Time Course Of Transients Research Articles

A dominant role for the beta 4 nicotinic receptor subunit in nicotinic modulation of glomerular microcircuits in the mouse olfactory bulb

Nicotinic acetylcholine receptors (nAChRs) regulate information transfer across the main olfactory bulb by instituting a high-pass intensity filter allowing for the filtering out of weak inputs. Excitation-driven inhibition of the glomerular microcircuit via GABA release from periglomerular cells appears to underlie this effect of nAChR activation. The multiplicity of nAChR subtypes and cellular locations raises questions about their respective roles in mediating their effects on the glomerular output. In this study, we address this issue by targeting heteromeric nAChRs using receptor knockouts (KOs) for the two dominant nAChR β-subunit genes known to be expressed in the central nervous system. KOs of the β2-nAChR subunit did not affect nAChR currents from mitral cells (MCs) but attenuated those from the external tufted (ET) cells. In slices from these animals, activation of nAChRs still effectively inhibited excitatory postsynaptic currents (EPSCs) and firing on MCs evoked by the olfactory nerve (ON) stimulation, thereby indicating that the filter mechanism was intact. On the other hand, recordings from β4-KOs showed that nAChR responses from MCs were abolished and those from ET cells were attenuated. Excitation-driven feedback was abolished as was the effect of nAChR activation on ON-evoked EPSCs. Experiments using calcium imaging showed that one possible consequence of the β2-subunit activation might be to alter the time course of calcium transients in juxtaglomerular neurons suggesting a role for these receptors in calcium signaling. Our results indicate that nAChRs containing the β4-subunit are critical in the filtering of odor inputs and play a determinant role in the cholinergic modulation of glomerular output. NEW & NOTEWORTHY In this study, using receptor gene knockouts we examine the relative contributions of heteromeric nAChR subtypes located on different cell types to this effect of receptor activation. Our results demonstrate that nAChRs containing the β4-subunit activate MCs resulting in feedback inhibition from glomerular interneurons. This period of inhibition results in the selective filtering of weak odor inputs providing one mechanism by which nAChRs can enhance discrimination between two closely related odors.

Editorial: Determinants of Synaptic Information Transfer: From Ca(2+) Binding Proteins to Ca(2+) Signaling Domains.

Editorial: Determinants of Synaptic Information Transfer: From Ca(2+) Binding Proteins to Ca(2+) Signaling Domains.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Detecting calcium in cardiac muscle: fluorescence to dye for.

THE IMPORTANCE OF Ca 2 as a second messenger in general and as a mediator between electrical excitation and cellular contraction in the heart is well accepted. Accordingly, numerous Ca 2 -sensitive fluorescent dyes are available nowadays, rendering the choice of the appropriate dye for a particular application a challenge. It appears to be important to provide calibrated Ca 2 signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca 2 transients. Additionally, we discuss how major properties of the fluorescence dyes should be considered with a special emphasis on their Ca 2 dissociation properties. Furthermore, we highlight putative risks of tripping, especially in the context of multicellular recordings, and how to circumvent them. As an ubiquitous intracellular messenger, Ca 2 ions play an important role in almost all living cells (4). A particularly dynamic role has been assigned to Ca 2 in muscle cells because here they not only control gene expression and other slower signaling processes but serve as a direct transducer of the electrical excitation of the plasma membrane into the activation of the physiological response of the cell, the contraction, by activation of the contractile machinery (5). Almost 50 years ago, Jobsis and O’Connor (10) reported the measurements of intracellular Ca 2 transients in skeletal muscle using the Ca 2 -sensitive dye murexide, which changes its absorbance upon binding of Ca 2 ions. Later, these absorbance dyes were improved and an important member of this family, arsenazo III, was widely used to study intracellular Ca 2 in

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

Nerve‐evoked purinergic signalling suppresses action potentials, Ca2+ flashes and contractility evoked by muscarinic receptor activation in mouse urinary bladder smooth muscle

