Low-affinity Dye Research Articles

Quantitation and Simulation of Single Action Potential-Evoked Ca2+ Signals in CA1 Pyramidal Neuron Presynaptic Terminals.

Presynaptic Ca2+ evokes exocytosis, endocytosis, and synaptic plasticity. However, Ca2+ flux and interactions at presynaptic molecular targets are difficult to quantify because fluorescence imaging has limited resolution. In rats of either sex, we measured single varicosity presynaptic Ca2+ using Ca2+ dyes as buffers, and constructed models of Ca2+ dispersal. Action potentials evoked Ca2+ transients with little variation when measured with low-affinity dye (peak amplitude 789 ± 39 nM, within 2 ms of stimulation; decay times, 119 ± 10 ms). Endogenous Ca2+ buffering capacity, action potential-evoked free [Ca2+]i, and total Ca2+ amounts entering terminals were determined using Ca2+ dyes as buffers. These data constrained Monte Carlo (MCell) simulations of Ca2+ entry, buffering, and removal. Simulations of experimentally-determined Ca2+ fluxes, buffered by simulated calbindin28K well fit data, and were consistent with clustered Ca2+ entry followed within 4 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca2+ to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while local diffusion dominated. Thus, Ca2+ within tens of nanometers of entry, did not accumulate. A model of synaptotagmin1 (syt1)-Ca2+ binding indicates that even with 10 µM free varicosity evoked Ca2+, syt1 must be within tens of nanometers of channels to ensure occupation of all its Ca2+-binding sites. Repetitive stimulation, evoking short-term synaptic enhancement, does not modify probabilities of Ca2+ fully occupying syt1’s C2 domains, suggesting that enhancement is not mediated by Ca2+-syt1 interactions. We conclude that at spatiotemporal scales of fusion machines, Ca2+ necessary for their activation is diffusion dominated.

Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

BackgroundMutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca2+ release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca2+ handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n′,n′-tetraacetic acid (EGTA) to measure Ca2+ fluxes. Muscles were subjected to both current and voltage clamp conditions.MethodsStandard and confocal fluorescence microscopy was used to study Ca2+ release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student’s test with significance set at p < 0.05.ResultsWe found that the peak value of Ca2+ fluxes elicited by single action potentials was significantly reduced by 15–20 % in C3KO fibers, but the kinetics was unaltered. Ca2+ release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca2+ release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca2+ release in C3KO mice but reduced peak Ca2+ fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca2+ release at all triads consistent with a homogenous reduction of functional voltage activated Ca2+ release sites.ConclusionsOverall, these results suggest that decreased Ca2+ release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

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

The role of dye affinity in optical measurements of Cai(2+) transients in cardiac muscle.

Previous experiments in cultures of neonatal rat myocytes demonstrated that the shape of Cai(2+) transients measured using high-affinity Ca(2+)-sensitive dyes may be misrepresented. The purpose of this study was to examine the role of dye affinity in Cai(2+) measurements in intact adult cardiac tissue by comparing optical recordings obtained with high- and low-affinity dyes. Experiments were carried out in porcine left ventricular (LV) wedge preparations stained locally by intramural injection via microcapillaries (diameter = 150 μm) with a low-affinity Ca(2+)-sensitive dye Fluo-4FF or Fluo-2LA (nominal Kd, ~7-10 μmol/l), high-affinity dye Rhod-2 (Kd = 0.57 μmol/l), and Fluo-4 or Fluo-2MA (Kd, ~0.4 μmol/l); in addition, tissue was stained with transmembrane potential (Vm)-sensitive dye RH-237. Optical recordings of Vm and Cai(2+) were made using optical fibers (diameter = 325 μm) glued with the microcapillaries. The durations of Cai(2+) transients measured at 50% level of recovery (CaD50) using high-affinity Fluo-4/Fluo-2MA dyes were up to ~81% longer than those measured with low-affinity Fluo-4FF/Fluo-2LA at long pacing cycle lengths (CL). In Fluo-4/Fluo-2MA measurements at long CLs, Cai(2+) transients often (~50% of cases) exhibited slow upstroke rise and extended plateau. In Rhod-2 measurements, CaD50 was moderately longer (up to ~35%) than in Fluo-4FF recordings, but Cai(2+) transient shapes were similar. In all series of measurements, mean action potential duration values were not significantly different (P > 0.05). The delays between Vm and Cai(2+) upstrokes were comparable for low- and high-affinity dyes (P > 0.05). In conclusion, measurements of Cai(2+) transient in ventricular myocardium are strongly affected by the affinity of Ca(2+) dyes. The high-affinity dyes may overestimate the duration and alter the shape of Cai(2+) transients.

Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non‐fast‐spiking GABAergic hippocampal interneurons using two‐photon laser microscopy and high‐ and low‐affinity dyes

Using two-photon laser microscopy, high- and low-affinity dyes and patch clamp electrophysiology, we successfully measured somatic stimulation-evoked Ca(2+) transients simultaneously in the dendrites and axonal boutons of the same non-fast-spiking GABAergic interneurons in acute slice preparations obtained from hippocampal area CA1. The advantage of the acute preparation is that both neuronal connections and anatomy are maintained. Calculated as unperturbed values, the amplitudes of Ca(2+) transients and changes in [Ca(2+)]i in response to somatic single or burst stimulation were much higher in boutons (428 nM/AP) than in dendrites (49 nM/AP), leading to the conclusion that the much greater influx of Ca(2+) observed in terminals might be due to a higher density of N-type voltage-sensitive Ca(2+) channels compared to the L-type channels present in dendrites. Whereas the decay of Ca(2+) transients recorded in dendrites was primarily mono-exponential, the decay in boutons was bi-exponential, as indicated by an initial fast phase, followed by a much slower reduction in fluorescence intensity. The extrusion of Ca(2+) was much faster in boutons than in dendrites. To avoid saturation effects and the flawed conversion of fluorescence measures of [Ca(2+)]i, we assessed the limits of [Ca(2+)] measurements (which ranged between 6 and 82% of the applied dye saturation) when high- and low-affinity dyes were applied at different concentrations. When two APs were delivered at a high frequency (>3 Hz) of stimulation, the low-affinity indicators OGB-6F (KD = 3.0 μM) and OGB-5N (KD = 20 μM) were able to accurately reflect the changes in ΔF/F produced by the consecutive APs. There was no difference in the endogenous buffer capacity (κE), which can shape Ca(2+) signals, calculated in dendrites (κE = 354) or boutons (κE = 458).

Flux in Artificial Ca Sparks Generated by 2-Photon Release from a Novel Cage Confocally Imaged at Microsecond Resolution

Control of calcium signaling in striated muscle relies on concurrent actions of calcium ions to promote and inhibit release channel opening. To understand these actions we developed artificial Ca sparks generated by 2-photon (2P) release from NDBF-EGTA (Momotake, Nature Methods 2006) as quantifiable local stimuli. A “Dual Scanner” (Zeiss) delivers IR laser flashes through a LSM 510 scanner, while rapidly imaging fluorescence of a [Ca2+] monitor via a slit scanner (5-LIVE; ca 100 μs/line). Ca sparks of 0.1 to 10 μM (A, B) are elicited in a droplet after microseconds of 2P irradiation at 720 nm and imaged with the low affinity dye fluo 4FF. Reaction-diffusion analysis (Rios, JGP 1999) yields the flux of Ca photorelease (C). This flux, which initially reaches several hundred mM/s, decays with τ of 2-3 ms. The technique is used to measure physico-chemical properties of calcium ligands, including bio-sensors. Applied inside muscle fibers (Figueroa, this meeting) it serves to quantitatively characterize calcium control in cells.Instrument purchased with a S10 NCRR award and Hasterlik Family matching funds. Supported by NIAMS, NHLBI/NIH and MDA.View Large Image | View Hi-Res Image | Download PowerPoint Slide

CICR and Calcium-Dependent Inactivation, Quantified Through the Response to Artificial Ca Sparks in Single Muscle Cells

Local calcium stimuli (artificial sparks) generated by 2-photon breakdown of the cage NDBF-EGTA were applied to evoke Ca release from the SR in single skeletal or cardiac muscle cells undergoing fast Ca imaging with the low affinity dye fluo 4FF. The figure shows selected sequential images of the Ca transient generated by a frog skeletal muscle fiber with permeabilized plasmalemma, in response to a spark (elicited outside the fiber to avoid photodamage). Two types of responses were observed: (i) an all-or-none wave -shown- that propagates over the entire cell and (ii) graded responses, which fail to propagate. Release analysis (Rios, JGP 1999; Figueroa, this meeting) separates SR release from simple diffusion of photo-released Ca into cells. The technique yields a sensitive measure of threshold [Ca2+] for release activation, which in the example (0.3 mM [Mg2+]cyto) was 1 μM, and can monitor inactivation by combining multiple stimuli. Modeling of these responses aims at describing quantitatively the properties of activation, as well as the roles of inactivation and depletion in the control of Ca release. Other details and acknowledgments are presented elsewhere (Figueroa, this meeting.)View Large Image | View Hi-Res Image | Download PowerPoint Slide

Details of Ca2+ dynamics matter

Details of Ca²⁺ dynamics matter

Efficient Ca2+ buffering in fast-spiking basket cells of rat hippocampus.

