mouse dorsal root ganglion neurons and Helisoma neurons is arrested when the cells are electrically stimulated (Cohan and Kater, 1986; Fields et al., 1990a). Furthermore, in the case of Helisoma neurons, this arrest has been attributed to a rise in the calcium concentration in the growth cones (Cohan et al., 1987). To test the generality of these results, we examined the response of cultured rat superior cervical ganglion (SCG) neurons to electrical stimulation and changes in cytoplasmic calcium. Suprathreshold electrical stimulation of SCG neurons at 10 Hz by extracellular patch electrodes for periods of up to 1 hr had no measurable effect on their rate of growth. In agreement with previous studies, electrical stimulation was accompanied by a rise in the internal calcium concentration: when measured by the fluorescence of fura-2, growth cone calcium levels rose from about 100 nM to greater than 500 nM and then settled to a plateau value of about 350 nM. Despite this increase, however, growth of SCG neurons' processes continued. Our results show that electrical activity is not a universal signal for neurons to stop growing and that a rise in internal calcium does not always arrest the migration of growth cones.

Animals↗