Contraction of urinary bladder smooth muscle (UBSM) is caused by the release of ATP and ACh from parasympathetic nerves. Although both purinergic and muscarinic pathways are important to contraction, their relative contributions and signalling mechanisms are not well understood. Here, the contributions of each pathway to urinary bladder contraction and the underlying electrical and Ca(2+) signalling events were examined in UBSM strips from wild type mice and mice deficient in P2X1 receptors (P2X1(-/-)) before and after pharmacological inhibition of purinergic and muscarinic receptors. Electrical field stimulation was used to excite parasympathetic nerves to increase action potentials, Ca(2+) flash frequency, and force. Loss of P2X1 function not only eliminated action potentials and Ca(2+) flashes during stimulation, but it also led to a significant increase in Ca(2+) flashes following stimulation and a corresponding increase in the force transient. Block of muscarinic receptors did not affect action potentials or Ca(2+) flashes during stimulation, but prevented them following stimulation. These findings indicate that nerve excitation leads to rapid engagement of smooth muscle P2X1 receptors to increase action potentials (Ca(2+) flashes) during stimulation, and a delayed increase in excitability in response to muscarinic receptor activation. Together, purinergic and muscarinic stimulation shape the time course of force transients. Furthermore, this study reveals a novel inhibitory effect of P2X1 receptor activation on subsequent increases in muscarinic-driven excitability and force generation.

Synaptic Vesicles in Mature Calyx of Held Synapses Sense Higher Nanodomain Calcium Concentrations during Action Potential-Evoked Glutamate Release

During development of the calyx of Held synapse, presynaptic action potentials (APs) become substantially faster and briefer. Nevertheless, this synapse is able to upregulate quantal output triggered by arriving APs. Briefer APs lead to less effective gating of voltage-gated Ca(2+) channels (VGCCs). Therefore, mechanisms downstream of Ca(2+) entry must effectively compensate for the attenuated Ca(2+) influx associated with shorter APs in more mature calyces. This compensation could be achieved by tighter spatial coupling between VGCCs and synaptic vesicles, so that the latter are exposed to higher intracellular Ca(2+) concentration ([Ca(2+)](i)). Alternatively or additionally, the Ca(2+) sensitivity of the release apparatus may increase during synapse development. To differentiate between these possibilities, we combined paired patch-clamp recordings with Ca(2+) imaging and flash photolysis of caged Ca(2+) and estimated the [Ca(2+)](i) requirements for vesicle release in the developing mouse calyx of Held synapse. Surprisingly, the dose-response relationship between [Ca(2+)](i) and release rate was shifted slightly to the right in more mature calyces, rendering their vesicles slightly less sensitive to incoming Ca(2+). Taking into account the time course and peak rates of AP-evoked release transients for the corresponding developmental stages, we estimate the local [Ca(2+)](i)"seen" by the Ca(2+) sensors on synaptic vesicles to increase from 35 to 56 mum [from postnatal day 9 (P9)-P11 to P16-P19]. Our results reinforce the idea that developmental tightening of the spatial coupling between VGCCs and synaptic vesicles plays a predominant role in enhancing quantal output at this synapse and possibly other central synapses.

Accumulation of cytoplasmic calcium, but not apamin‐sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells

The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s(-1), the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s(-1) the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They suggest that the calcium is present at the SK channel for a very short time after each action potential, and the current decays at a rate set by the deactivation kinetics of the SK channel. At high rates, repetitive firing was governed by a fast apamin-insensitive AHP current that did not accumulate, but rather showed depression with increases in activation frequency. A slowly accumulating AHP current, also insensitive to apamin, was extremely small at low rates but became significant with higher firing rates.

