Calcium Indicator Dye Research Articles

Measuring Ca 2+-signalling at fertilization in the sea urchin Psammechinus miliaris: Alterations of this Ca 2+-signal by copper and 2,4,6-tribromophenol

During fertilization, eggs undergo a temporary rise in the intracellular concentration of free Ca 2+ ions. Using the membrane permeable acetoxymethylester of the fluorescent calcium indicator dye Fura-2, Fura-2 AM, the Ca 2+-signal at fertilization was not detectable in eggs of the sea urchin Psammechinus miliaris. However, after treatment of the eggs with Fura-2 AM in combination with MK571, an inhibitor for multidrug resistance associated proteins, clear Ca 2+-signals at fertilization could be measured without microinjection of the dye. We used this methodology to detect possible alterations of Ca 2+-signalling at fertilization by exposure of eggs to environmental pollutants. For this purpose, the heavy metal copper, the bromophenol 2,4,6-tribromophenol, the organic compound bisphenol A and the polycyclic aromatic hydrocarbon phenanthrene were tested for their potential to inhibit fertilization success of P. miliaris. Copper and 2,4,6-tribromophenol showed a dose-dependent effect on fertilization rates of P. miliaris and significantly inhibited fertilization at 6.3 µM Cu 2+ and 1 µM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 µM while phenanthrene had no effect up to 56 µM. 6.3 µM copper and 100 µM 2,4,6-tribromophenol significantly increased the Ca 2+-signal at fertilization. This alteration may contribute to the reduced fertilization rates of P. miliaris after exposure to copper and 2,4,6-tribromophenol.

Imaging the Glucagon-Secreting Alpha Cells

Pancreatic islets secrete insulin and glucagon, two critical hormones for the blood glucose homeostasis. Islets are composed mainly of insulin-secreting beta-cells (∼80%) and glucagon-secreting alpha-cells (10-15%). Typically, insulin is secreted after a meal to reduce blood glucose levels, whereas glucagon is released in times of starvation. In order for insulin and glucagon to exert their opposite effects, glucose must also have an inverse effect on the secretory function of the two cell types. While the mechanisms of glucose-stimulated insulin secretion by beta-cells are fairly well understood, the study of the mechanisms underlying glucagon secretion has been hindered by the lack of reliable methods to distinguish between alpha- and beta-cells. Thus, it is not clear how glucose suppresses glucagon secretion. There is still little agreement as to whether this inhibition is a direct effect on alpha-cells or mediated by a paracrine inhibition from beta-cells via secretion of molecules such as insulin, Zn2+, GABA.Recently, a fragment of a rat glucagon promoter has been successfully used to specifically drive the expression of fluorescent proteins in mouse alpha-cells. Thus, it is now possible to easily identify glucagon-secreting cells within isolated islets or to flow sort a purified population of alpha-cells. Here, we report the use of dynamic fluorescence imaging techniques on these transgenic islets and cells. In particular, we investigated how glucose affects 1) alpha-cell metabolism via measurement of NAD(P)H autofluorescence by two-photon excitation microscopy; 2) alpha-cell intracellular calcium concentrations, via the loading of alpha-cells expressing red fluorescent proteins (tdRFP) with the calcium indicator dye Fluo4-AM; and 3) alpha-cell membrane potential, via application of the fluorescent probe DiSBAC2(3). The results of these biophysical measurements will also be compared to parallel glucagon secretion in response to glucose from both intact islets and sorted alpha-cells.

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Functional analysis of identified interneurons in the mouse visual cortex

Event Abstract Back to Event Functional analysis of identified interneurons in the mouse visual cortex GABAergic interneurons are integral to cortical circuit structure and function but the breadth of functions subserved by each of subtypes of this highly diverse cortical neuron class is less understood in an in vivo preparation. Conventional extracellular electrophysiology falls short in distinguishing interneuron subtypes. With a custom built two photon scanning microscope and in vivo calcium indicator dye imaging we are studying tuning properties of genetically identified subtypes of cortical interneuron in anesthetized mouse V1 to moving grating stimuli. Genetically identified neuronal types in cortex can be reliably labeled with fluorescent reporters which persists throughout the development of the organism. Therefore, our goal is to study a few selected interneuron subtypes at time points around developmentally important critical period for ocular dominance plasticity in V1. In our preparation, mice are anesthetized and ventilated with 0.7% isoflurane in a 20:80, O2:N2 mixture. We bulk-load calcium indicator dye, Oregon Green BAPTA-1 (OGB), using patch pipettes into layer 2/3 of the V1. Anesthetized mice were presented with moving gratings visual stimuli at 2Hz temporal frequency, 0.02 cycles per degree spatial frequency, six orientations and twelve directions. We were able to distinguish GFP (or red tdTomato) labeled cell from the OGB signal by using two different 2-photon excitation wavelengths i.e., 800nm and 950nm respectively (Sohya 2007). Preliminary analysis of somatostatin positive interneurons in 28-days (P28) old GIN (GFP labeled interneuron) mice suggest that these neurons (n = 6 cells) are sparse in layer 2/3 and show only very modest calcium dye response amplitudes compared to the other nearby cortical neurons. The weakly evoked responses were non-selective to orientation and directions of gratings compared to 10% of nearby neurons (n = 54 cells) showing responses selectively tuned to an orientation and/or direction. The lack of response in these cells could be attributed to non-optimal visual stimulation. To overcome this we used a contrast invariant Gaussian noise movie (Niell 2008) which in principle drives most visually tuned V1 neurons (though not necessarily optimally). The noise stimuli was successful in driving a large number of neurons but the calcium responses from GIN neurons were at the best weak and sparse. These results are in contrast with recent reports of synaptically driven spiking and calcium transients of Martinotti cells (GIN cells) in response to activity in neighboring pyramidal neurons in cortical slices (Kaiser 2004, Fanselow 2008) . We are duplicating our study in P21 (+2) and P56 GIN mice in order to identify developmentally relevant changes. We are also performing a comparative study with parvalbumin and GAD-positive interneurons. Support: Allen Institute founders, Paul G. Allen and Jody Patton

