Calcium Green-5N Research Articles

High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations

We measured glutamate-stimulated increases in intracellular free Ca 2+ concentrations ([Ca 2+] i) in cultured rat forebrain neurons loaded with both a high- and a low-affinity Ca 2+ indicator. In these dual-dye studies, the high-affinity indicators Fluo-3 and Fura-2 gave both qualitatively and quantitatively different results than the low-affinity indicators Mag-fura-2 and Calcium Green-5N. The glutamate-stimulated peak [Ca 2+] i reported using Fluo-3 and Fura-2 were less than 6 μM while the low-affinity indicators Mag-fura-2 and Calcium Green-5N indicated [Ca 2+] i responses were more than 12 μM. The shapes of the responses obtained with the two types of dyes were also different, and only the low-affinity indicators effectively demonstrated that [Ca 2+] i continues to rise during prolonged (5-min) stimulations. These dual-dye studies also revealed that kainate- and depolarization-induced [Ca 2+] i responses could be differentiated from glutamate responses only with the low-affinity indicators. These results suggest that Fluo-3 and Fura-2 underestimate [Ca 2+] i induced by excitotoxic glutamate stimuli and that these responses are greater than have previously been reported. These studies also reveal, in contrast to previous reports, that excitotoxic stimuli do indeed cause increases in [Ca 2+] i that are greater than those produced by non-toxic stimuli.

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.

Rapid Coupling of Calcium Release to Depolarization inLimulus polyphemusVentral Photoreceptors as Revealed by Microphotolysis and Confocal Microscopy

Microphotolysis and confocal microscopy were used to investigate the timing of calcium release and of the electrical response in Limulus polyphemus ventral photoreceptors. The fluorescent dyes Fluo-3 and Calcium Green-5N were used to monitor local Ca2+ elevations. Photolysis of caged inositol trisphosphate (InsP3) close to the plasma membrane of the light-sensitive rhabdomeral (R-) lobe resulted in Ca2+ elevation within 10-20 msec, 20-45 msec before the physiological response to light normally would be detected. Inward ionic current flow and depolarization followed InsP3-induced calcium release within 2.5 +/- 3.3 msec. Voltage-clamping the cells and removal of extracellular Ca2+ did not affect the timing of the Ca2+ elevation that followed the photolysis of caged InsP3 or its relationship to the electrical response. In contrast to the physiological response to light, which only released calcium within the R-lobe, photolysis of InsP3 elevated Cai in both lobes, although with much greater effect in the R-lobe, as compared with the bulk of the A-lobe, suggesting the presence of InsP3-sensitive calcium stores in both lobes. Photolysis of caged calcium [o-nitrophenyl EGTA (NPE)] at the edge of the R-lobe activated an inward ionic current within 1.8 +/- 0.7 msec. This NPE-induced current reversed at a membrane potential of 10 +/- 6 mV in the range typical of that of the light-activated current under physiological conditions. Calcium release, therefore, activates an inward current rapidly enough to contribute to the electrical response to light.

Monitoring calcium in turtle hair cells with a calcium-activated potassium channel.