Multimodality of Ca2+ Signaling in Rat Atrial Myocytes

It has been suggested that the multiplicity of Ca(2+) signaling pathways in atrial myocytes may contribute to the variability of its function. This article reports on a novel Ca(2+) signaling cascade initiated by mechanical forces induced by "puffing" of solution onto the myocytes. Ca(i) transients were measured in fura-2 acetoxymethyl (AM) loaded cells using alternating 340- and 410-nm excitation waves at 1.2 kHz. Pressurized puffs of bathing solutions, applied by an electronically controlled micro-barrel system, activated slowly (approximately 300 ms) developing Ca(i) transients that lasted 1,693 +/- 68 ms at room temperature. Subsequent second and third puffs, applied at approximately 20 s intervals activated significantly smaller or no Ca(i) transients. Puff-triggered Ca(i) transients could be reactivated once again following caffeine (10 mM)-induced release of Ca(2+) from sarcoplasmic reticulum (SR). Puff-triggered Ca(i) transients were independent of [Ca(2+)](o), and activation of voltage-gated Ca(2+) or cationic stretch channels or influx of Ca(2+) on Na(+)/Ca(2+)exchanger, because puffing solution containing no Ca(2+), 10 microM diltiazem, 1 mM Cd(2+), 5 mM Ni(2+), or 100 microM Gd(3+) failed to suppress them. Puff-triggered Ca(i) transients were enhanced in paced compared to quiescent myocytes. Electrically activated Ca(i) transients triggered during the time course of puff-induced transients were unaltered, suggesting functionally separate Ca(2+) pools. Contribution of inositol 1,4,5-triphosphate (IP(3))-gated or mitochondrial Ca(2+) pools or modulation of SR stores by nitric oxide/nitric oxide synthase (NO/NOS) signaling were evaluated using 0.5 to 500 microM 2-aminoethoxydiphenyl borate (2-APB) and 0.1 to 1 microM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 1 mM Nomega-Nitro-L-arginine methyl ester (L-NAME) and 7-nitroindizole, respectively. Only FCCP appeared to significantly suppress the puff-triggered Ca(i) transients. It was concluded that neither Ca(2+) influx nor depolarization was required for activation of this signaling pathway. These studies suggest that pressurized puffs of solutions activate a mechanically sensitive receptor, which signals in turn the release of Ca(2+) from a limited Ca(2+) store of mitochondria. How mechanical forces are sensed and transmitted to mitochondria to induce Ca(2+) release and what role such a Ca(2+) signaling pathway plays in the physiology or pathophysiology of the heart remain to be worked out.

Calbindin in Cerebellar Purkinje Cells Is a Critical Determinant of the Precision of Motor Coordination

Long-term depression (LTD) of Purkinje cell-parallel fiber synaptic transmission is a critical determinant of normal cerebellar function. Impairment of LTD through, for example, disruption of the metabotropic glutamate receptor-IP3-calcium signaling cascade in mutant mice results in severe deficits of both synaptic transmission and cerebellar motor control. Here, we demonstrate that selective genetic deletion of the calcium-binding protein calbindin D-28k (calbindin) from cerebellar Purkinje cells results in distinctly different cellular and behavioral alterations. These mutants display marked permanent deficits of motor coordination and sensory processing. This occurs in the absence of alterations in a form of LTD implicated in the control of behavior. Analysis of synaptically evoked calcium transients in spines and dendrites of Purkinje cells demonstrated an alteration of time course and amplitude of fast calcium transients after parallel or climbing fiber stimulation. By contrast, the delayed metabotropic glutamate receptor-mediated calcium transients were normal. Our results reveal a unique role of Purkinje cell calbindin in a specific form of motor control and suggest that rapid calcium buffering may directly control behaviorally relevant neuronal signal integration.

Different effects of endothelin-1 on calcium and potassium currents in canine ventricular cells

Effects of endothelin-1 (ET-1) on the L-type calcium current (ICa) and delayed rectifier potassium current (IK) were studied in isolated canine ventricular cardiomyocytes using the whole-cell configuration of the patch-clamp technique. ET-1 (8 nM) was applied in three experimental arrangements: untreated cells, in the presence of 50 nM isoproterenol, and in the presence of 250 microM 8-bromo-cAMP. In untreated cells, ET-1 significantly decreased the peak amplitude of ICa by 32.3+/-4.8% at +5 mV (P<0.05) without changing activation or inactivation characteristics of ICa. ET-1 had no effect on the amplitude of IK, Ito (transient outward current) or IK1 (inward rectifier K current) in untreated cells; however, the time course of recovery from inactivation of Ito was significantly increased by ET-1 (from 26.5+/-4.6 ms to 59.5+/- 1.8 ms, P < 0.05). Amplitude and time course of intracellular calcium transients, recorded in voltage-clamped cells previously loaded with the fluorescent calcium indicator dye Fura-2, were not affected by ET-1. ET-1 had no effect on force of contraction in canine ventricular trabeculae. Isoproterenol increased the amplitude of ICa to 263+/-29% of control. ET-1 reduced ICa also in isoproterenol-treated cells by 17.8+/-2% (P<0.05); this inhibition was significantly less than obtained in untreated cells. IK was increased by isoproterenol to 213+/-18% of control. This effect of isoproterenol on IK was reduced by 31.8+/-4.8% if the cells were pretreated with ET-1. Similarly, in isoproterenol-treated cells ET-1 decreased IK by 16.2+/-1.5% (P<0.05). Maximal activation of protein kinase A (PKA) was achieved by application of 8-bromo-cAMP in the pipette solution. In the presence of 8-bromo-cAMP ET-1 failed to alter ICa or IK It was concluded that differences in effects of ET-1 on ICa and IK may be related to differences in cAMP sensitivity of the currents.