Fast-spiking parvalbumin-expressing basket cells (BCs) represent a major type of inhibitory interneuron in the hippocampus. These cells inhibit principal cells in a temporally precise manner and are involved in the generation of network oscillations. Although BCs show a unique expression profile of Ca(2+)-permeable receptors, Ca(2+)-binding proteins and Ca(2+)-dependent signalling molecules, physiological Ca(2+) signalling in these interneurons has not been investigated. To study action potential (AP)-induced dendritic Ca(2+) influx and buffering, we combined whole-cell patch-clamp recordings with ratiometric Ca(2+) imaging from the proximal apical dendrites of rigorously identified BCs in acute slices, using the high-affinity Ca(2+) indicator fura-2 or the low-affinity dye fura-FF. Single APs evoked dendritic Ca(2+) transients with small amplitude. Bursts of APs evoked Ca(2+) transients with amplitudes that increased linearly with AP number. Analysis of Ca(2+) transients under steady-state conditions with different fura-2 concentrations and during loading with 200 microm fura-2 indicated that the endogenous Ca(2+)-binding ratio was approximately 200 (kappa(S) = 202 +/- 26 for the loading experiments). The peak amplitude of the Ca(2+) transients measured directly with 100 microm fura-FF was 39 nm AP(-1). At approximately 23 degrees C, the decay time constant of the Ca(2+) transients was 390 ms, corresponding to an extrusion rate of approximately 600 s(-1). At 34 degrees C, the decay time constant was 203 ms and the corresponding extrusion rate was approximately 1100 s(-1). At both temperatures, continuous theta-burst activity with three to five APs per theta cycle, as occurs in vivo during exploration, led to a moderate increase in the global Ca(2+) concentration that was proportional to AP number, whereas more intense stimulation was required to reach micromolar Ca(2+) concentrations and to shift Ca(2+) signalling into a non-linear regime. In conclusion, dentate gyrus BCs show a high endogenous Ca(2+)-binding ratio, a small AP-induced dendritic Ca(2+) influx, and a relatively slow Ca(2+) extrusion. These specific buffering properties of BCs will sharpen the time course of local Ca(2+) signals, while prolonging the decay of global Ca(2+) signals.

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca2+ inside the ER ([Ca2+]ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca2+]ER (EC50) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca2+]ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 µM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 µM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca2+. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland.

Measurement of Ca2+signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

Na+-Dependent Sources of Intra-Axonal Ca2+Release in Rat Optic Nerve duringIn VitroChemical Ischemia

The contribution of intracellular stores to axonal Ca2+ overload during chemical ischemia in vitro was examined by confocal microscopy. Ca2+ accumulation was measured by fluo-4 dextran (low-affinity dye, KD approximately 4 microM) or by Oregon Green 488 BAPTA-1 dextran (highaffinity dye, KD approximately 450 nM). Axonal Na+ was measured using CoroNa Green. Ischemia in CSF containing 2 mM Ca2+ caused an approximately 3.5-fold increase in fluo-4 emission after 30 min, indicating a large axonal Ca2+ rise well into the micromolar range. Axonal Na+ accumulation was enhanced by veratridine and reduced, but not abolished, by TTX. Ischemia in Ca2+-free (plus BAPTA) perfusate resulted in a smaller but consistent Ca2+ increase monitored by Oregon Green 488 BAPTA-1, indicating release from intracellular sources. This release was eliminated in large part when Na+ influx was reduced by replacement with N-methyl-D-glucamine (NMDG+; even in depolarizing high K+ perfusate), Li+, or by the application of TTX and significantly increased by veratridine. Intracellular release also was reduced significantly by neomycin or 1-(6-[(17beta-methoxyestra-1,3,5 [10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank]) (phospholipase C inhibitors), heparin [inositol trisphosphate (IP3) receptor blocker], or 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; mitochondrial Na+/Ca2+ exchange inhibitor) as well as ryanodine. Combining CGP37157 with U73122 [GenBank] or heparin decreased the response more than either agent alone and significantly improved electrophysiological recovery. Our conclusion is that intra-axonal Ca2+ release during ischemia in rat optic nerve is mainly dependent on Na+ influx. This Na+ accumulation stimulates three distinct intra-axonal sources of Ca2+: (1) the mitochondrial Na+/Ca2+ exchanger driven in the Na+ import/Ca2+ export mode, (2) positive modulation of ryanodine receptors, and (3) promotion of IP3 generation by phospholipase C.