1. An apamin-sensitive Ca(2+)-activated K+ channel was characterized in turtle hair cells and utilized to monitor submembranous intracellular Ca2+ and to evaluate the concentration of the mobile endogenous calcium buffer. 2. Isolated hair cells were voltage clamped with whole-cell patch electrodes filled with a Cs(+)-based intracellular solution to block the large-conductance Ca(2+)-activated K+ (BK) channel. Ca2+ currents evoked by depolarization were followed by inward tail currents lasting several hundred milliseconds. Both the Ca2+ current and slow tail current were abolished by nifedipine. 3. The tail current was carried by K+ and Cs+ (relative permeabilities PCa/PK = 0.22), and was fully blocked by 0.1 microM apamin and half blocked by 5 mM external TEA. These properties suggest the tail current flows through a Ca(2+)-activated K+ channel distinct from the BK channels. 4. Intracellular Ca2+ was imaged with a confocal microscope in hair cells filled with the indicator Calcium Green-5N introduced via the patch pipette. Increases in Ca2+ evoked by depolarization were localized to hotspots on the basolateral surface of the cell. The time course of the tail current closely matched the fast component of the fluorescenece monitored at a hotspot. 5. Ca(2+)-ATPase pump inhibitors thapsigargin, 2,4-di-(t-butyl)hydroquinone (BHQ) and vanadate, which are known to influence calcium regulation in turtle hair cells, prolonged the time course of the tail current, supporting the idea that the channel monitors cytoplasmic Ca2+. 6. The mobile endogenous buffer was estimated by combining perforated-patch and whole-cell recordings on a single cell. After recording tail currents with an amphotericin-perforated patch, the patch was ruptured to obtain the whole-cell mode, thus allowing washout of soluble cytoplasmic proteins and exchange with pipette buffers. By varying the concentration of Ca2+ buffer in the pipette, the mobile endogenous buffer was found to be equivalent to about 1 mM BAPTA.

Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization.

The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, indicating that InsP3 binding did not cause desensitization. When Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP3 caused rapid, partial decreases in fluorescence ratios. Subsequent addition of a maximal InsP3 concentration caused the fluorescence to fall to within 5% of that recorded after ionomycin. Addition of all but the lowest concentrations of InsP3 to stores loaded with the lower affinity indicator, Calcium Green-5N, caused almost complete emptying of the stores at rates that increased with InsP3 concentration. The lowest concentration of InsP3 (10 nM) slowly emptied approximately 80% of the stores, but within 3 min the rate of Ca2+ release slowed leaving approximately 7 microM Ca2+ within the stores, which was then rapidly released by a maximal InsP3 concentration. In stores co-loaded with both indicators, InsP3-evoked Ca2+ release appeared quantal with Fura 2 and largely non-quantal with Calcium Green-5N; the discrepancy is not, therefore, a direct effect of the indicators. The fall in luminal [Ca2+] after activation of InsP3 receptors may, therefore, cause their inactivation, but only after the Ca2+ content of the stores has fallen by approximately 95% to < or = 10 microM.

Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles).

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radio-active filter technique. Trypsin treatment of vesicles resulted in a 3-fold activation of Ca2+ uptake and loss of calmodulin sensitivity. Immunoblotting experiments with an antiserum raised against the Ca2+-ATPase showed that the trypsin activation was accompanied by a decrease in the amount of intact Ca2+-ATPase (111 kD) and by successive appearances of polypeptides of 102 and 99 to 84 kD. 125I-Calmodulin overlays showed that only the intact Ca2+-ATPase bound calmodulin. Removal of the calmodulin-binding domain (about 9 kD) was not enough to obtain full activation. Trypsin proteolysis resulted in a Ca2+ concentration necessary for half-maximal activity of 0.5 [mu]M, whereas a value of about 2 [mu]M was obtained with untreated membranes in the presence of calmodulin. Without trypsin treatment or calmodulin the activity was not saturated even at 57 [mu]M free Ca2+. The data suggest that trypsin digestion and calmodulin activate the cauliflower Ca2+-ATPase by at least partly different mechanisms.

Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat.

