Oregon Green BAPTA-1 Research Articles

Activation of Metabotropic Glutamate Receptor mGlu5 on Nuclear Membranes Mediates Intranuclear Ca2+ Changes in Heterologous Cell Types and Neurons

Nuclear Ca2+ plays a critical role in many cellular functions although its mode (s) of regulation is unclear. This study shows that the metabotropic glutamate receptor, mGlu5, mobilizes nuclear Ca2+ independent of cytosolic Ca2+ regulation. Immunocytochemical, ultrastructural, and subcellular fractionation techniques revealed that the metabotropic glutamate receptor, mGlu5, can be localized to nuclear membranes in heterologous cells as well as midbrain and cortical neurons. Nuclear mGlu5 receptors derived from HEK cells or cortical cell types bound [3H]quisqualate. When loaded with Oregon Green BAPTA, nuclei isolated from mGlu5-expressing HEK cells responded to the addition of glutamate with rapid, oscillatory [Ca2+] elevations that were blocked by antagonist or EGTA. In contrast, carbachol-activation of endogenous muscarinic receptors led to cytoplasmic but not nuclear Ca2+ responses. Similarly, activation of mGlu5 receptors expressed on neuronal nuclei led to sustained Ca2+ oscillatory responses. These results suggest mGlu5 may mediate intranuclear signaling pathways.

Calcium Dynamics, Buffering, and Buffer Saturation in the Boutons of Dentate Granule-Cell Axons in the Hilus

The axons of dentate gyrus granule cells form synapses in the hilus. Ca(2+) signaling was investigated in the boutons of these axons using confocal fluorescence imaging. Boutons were loaded with various concentrations of the Ca(2+) indicator Oregon Green BAPTA-1 by patch-clamping the cell bodies and allowing the dye to diffuse into the axon. Resting free [Ca(2+)] started at 74 nm, rose to approximately 1 microm immediately after an action potential, and then decayed to rest with a time constant of 43 msec (all extrapolated to a dye concentration of zero). Action potential-induced [Ca(2+)] rises were smaller in larger boutons, consistent with a size-independent Ca(2+) channel density of 45/microm(2). Action potential-induced [Ca(2+)] changes varied with dye concentration in a manner consistent with kappa(E) approximately 20 for the ratio of endogenous buffer-bound Ca(2+) to free Ca(2+). During trains of action potentials, [Ca(2+)] increments summed supralinearly by more than that expected from dye saturation. The amount of endogenous Ca(2+) buffering declined as [Ca(2+)] rose, and this saturation indicated a buffer with a dissociation constant of approximately 500 nm and a concentration of approximately 130 microm. This is similar to the dissociation constant of calbindin-D28K, a Ca(2+)-binding protein that is abundant in dentate granule cells. Thus, calbindin-D28K is a good candidate for the Ca(2+) buffer revealed by these experiments. The saturation of endogenous buffer can generate short-term facilitation by amplifying [Ca(2+)] changes during repetitive activity. Buffer saturation may also be relevant to the presynaptic induction of long-term potentiation at synapses formed by dentate granule cells.

Imaging of changes in sarcoplasmic reticulum [Ca2+] using Oregon Green BAPTA 5N and confocal laser scanning microscopy

We describe experiments in which the low affinity indicator Oregon Green BAPTA 5N was used to record the spatially resolved changes in [Ca2+] from intracellular stores in rat gastric myocytes. Cells were loaded with the membrane permeant form of the indicator and imaged using a confocal scanning laser microscope. In doubly stained cells the Oregon Green signal colocalized with BIODIPY 558/568 Brefeldin A, a label for the endo/sarcoplasmic reticulum (SR) and Golgi apparatus. Oregon Green BAPTA 5N was calibrated in gastric myocytes, giving an in situ Kd of 90μM. The resting free [Ca2+] within the SR averaged 65μM. A reversible decrease in Oregon Green fluorescence was observed on bath application of Inositol triphosphate (IP3) (10μM) to permeabilized cells. Similar changes were also observed when cyclopiazonic acid (5μM) was applied to intact myocytes, again with recovery of store [Ca2+] following drug washout. Identical patterns of Ca2+ depletion were seen when caffeine (1μM) and carbachol (10μM) were applied sequentially to the same cells, suggesting that activation of ryanodine and IP3-sensitive channels can result in the release of Ca2+ from the same regions of the SR.

Integration of calcium signals by calmodulin in rat sensory neurons.

We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.

Stimulation-Evoked Increases in Cytosolic [Ca2+] in Mouse Motor Nerve Terminals Are Limited by Mitochondrial Uptake and Are Temperature-Dependent

Increases in cytosolic [Ca(2+)] evoked by trains of action potentials (20-100 Hz) were recorded from mouse and lizard motor nerve terminals filled with a low-affinity fluorescent indicator, Oregon Green BAPTA 5N. In mouse terminals at near-physiological temperatures (30-38 degrees C), trains of action potentials at 25-100 Hz elicited increases in cytosolic [Ca(2+)] that stabilized at plateau levels that increased with stimulation frequency. Depolarization of mitochondria with carbonylcyanide m-chlorophenylhydrazone (CCCP) or antimycin A1 caused cytosolic [Ca(2+)] to rise to much higher levels during stimulation. Thus, mitochondrial Ca(2+) uptake contributes importantly to limiting the rise of cytosolic [Ca(2+)] during repetitive stimulation. In mouse terminals, the stimulation-induced increase in cytosolic [Ca(2+)] was highly temperature-dependent over the range 18-38 degrees C, with greater increases at lower temperatures. At the lower temperatures, application of CCCP continued to depolarize mitochondria but produced a much smaller increase in the cytosolic [Ca(2+)] transient evoked by repetitive stimulation. This result suggests that the larger amplitude of the stimulation-induced cytosolic [Ca(2+)] transient at lower temperatures was attributable in part to reduced mitochondrial Ca(2+) uptake. In contrast, the stimulation-induced increases in cytosolic [Ca(2+)] measured in lizard motor terminals showed little or no temperature-dependence over the range 18-33 degrees C.