In-Depth Activation of Channelrhodopsin 2-Sensitized Excitable Cells with High Spatial Resolution Using Two-Photon Excitation with a Near-Infrared Laser Microbeam

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.

Calmodulin Activation by Calcium Transients in the Postsynaptic Density of Dendritic Spines

The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM) and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.

T1876 Lubiprostone Activation of Cl − Currents Does Not Involve CA 2+, cAMP, or PKA

Introduction Lubiprostone stimulates electrogenic Cltransport in human intestinal T84 cells and Clcurrents in HEK293 cells stably transfected with recombinant human ClC-2 (hClC-2) with an EC50 of approximately 20 nM [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether lubiprostone (over the concentration range that activates electrogenic Cltransport) causes changes in [Ca]i, cAMP or PKA mediated phosphorylation. Methods T84 cells, HEK293 cells and HEK293 cells stably expressing hClC-2 or mutant hClC-2 were used. T84 cell [Ca]i, was measured using the calcium indicator dye indo-1/AM and intracellular [cAMP] was determined using a commercial ELISA kit. A commercial FLIPER assay was used to measure [Ca]i, and [cAMP] in HEK293 cells. Cl currents were measured by whole cell patch clamp. Results Lubiprostone at 1, 10 or 100 nM did not increase [Ca]i, in T84 cells and only slightly increased [cAMP] from 0.56 ± 0.02 to 4.0 ± 0.3 pmole/105 cells (n=4, P<0.001) at 100 nM (5 times the EC50 for lubiprostone activation of Cl currents). 1 μM PGE1 significantly (P<0.0005) increased [Ca]i from 80.8 ± 2.4 nM (control) to 1175.0 ± 18.7 nM (n=3) and greatly increased [cAMP] to 37.6 ± 0.6 pmole/105 cells (n=4) (P<0.0005). In contrast, 100 nM lubiprostone had no effect on [Ca]i or cAMP in HEK293 cells. Nevertheless hClC-2 in HEK293 cells is activated by lubiprostone, thereby demonstrating that neither [Ca]i or cAMP signaling are involved in lubiprostone activation of hClC-2. To rule out involvement of PKA phosphorylation of hClC-2 in lubiprostone activation of Clcurrents, a mutant hClC-2 Clchannel lacking the two unique PKA consensus phosphorylation sites (RRAT and RGET) essential for PKA activation of ClC-2, was used [Cuppoletti, et al JBC 279:21849, 2004]. In wild-type hClC-2 expressing HEK293 cells, control Clcurrents at -140 mV (n=6) of -27.2 ± 3.5 pA/pF, significantly increased (P<0.0005) to -100.9 ± 6.5 pA/pF with 20 nM lubiprostone and subsequently were reduced with 500 μM CdCl2 to control levels, -29.9 ± 5.6 pA/pF (P<0.0005). Cl currents in mutant hClC-2 expressing HEK293 cells was also significantly (P<0.0005) increased with 20 nM lubiprostone and inhibited by CdCl2 (n=6): control, -27.1 ± 6.9 pA/pF; with lubiprostone, -81.9 ± 5.1 (P<0.0005); after CdCl2, -29.4 ± 3.7 pA/pF (P<0.0005). Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved. Supported by Sucampo Pharmaceuticals, Inc. E001288 Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved.