Simultaneous optical imaging of membrane potential and intracellular calcium

Imaging of membrane potential (V m) and intracellular calcium (Ca i 2+) is important for studying mechanisms of cardiac excitation, arrhythmias and defibrillation. We have developed an optical technique for simultaneous mapping of V m and Ca i 2+ in cultured cell monolayers using fluorescent V m- and Ca 2+-sensitive dyes. Cultures of neonatal rat myocytes were double-stained with dyes RH-237 (V m) and an analog of Fluo-3 or Rhod-2 dyes (Ca 2+). These dyes have overlapping excitation spectra, allowing simultaneous excitation at the same wavelength range, and separate emission spectra allowing division of the fluorescent light into components sensitive to V m and Ca i 2+, which were measured with two 16 x 16 photodiode arrays. It was found that Ca i 2+ measurements were strongly dependent on the properties of Ca 2+-sensitive dyes. Thus, high-affinity Ca 2+ dyes such as Fluo-4 and Rhod-2 reported Ca i 2+ transients approximately twice as long as those reported by low-affinity dyes Fluo-4FF and Rhod-FF. In addition, dyes with different affinities resulted in different measurements of Ca i 2+ responses to electrical shocks. When shocks were applied during the early plateau phase of the action potential, low-affinity dyes reported transient Ca i 2+ decreases at sites of both negative and positive V m changes. In contrast, high-affinity Ca 2+ dyes reported only a negligible change of plateau Ca i 2+ and a large elevation of diastolic Ca i 2+. These discrepancies between high- and low-affinity dyes were explained by a model of dye-ion interaction, which indicated that apparently long Ca i 2+ transients and Ca i 2+ responses to electrical shocks measured with high-affinity dyes were due to their non-linear response. These results indicate that optical measurements of Ca i 2+ transient duration and shock-induced Ca i 2+ changes require the use of low-affinity Ca i 2+ dyes.

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca2+, which is mediated by Ca2+-ATPase (SERCA), and passive leakage of Ca2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca2+ in the ER ([Ca2+]ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca2+ concentration in the cytoplasm ([Ca2+] i ) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca2+ from the ER; this is confirmed by a gradual drop in the [Ca2+]ER. Such a leakage of Ca2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca2+ from the ER. This effect did not depend on the functioning of SERCA, InsP3R, or RyR. Therefore, passive leakage of Ca2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane.

The acrosomal vesicle of mouse sperm is a calcium store.

Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.

Cross-tolerance to otherwise lethal N-methyl- D-aspartate and oxygen–glucose deprivation in preconditioned cortical cultures

In vitro ischemic preconditioning induced by subjecting rat cortical cultures to nonlethal oxygen–glucose deprivation protects against a subsequent exposure to otherwise lethal oxygen–glucose deprivation. We provide evidence that attenuation of the postsynaptic N-methyl- D-aspartate (NMDA) receptor- and Ca 2+-dependent neurotoxicity underlies oxygen–glucose deprivation tolerance. It is demonstrated that extended tolerance to otherwise lethal NMDA or oxygen–glucose deprivation can be induced by either of their sublethal forms of preconditioning. These four pathways are linked, since NMDA receptor blockade during preconditioning by oxygen–glucose deprivation eliminates tolerance. These results suggest that NMDA tolerance, induced by nonlethal activation of these receptors during oxygen–glucose deprivation preconditioning, underlies oxygen–glucose deprivation tolerance. Several neurotoxic downstream Ca 2+-dependent signaling events specifically linked to NMDA receptor activation are attenuated during otherwise lethal oxygen–glucose deprivation in preconditioned cultures. Specifically, calpain activation, as well as degradation of microtubule-associated protein-2 and postsynaptic density-95, are attenuated 2 h following otherwise lethal NMDA treatment alone or oxygen–glucose deprivation in preconditioned cultures. Formation of microtubule-associated protein-2-labeled dendritic varicosities is also attenuated in preconditioned cultures within 1 h of lethal oxygen–glucose deprivation or NMDA application. Intracellular Ca 2+ levels, measured using the high- or low-affinity dyes Fluo-4 ( K d≅345 nM) or Fluo-4FF ( K d≅9.7 μM) respectively, are markedly attenuated during lethal oxygen–glucose deprivation in preconditioned cultures. Collectively, the results suggest the attenuation of the postsynaptic NMDA-mediated component of otherwise lethal oxygen–glucose deprivation through the suppression of Ca 2+-dependent neurotoxic signaling, a mechanism that is initially induced by transient nonlethal activation of this receptor during ischemic preconditioning.