A calcium antagonist protects against doxorubicin-induced impairment of calcium handling in neonatal rat cardiac myocytes.

The effects of doxorubicin (DOX) on intracellular calcium transients and the cardioprotective effects of a calcium antagonist on DOX-induced impairment of calcium handling were examined in neonatal rat cultured cardiac myocytes. Cultured cardiac myocytes isolated from neonatal Wistar-Kyoto rats were treated with DOX for 24 h. Field-stimulated calcium transients in single myocytes were measured in the presence or absence of isoproterenol using fura-2/AM. Calcium transients were also measured after the addition of DOX to myocytes pretreated with a calcium antagonist, benidipine. DOX reduced the amplitude, maximum velocity of increase and decrease of calcium transients and prolonged the time course of calcium transients and impaired the beta-adrenoceptor responsiveness of calcium transients in a concentration-dependent manner. The DOX-induced impairment of calcium transients and beta-adrenoceptor responsiveness was improved by 10(-8) mol/L of benidipine. However, these improvements decreased with increasing concentrations of benidipine. DOX impaired both the mobilization and removal of intracellular calcium ions in contraction-relaxation cycles and the response of calcium transients to beta-adrenoceptor stimulation. Appropriate concentration of benidipine ameliorated DOX-induced impairment of calcium dynamics, suggesting that benidipine, a long-acting calcium antagonist, has potential clinical usefulness on DOX-induced abnormal calcium handling.

An angiotensin-converting enzyme inhibitor protects against doxorubicin-induced impairment of calcium handling in neonatal rat cardiac myocytes.

1. The effects of doxorubicin (DOX) on intracellular calcium transients were examined in neonatal rat cultured cardiac myocytes, as were the cardioprotective effects of an angiotensin-converting enzyme (ACE) inhibitor on DOX-induced impairment of calcium handling. 2. Cultured cardiac myocytes isolated from neonatal Wistar-Kyoto rats were treated with DOX for 24 h. Field-stimulated calcium transients in single myocytes were measured in the presence or in the absence of isoproterenol using fura-2/AM. Calcium transients were also measured after the addition of DOX to myocytes pretreated with M-I (an active metabolite of delapril HCL, an ACE inhibitor. 3. Doxorubicin reduced the amplitude and maximum velocity of increase and decrease of calcium transients, prolonged the time-course of calcium transients and impaired the beta-adrenoceptor responsiveness of calcium transients in a dose-dependent manner. The DOX-induced impairment of calcium transients and beta-adrenoceptor responsiveness was improved by M-I. 4. Doxorubicin impaired both the mobilization and sequestration of intracellular calcium ions in contraction-relaxation cycles and the response of calcium transients to beta-adrenoceptor stimulation. The ACE inhibitor ameliorated DOX-induced impairment of calcium dynamics, suggesting ihat M-I, an active metabolite of delapril, protects against DOX-induced abnormal calcium handling leading to cardiac dysfunction.

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.

We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50-200 microM), short duration (< 100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.

Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses

The effect of the membrane-permeant calcium chelator 1,2- bis-(2-aminophenoxy)ethane-N,N,N′,N−'tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca 2+] i, we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 μM BAPTA/AM reduced the number of cells showing deregulation of [Ca 2+] i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCI were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3–30 μM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 μM BAPTA/AM, the time constant of decline of [Ca 2+] i was increased by a factor of 4.1 ± 2.4 ( n = 14) and the amplitude was reduced to about 50%. With 30 μM BAPTA/AM, the K +-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca 2+] i-induced cell damage, however, at the expense of a disturbed calcium signalling.