Estimating Intracellular Calcium Concentrations and Buffering without Wavelength Ratioing

We describe a method for determining intracellular free calcium concentration ([Ca 2+]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca 2+] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca 2+] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca 2+] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid). Resting [Ca 2+] was 32–59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca 2+] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44–80) and peak [Ca 2+] changes at zero added buffer (178–312 nM). In young animals (postnatal days 14–17) our results were comparable to previous estimates obtained by ratiometric methods (Helmchen et al., 1996, Biophys. J. 70:1069–1081), and no significant differences were seen in older animals (P24–28). We expect our method to be widely applicable to measurements of [Ca 2+] and [Ca 2+]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.

Fiber optic pH/Ca 2+ fluorescence microsensor based on spectral processing of sensing signals

In this paper, we describe the fabrication of a dual function pH/Ca 2+ fluorescence microsensor. Dextran conjugates of the fluorescence indicators Oregon Green BAPTA-1, a calcium ion probe, and SNARF (seminaphtorhodafluor)-1, a ratiometric pH probe, are immobilized in a 2-hydroxyethyl methacrylate (PHEMA) matrix support adsorbed to the distal end of an optical fiber. Both dyes are excited at 488 nm. The emission peak maximum of Oregon Green Bapta-1 is at 520 nm, and the emission peak maxima of SNARF-1 are at 584 and 630 nm. The pH and [Ca 2+] in the analyte sample are determined by spectral processing of the fluorescence sensing signals. The sensor covers a pH range between pH 6 and pH 8, and calcium ion concentrations between 0.03 and 1.5 μM. The response times of the sensor are about 1 s for pH and 5 s for [Ca 2+] changes. The sensor is highly reversible and maintains its function for over 10 days of continuous use. The sensor is stored for 3 months in a dry, light tight environment, at room temperature with no significant degradation of its analytical properties.

Single-bouton-mediated synaptic transmission: postsynaptic conductance changes in their relationship with presynaptic calcium signals.

Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals. This paper describes a method that was designed to activate transmitter release from solitary synaptic boutons in culture. Neurons from the embryonic rat superior colliculus were grown at low density and double-loaded with a fluorescent marker of synaptic vesicles (FM1-43 or RH414) and a fluorescent Ca2+ indicator (Fura-2, Mag-fura-2, Oregon Green BAPTA-1 or Oregon Green BAPTA-5N). Action potential generation was blocked by tetrodotoxin. Appropriate synaptic boutons were selected under phase-contrast and fluorescence illumination at a magnification of 1000. They were activated by short electrical pulses via a fine-tipped glass pipette filled with bath solution. Presynaptic Ca2+ transients were measured in a region delineated by the FM1-43/RH414 fluorescence. By simultaneous presynaptic Ca2+ imaging and whole-cell recording of postsynaptic responses to single depolarizing pulses, the quantitative relationships between pre- and postsynaptic parameters of synaptic strength in a small synapse of central origin could, for the first time, be analysed. The experiments showed that the average postsynaptic currents depend strongly on the size of the presynaptic Ca2+ transients. However, at any level of presynaptic Ca2+ concentration postsynaptic responses fluctuated in amplitude.

Two-photon laser-scanning microscopy: tests of objective lenses and Ca 2+ probes

The characteristics of objective lenses and Ca 2+-sensitive probes were examined for imaging with a two-photon laser-scanning microscope (TP-LSM). The brightness of the images of beads taken by different objectives greatly varied and depended predominantly on their numerical aperture ( NA) and less on transmittance and chirping effects. Lateral and axial resolutions, dx and dz, defined as the half decay length of fluorescence intensity of the image of a spherical bead (0.3 m) were 0.12 and 0.42 gm (objective; 40 × /0.75). They are far better than those of confocal microscopes (0.3 and 1.5 μm respectively) measured similarly (Kuba et al., 1994). dx linearly increased with an increase in 1 NA , while dz linearly increased with an increase in n (NA) 2 ( n, refractive index) except for an objective of large NA (1.3). The coverslip compensation of objective lenses greatly affected the shape of the X– Z scanned images of 5.0 μm beads as well as resolutions, indicating a large effect of spherical aberration. Two-photon excitation spectra of Ca 2+-sensitive fluorescent probes, indo-1, fura-2 and Oregon Green BAPTA-1, lied in a wavelength range shorter than twice that activated by one-photon absorption, while emission spectra were unchanged. Three-dimensional images of a cultured hippocampal neurone loaded with Oregon Green BAPTA-1 showed fine structures of spines, dendrites and axons, while imaging with FM1-43 localized presynaptic boutons and demonstrated synaptic vesicle turnover. Dyes bleached little during the recording of 100 sectioned images. These characteristics of TP-LSM as well as its ability to image deeper tissues provide excellent means to study dynamic, spatial changes in intracellular substances and structures. To achieve the good performance of a TP-LSM, however, the relevant usage of appropriate objectives and fluorescent probes are required.

Evidence that mitochondria buffer physiological Ca2+ loads in lizard motor nerve terminals.

1. Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG-5N). Mitochondrial [Ca2+] was measured using rhod-2, bath loaded as dihydrorhod-2. 2. In response to 100-250 stimuli at 25-100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation. 3. When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+]. 4. After addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1-2 microM) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+]. 5. These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.