Induction of spontaneous recurrent epileptiform discharges causes long-term changes in intracellular calcium homeostatic mechanisms

Calcium and calcium-dependent systems have been long implicated in the induction of epilepsy. We have previously observed that intracellular calcium ([Ca2+]i) levels remain elevated in cells undergoing epileptogenesis in the hippocampal neuronal culture (HNC) model. In this study, we employed the hippocampal neuronal culture (HNC) model of in vitro ‘epilepsy’ which produces spontaneous recurrent epileptiform discharges (SREDs) for the life of the neurons in culture to investigate alterations in [Ca2+]ihomeostatic mechanisms that may be associated with the ‘epileptic’ phenotype. [Ca2+]iimaging fluorescence microscopy was performed on control and ‘epileptic’ neurons with two different fluorescent dyes ranging from high to low affinities for [Ca2+]i. We measured baseline [Ca2+]ilevels and the ability to restore resting [Ca2+]ilevels after a brief 2-min exposure to the excitatory amino acid glutamate in control neurons and neurons with SREDs. Neurons manifesting SREDs had statistically significantly higher baseline [Ca2+]ilevels that persisted for the life of the culture. In addition, the ‘epileptic’ phenotype was associated with an inability to rapidly restore [Ca2+]ilevels to baseline following a glutamate induced [Ca2+]iload. The use of the low affinity dye Fura-FF demonstrated that the difference in restoring baseline [Ca2+]ilevels was not due to saturation of the high affinity dye Indo-1, which was utilized for evaluating the [Ca2+]ikinetics at lower [Ca2+]ilevels. Peak [Ca2+]ilevels in response to glutamate were the same in both ‘epileptic’ and control neurons. While [Ca2+]ilevels recovered in approximately 30min in control cells, it took more than 90min to reach baseline levels in cells manifesting SREDs. Alterations of [Ca2+]ihomeostatic mechanisms observed with the ‘epileptic’ phenotype were shown to be independent of the presence of continuous SREDs and persisted for the life of the neurons in culture. Epileptogenesis was shown not to affect the degree or duration of glutamate induced neuronal depolarization in comparing control and ‘epileptic’ neurons. The results indicate that epileptogenesis in this in vitro model produced long-lasting alterations in [Ca2+]iregulation that may underlie the ‘epileptic’ phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.

Action potential-evoked Ca2+ signals and calcium channels in axons of developing rat cerebellar interneurones.

Axonal [Ca2+] transients evoked by action potential (AP) propagation were studied by monitoring the fluorescence of the high-affinity calcium-sensitive dye Oregon Green 488 BAPTA-1, introduced through whole-cell recording pipettes in the molecular layer of interneurones from cerebellar slices of young rats. The spatiotemporal profile of Ca2+-dependent fluorescence changes was analysed in well-focused axonal stretches a few tens of micrometres long. AP-evoked Ca2+ signals were heterogeneously distributed along axons, with the largest and fastest responses appearing in hot spots on average approximately 5 microm apart. The spatial distribution of fluorescence responses was independent of the position of the focal plane, uncorrelated to basal dye fluorescence, and independent of dye concentration. Recordings using the low-affinity dye mag-fura-2 and a Cs+-based intracellular solution revealed a similar pattern of hot spots in response to depolarisation, ruling out measurement artefacts or possible effects of inhomogeneous dye distribution in the generation of hot spots. Fluorescence responses to a short train of APs in hot spots decreased by 41-76 % after bath perfusion of omega-conotoxin MVIIC (5-6 microM), and by 17-65 % after application of omega-agatoxin IVA (500 nM). omega-Conotoxin GVIA (1 microM) had a variable, small effect (0-31 % inhibition), and nimodipine (5 microM) had none. Somatically recorded voltage-gated currents during depolarising pulses were unaffected in all cases. These data indicate that P/Q-type Ca2+ channels, and to a lesser extent N-type channels, are responsible for a large fraction of the [Ca2+] rise in axonalhot spots. [Ca2+] responses never failed during low-frequency (<= 0.5 Hz) stimulation, indicating reliable AP propagation to the imaged sites. Axonal branching points coincided with a hot spot in approximately 50 % of the cases. The spacing of presynaptic varicosities, as determined by a morphological analysis of Neurobiotin-filled axons, was approximately 10 times larger than the one measured for hot spots. The latter is comparable to the spacing reported for varicosities in mature animals. We discuss the nature of hot spots, considering as the most parsimonious explanation that they represent functional clusters of voltage-dependent Ca2+ channels, and possibly other [Ca2+] sources, marking the position of developing presynaptic terminals before the formation of en passant varicosities.