Time course of calcium transients derived from Fura-2 fluorescence measurements in single fast twitch fibres of adult mice and rat myotubes developing in primary culture

In this study, we applied a method to correct for the altered binding kinetics of Fura-2 for Ca 2+ in vivo, on Ca 2+ fluorescence transients (Ca 2+ F) measured using Fura-2 in single adult fast twitch skeletal muscle fibres of the mouse, which exhibit very fast [Ca 2+] responses, and rat myotubes developing in culture which exhibit slower [Ca 2+] responses (rise time [20–80% of peak] of Ca 2+ F transients: 1.81 ± 0.17 ms and 16.14 ± 2.60 ms, respectively). After correction, the [Ca 2+] transients (Ca 2+ C) measured in both the adult mouse fibres and the myotubes rose more rapidly (mean rise time of Ca 2+ C transients: adult mouse fibres, 0.76 ± 0.12 ms; rat myotubes, 8.25 ± 2.83 ms) and often exhibited a Ca 2+ spike which exceeded the peak of the Ca 2+ F transient. In the adult mouse fibres, correction increased the mean peak [Ca 2+] of the Ca 2+ F transients by a factor of 7 from 0.53 ± 0.08 μM to 3.76 ± 0.71 μM The accuracy of the time course of the corrected Ca 2+ transients was confirmed by comparison to the time course of Ca 2+ transients measured with Mag-Fura-5, which had a similar mean rise time (0.94 ± 0.10 ms, t-test, P = 0.80). The more slowly rising Ca 2+ transients measured in the rat myotubes were less affected by the correction process, increasing in mean peak [Ca 2+] by a factor of only 1.2 from 0.82 ± 0.17 μM to 0.97 ± 0.15 μM. During the decay phase of the Ca 2+ transients elicited in the adult mouse fibres and the myotubes, the corrected Ca 2+ C signal largely followed the unmodified Ca 2+ F transient. The correction process was found to have little effect on Ca 2+ transients with rise time values greater than 10 ms, which included most of the Ca 2+ transients measured in the myotubes.

Presynaptic Calcium Dynamics and Transmitter Release Evoked by Single Action Potentials at Mammalian Central Synapses

The relationship between presynaptic calcium transients ([Ca2+]t) and transmitter release evoked by a single stimulus was both investigated experimentally and modeled at a mammalian central synapse, the CA3 to CA1 pyramidal cell synapse in guinea pig hippocampal slices. In the present study, we compared the low-affinity calcium indicator furaptra with the higher-affinity indicator fura-2. The 10-90% rise time of the furaptra transient was 2.4 ms compared to 7.8 ms with fura-2; the half-decay time (tau 1/2) was 30 ms for furaptra, compared to 238 ms for fura-2. The half-width of the calcium influx was 1.8 ms with furaptra, which provides an upper limit to the duration of the calcium current (ICa) evoked by an action potential. Modeling the decay time course of the furaptra transients led to the conclusion that the predominant endogenous calcium buffer in these terminals must have relatively slow kinetics (kon < 10(5)/M.s), although the presence of small amounts of fast buffers cannot be excluded. The relationship between the [Ca2+]t measured with furaptra and the postsynaptic response was the same as previously observed with fura-2: the postsynaptic response was proportional to about the fourth power (m approximately 4) of the amplitude of either [Ca2+]t or calcium influx. Thus, although fura-2 may be locally saturated by the high local [Ca2+] responsible for transmitter release, the volume-averaged fura-2 signal accurately reflects changes in this local concentration. The result that both indicators gave similar values for the power m constrains the amplitude of calcium influx in our model: Ica < 1 pA for 1 ms.

Presynaptic calcium dynamics at the frog retinotectal synapse.