1. Simultaneous whole-cell recordings in a rat brain slice preparation are described from presynaptic terminals (calyces of Held) and postsynaptic somata which form an axosomatic synapse in the medial nucleus of the trapezoid body (MNTB). 2. Presynaptic action potentials evoked suprathreshold excitatory postsynaptic potentials (EPSPs). The minimum synaptic delay was around 0.4 ms at 36 degrees C and 0.9 ms at 23-24 degrees C. The amplitude of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated component of the excitatory postsynaptic currents (EPSCs) was 2-13 nA (at -80 mV). 3. Current-voltage relations showed that presynaptic Ca2+ channels were of the high voltage-activated type. 4. A single action potential evoked a presynaptic fluorescence transient that decayed with a time constant of 0.3-0.7 s, depending on the concentration (60-200 microM) of the Ca2+ indicator Calcium Green-5N (CG-5N). The peak amplitude of the [Ca2+]i transient was severalfold larger in the terminal than in the preterminal axon. 5. EPSC peak amplitudes were stable for more than 30 min after establishing the whole-cell configuration in the presynaptic terminal when the pipette contained 50 microM BAPTA. In contrast, with 1 mM BAPTA, peak amplitudes of EPSCs were reduced to one-third. 6. Trains of presynaptic action potentials evoked EPSCs with progressively smaller amplitudes. Little change was observed in the depression when the terminals were dialysed with 50 microM BAPTA, whereas depression was reduced with 1 mM BAPTA. 7. In low (1 mM) [Ca2+]o, facilitation instead of depression of EPSCs was observed. 8. The effects of presynaptic BAPTA suggest that the endogenous mobile Ca2+ buffer capacity of giant presynaptic terminals in the MNTB is lower than in other terminals of fast transmitting synapses.

Modulation of Na +,Ca 2+ exchange current by EGTA calcium buffering in giant cardiac membrane patches

Effects of calcium buffering by EGTA were examined on sodium-calcium exchange currents ( I Naca) in inside-out giant cardiac membrane patches. Free calcium concentrations (Ca 2+) were monitored with a calcium electrode and a fluorescent calcium indicator (Calcium Green-5N). With 1.8 μM cytoplasmic Ca 2+, inward I Naca increased 2-fold at −120 mV when EGTA concentration was increased from 0.1 mM to 10 mM (37°C and 140 mM extracellular sodium). Stimulation by EGTA was decreased or abolished under conditions of attenuated exchanger turnover rate: temperature < 30°C, extracellular sodium < 70 mM, and membrane potential > + 60 mV. EGTA concentration had no effect on outward I Naca with 100 mM cytoplasmic Na + and 0.8 μM cytoplasmic Ca 2+, conditions under which the current inactivated by about 70%. EGTA (0.1–10 mM) and BAPTA (10 mM) inhibited the current by about 80% when the outward I Naca was stimulated by 2 mM cytoplasmic ATP or by phosphatidylserine. The apparent K i for EGTA was 0.2 mM. The electroneutral calcium ionophore, A23187, activated outward I Naca even in presence of 10 mM EGTA. Our results are consistent with EGTA acting as a simple calcium buffer with no direct effect on the exchanger. At low concentrations of EGTA, inhibition of the inward I Naca is expected due to submembrane calcium depletion by the exchanger; enhancement of the outward I Naca at low EGTA concentrations is expected because submembrane calcium accumulates and activates I Naca via regulatory calcium binding sites.

Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.

Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a single synaptic climbing fiber response, a so-called complex spike, resulted in a transient elevation of [Ca2+]i that showed distinct differences among various subcellular compartments. With conventional imaging, the Ca2+ signals were prominent in the dendrites and almost absent in the soma. Confocal recordings from the somatic region, however, revealed steep transient increases in [Ca2+]i that were confined to a submembrane shell of 2- to 3-microns thickness. In the central parts of the soma [Ca2+]i increases were much slower and had smaller amplitudes. The kinetics and amplitudes of the changes in [Ca2+]i were analyzed in more detail by using the fast, low-affinity Ca2+ indicator Calcium Green-5N (Kd approximately 17 microM). We found that brief depolarizing pulses produced [Ca2+]i increases in a narrow somatic submembrane shell that resembled those seen in the dendrites. These results provide direct experimental evidence that the surface-to-volume ratio is a critical determinant of the spatiotemporal pattern of Ca2+ signals evoked by synaptic activity in neurons.