Presynaptic Ca(2+) influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution.

Presynaptic calcium influx at the inhibitor of the crayfish neuromuscular junction was investigated by measuring fluorescence transients generated by calcium-sensitive dyes. This approach allowed us to correlate presynaptic calcium influx with transmitter release at a high time resolution. Systematic testing of the calcium indicators showed that only low-affinity dyes, with affinities in the range of micromolar, should be used to avoid saturation of dye binding and interference with transmitter release. Presynaptic calcium influx was regulated by slowly increasing the duration of the action potential through progressive block of potassium channels. The amplitude of the calcium transient, measured from a cluster of varicosities, was linearly related to the duration of the action potential with a slope of 1.2. Gradual changes in potassium channel block allowed us to estimate the calcium cooperativity of transmitter release over a 10-fold range in presynaptic calcium influx. Calcium cooperativity measured here exhibited one component with an average value of 3.1. Inspection of simultaneously recorded presynaptic calcium transients and inhibitory postsynaptic currents (IPSCs) showed that prolonged action potentials were associated with a slow rising phase of presynaptic calcium transients, which were matched by a slow rate of rise of IPSCs. The close correlation suggests that fluorescence transients provide information on the rate of calcium influx. Because there is an anatomic mismatch between the presynaptic calcium transient, measured from a cluster of varicosities, and IPSC, measured with two-electrode voltage clamp, macropatch recording was used to monitor inhibitory postsynaptic responses from the same cluster of varicosities from which the calcium transient was measured. Inhibitory postsynaptic responses recorded with the macropatch method exhibited a faster rising phase than that recorded with two-electrode voltage clamp. This difference could be attributed to slight asynchrony of transmitter release due to action potential conduction along fine branches. In conclusion, this report shows that fluorescence transients generated by calcium-sensitive dyes can provide insights to the properties of presynaptic calcium influx, and its correlation with transmitter release, at a high time resolution.

Stimulation-induced changes in [Ca2+] in lizard motor nerve terminals.

1. Motor axons were injected ionophoretically with one of five Ca(2+)-sensitive dyes (fluo-3, Calcium Green-2, Calcium Green-5N, fluo-3FF and Oregon Green BAPTA-5N). Changes in fluorescence (delta F/Frest) within motor terminal boutons following a single action potential and brief stimulus trains were monitored with high temporal resolution using a confocal microscope. 2. Stimulation-induced increases in delta F/Frest were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in delta F/Frest began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. delta F/Frest was graded with bath [Ca2+]. Both delta F/Frest and the EPP were reduced, but not eliminated, by omega-conotoxin GVIA (5-10 microM). 3. For dyes with lower affinity for Ca2+ (e.g. Oregon Green BAPTA-5N, Kd approximately 60 microM) stimulation-induced increases in delta F/Frest were measured in the presence of the K+ channel blocker 3,4-diaminopyridine (3,4-DAP, 100 microM). During brief stimulus trains (4 at 50 Hz) in 3,4-DAP, the EPP exhibited profound depression, but the fluorescence increase associated with each stimulus showed little decrement, suggesting that depression was not mediated by a reduction in Ca2+ entry. 4. For dyes with a higher affinity for Ca2+ (e.g. fluo-3, Kd approximately 0.5-1 microM) stimulation-induced increases in delta F/Frest could also be measured in normal physiological saline. Increases in delta F/Frest were much greater with 3,4-DAP present, but the amplitude decreased with successive stimuli due to partial dye saturation. 5. Calculations suggested that following a single action potential the average [Ca2+] within a bouton increased by up to 150 nM in normal saline and 940 nM in 3,4-DAP. With low affinity dyes the delta F/Frest measured near the membrane had a higher peak amplitude and a faster early decay than that measured in the centre of the bouton, suggesting that substantial spatial [Ca2+] gradients exist within boutons for at least 15 ms following stimulation.