1. We characterized the kinetics of presynaptic Ca2+ ion concentration in optic nerve fibers and terminals of the optic tectum in Rana pipiens with the use of microfluorimetry. Isolated frog brains were incubated with the membrane-permeant tetraacetoxymethyl ester (AM) of the Ca2+ indicator fura-2. An optic nerve shock caused a transient decrease in the 380-nm excited fluorescence in the optic tectum with a rise time of <15 ms and a recovery to prestimulus levels on a time scale of seconds. 2. In normal saline, the amplitude of the fluorescence transients was dependent on stimulus intensity and at all levels it was directly correlated with the amplitude of postsynaptic field potentials produced by activation of unmyelinated optic nerve fibers. In the presence of the non-N-methyl-D-aspartate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, the amplitude and time course of fluorescence transients remained essentially unchanged while postsynaptic field potential amplitude was greatly reduced. Replacing extracellular Ca2+ with Ba2+ blocked unfacilitated postsynaptic field potentials while fluorescence transients remained significant. In reduced-Ca2+ salines (<1 mM), the amplitude of fluorescence transients increased approximately linearly with extracellular [Ca2+], whereas the amplitude the corresponding field potential was nonlinearly related to the fluorescent transient amplitude (approximately 2.5 power). In thin sections of labeled tecta, fluorescence labeling was localized to 1-micron puncta in the termination zone of optic nerve fibers in the superficial layers. Taken together, these results provide strong evidence that the fluorescence transients correspond to an increase in Ca2+ in presynaptic terminals of unmyelinated optic nerve fibers. 3. During trains of optic nerve stimulation, the amplitude of fluorescence transients to succeeding action potentials became smaller. The decrement of the amplitudes was not observed in mag-fura-5-labeled tecta, when the intracellular Ca2+ buffering capacity of fura-2-labeled terminals was increased by incubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-AM, or in low-Ca2+ saline. We conclude that the Ca2+ influx per action potential is constant during the train and that the reduced response was produced by saturation of the fura-2. We provide a mathematical analysis of this saturation effect and use it to estimate the Ca2+ change per action potential. 4. Both BAPTA-AM and EGTA-AM reduced the overall amplitude of fura-2-measured Ca2+ transients and reduced the saturation effect in action potential trains. However, there was a qualitative difference in their effects on the shape of the transient. Incubation with the fast buffer BAPTA prolonged the decay to baseline. In contrast, the slow buffer EGTA (or EDTA) produced an initial decay faster than the control condition while also producing the slower subsequent phase observed with BAPTA. We demonstrate that these results are consistent with numerical simulations of Ca2+ dynamics in a single-compartment model where the fast initial decay is produced by the forward rate of Ca2+ binding to EGTA. 5. Ca2+ influx into tectal presynaptic structures, and also into unmyelinated axons in the isolated optic nerve, was diminished (60-70%) in the presence of the voltage-activated Ca2+ channel blocker omega-conotoxin GVIA, but was only weakly affected (approximately 10%) by omega-agatoxin IVA. 6. After 10- to 50-Hz stimulus trains, synaptic enhancement of unmyelinated fibers decayed with a characteristic time similar to fura-2 fluorescence decays. Incubation with EDTA-AM or EGTA-AM produced little effect on evoked release but reduced both the amplitude of the fura-2-measured Ca2+ transient and the amplitude of short-term synaptic enhancement.

Deformation of the Bowditch staircase in Ca(2+)-overloaded mammalian cardiac tissue--a calcium phenomenon?

Concentrations of 1-4 mumol l-1 isoproterenol cause both in right ventricular papillary muscles and in enzymatically isolated myocytes of the guinea-pig a Ca2+ overload-induced state which is functionally characterized by biphasic (multiphasic) twitches and biphasic (multiphasic) intracellular calcium transients, respectively, during excitation-contraction coupling. This state was stabilized in the in vitro experiments for some hours by a co-ordination of the interstimulus interval, the temperature of the superfusion fluid and the addition of calcium agonists. The functional stability is the precondition for the reproducibility of the experimental results particularly after the application of long-lasting stimulation programmes. Changes in the shape of biphasic contractions were compared with changes in the time course of biphasic intracellular calcium transients using three manipulations of a different kind: (1) the interruption of the steady pacing rhythm, (2) the variation of the interstimulus interval, (3) the addition of ryanodine. It was shown that: (1) The BOWDITCH staircase in calcium overloaded multicellular preparations is changed in that each individual component of the twitch passes through its own staircase. A homologous behaviour can be observed in the configuration of the phasic and tonic component of biphasic intracellular calcium transients. (2) At different driving frequencies the relative proportion of the two components of a biphasic twitch corresponds to the time integrals of the two components of biphasic intracellular calcium transients. (3) Ryanodine suppresses both the first component of the biphasic twitch and the phasic component of the biphasic intracellular calcium transient. The SR Ca(2+)-ATPase inhibitor thapsigargin increases the second component of the biphasic calcium transient. This supports the hypothesis that the size of the tonic component is in part determined by intracellular calcium reuptake. The results of both kinds of experiments would be compatible with the assumption that in calcium overloaded mammalian cardiac cells calcium reaches the contractile system directly as well as via two intracellular stores ('extended two-Ca-store concept').

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.