Measurement of cytosolic Ca2+ concentration in Limulus ventral photoreceptors using fluorescent dyes.

Several Ca-sensitive fluorescent dyes (fura-2, mag-fura-2 and Calcium Green-5N) were used to measure intracellular calcium ion concentration, Cai, accompanying light-induced excitation of Limulus ventral nerve photoreceptors. A ratiometric procedure was developed for quantification of Calcium Green-5N fluorescence. A mixture of Calcium Green-5N and a Ca-insensitive dye, ANTS, was injected in the cell and the fluorescence intensities of both dyes were used to calculate the spatial average of Cai within the light-sensitive R lobe of the photoreceptor. In dark-adapted photoreceptors, the initial Cai was 0.40 +/- 0.22 microM (SD, n = 7) as measured with fura-2. Cai peaked in the light-sensitive R lobe at 700-900 ms after the onset of an intense measuring light step, when the spatial average of Cai within the R lobe reached 68 +/- 14 and 62 +/- 37 microM (SD, n = 5) as measured with mag-fura-2 and Calcium Green-5N, respectively. The rate of Cai rise was calculated to be approximately 350 microM/s under the measuring conditions. The resting level of Mg2+ was estimated to be 1.9 +/- 0.9 mM, calculated from mag-fura-2 measurements. To investigate the effect of adapting light on the initial Cai level in the R lobe, a 1-min step of 420 nm background light was applied before each measurement. The first significant (P < 0.05) change in the initial level of Cai occurred even at the lowest adapting light intensity, which delivered approximately 3 x 10(3) effective photons/s. The relative sensitivity of the light-adapted photoreceptors was linearly related to the relative Cai on a double log plot with slope between -4.3 and -5.3. We were unable to detect a Cai rise preceding the light-activated receptor potential. The Cai rise, measured with Calcium Green-5N, lagged 14 +/- 5 ms (SD, n = 32) behind the onset of the receptor potential at room temperature in normal ASW. In the absence of extracellular Ca2+ and at 10 degrees C, this lag increased to 44 +/- 12 ms (SD, n = 17).

The light response of drosophila photoreceptors is accompanied by an increase in cellular calcium: Effects of specific mutations

Photoreceptors of dissociated Drosophila retinae were loaded with the fluorescent Ca 2+ indicators, fluo-3 and Calcium Green-5N. In fluo-3-loaded, wild-type photoreceptors, a rapid increase in fluorescence (Ca 2+ signal) accompanied the light-evoked inward current. Removal of extracellular Ca 2+ greatly reduced the Ca 2+ signal, indicating Ca 2+ influx as its major cause. In Calcium Green-5N-loaded trp mutants, which lack a large fraction of the Ca 2+ permeability underlying the light-evoked inward current, the Ca 2+ signal was smaller relative to wild-type photoreceptors. Fluo-3-loaded norpA mutant photoreceptors, which lack a light-activated phospholipase C, generated no light-evoked inward current and no Ca 2+ signal. The phosphoinositide pathway therefore appears necessary for both excitation and changes in cytosolic free Ca 2+ concentration.

Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca 2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons

We describe here the properties of calcium green-5N (CG5N), a novel Ca 2+-sensitive fluorescent probe. CG5N binds Ca 2+ with an affinity of 4.29 ± 0.67 μM. Ca 2+ binding is associated with a 14.7-fold increase in fluorescence. Mg 2+ binds to CG5N with an affinity of 5 mM but produces a very small change in fluorescence. Using CG5N [Ca 2+] i was recorded in single cultured neurons from fetal rat forebrain. Glutamate together with glycine increased [Ca 2+] i by 1.17 ± 0.15 μM. Pretreating cells with 5 mM potassium cyanide significantly potentiated the response to glutamate, resulting in a mean increase of 8.8 ± 2 μM. These results demonstrate that CG5N is a useful indicator for measuring very large changes in [Ca 2+] i in neurons.