A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum

For the analysis of Ca(2+)-dependent signaling, acetoxymethyl (AM)-derivatized ion indicators have become a popular tool. These indicators permeate membranes in an ion-insensitive form but, within cells, esterases hydrolyze these compounds to release ion-sensitive dyes. However, the properties of these indicators Limit their targeting to subcellular structures such as the endoplasmic reticulum, the dominant intracellular Ca2+ store. This study presents a novel approach for trapping fluorescent Ca2+ indicators in the ER. The method combines the selectivity of protein targeting with the biochemical advantages of synthetic Ca2+ indicators and allows direct, non-disruptive measurements of Ca(2+)-store dynamics with a high structural and temporal resolution. A recombinant carboxylesterase was targeted to the ER, providing a local esterase activity. After esterase-based dye loading, this additional esterase activity allowed improved trapping of Ca(2+)-sensitive forms of low-affinity Ca2+ indicators (e.g. Fluo5N) within the ER. The utility of the method was confirmed using different cell systems (293T, BHK21, cortical neurons) and activating different signaling pathways. In neurons, this approach enabled the detection of ER Ca2+ release with high resolution. In addition, the method allowed rapid confocal imaging of Ca2+ release from the ER, after activation of metabotropic glutamate receptors, in the presence of extracellular Ca2+.

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

Disturbed erythrocyte calcium homeostasis and adenine nucleotide dysregulation in canine phosphofructokinase deficiency

Muscle-type phosphofructokinase deficiency (PFKD) causes a hemolytic disorder and exertional myopathy in humans and dogs. In humans, PFKD is accompanied by a disturbed calcium homeostasis and associated adenine nucleotide dysregulation, which may potentiate the erythroenzymopathy associated with this inherited disorder. This study shows that canine PFKD also manifests these erythrocyte abnormalities. Compared to normal, healthy red cells, PFK-deficient erythrocytes contain lower concentrations of ATP and higher concentrations of IMP and calcium, the latter as per a calcium indicator dye. Adenosine monophosphate deaminase (AMPD) enriched 5000-fold from canine erythrocytes adsorbs to immobilized calcium–calmodulin and the interaction between these two proteins activates AMPD through a Kmapp effect. This behavior is similar to that of the human erythrocyte enzyme and provides a potential contributing mechanism for accelerated adenine nucleotide turnover in canine PFKD. We propose that adenine nucleotide replacement strategies could benefit the erythroenzymopathy in human and canine PFKD and that the dog model of this disorder is an appropriate vehicle for further elucidating this hypothesis.

Improved fluorescent (calcium indicator) dye uptake in brain slices by blocking multidrug resistance transporters

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that, also known as multidrug resistance proteins, transport a wide variety of substrates across biological membranes in an energy-dependent manner. Recently it has been shown that members of this protein family interfere with fluorescent (calcium indicator) dye uptake in taste buds of rat and in cells in the olfactory epithelium of larval Xenopus laevis, including olfactory receptor neurons. It has, however, not been resolved whether this effect only serves to extrude xenobiotics in sensory taste and olfactory cells, or alternatively, whether it is a more general feature of many central nervous system neurons. In the latter case blocking these transporters would improve fluorescent dye uptake in general. Here we show, by means of cell imaging, that also neurons of the olfactory bulb express multidrug resistance transporters, whereby a marked inhomogeneity among cells in the main and accessory olfactory bulb was observed. Blocking these transporters improved the net uptake of fluorescent dyes not only in cell somata of the olfactory bulb, but especially in fine neuronal structures such as individual dendrites or olfactory glomeruli, which consist of a tangle of tiny neuronal processes. We therefore suggest that the expression of multidrug resistance proteins may be common in cells of the central nervous system, and that the application of specific transport inhibitors could generally improve fluorescent dye uptake in brain slices, thereby improving calcium imaging conditions.

ACTIVATION OF DOMESTIC CAT OOCYTES USING THE SULFHYDRYL REAGENT THIMEROSAL

Domestic cats are useful as animal models in comparative medicine programs as they provide insight into disease mechanism and facilitate investigation of new therapeutic options. The application of cloning in these animals is also beneficial in terms of conserving the precious animal models. Although successful cat cloning by nuclear transfer has recently been reported, the technology is still extremely ineffective. One potential reason for the low efficiency is suboptimal embryo development after oocyte activation. During nuclear transfer, the calcium signals that are normally triggered by the fertilizing sperm must be reproduced artificially to initiate the development of the reconstructed embryos. Thimerosal is a chemical which is able to oxidize critical sulfhydryl groups on intracellular calcium release proteins and thus induce the release of calcium from the intracellular stores and trigger oocyte activation. The effects of thimerosal have previously been reported in a number of species; the objective of this study was to evaluate whether thimerosal can induce calcium release and initiate parthenogenetic embryo development in the oocyte of the domestic cat. Cat ovaries were obtained from a local veterinary clinic. Immature oocytes collected from the ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mM cysteine, 0.1 mM cysteamine, 1.0 IU/ml eCG, 2.0 IU/ml hCG, 25 ng/ml EGF, for 24 hours. For intracellular calcium measurements, cat oocytes were incubated in the presence of 2 μM fura-2 AM, a calcium indicator dye and 0.02% pluronic F-127, for 40 minutes. Individual oocytes were then transferred into TL-Hepes medium containing 200 μM thimerosal. Changes in the cytoplasmic free calcium levels were monitored using the InCyt Im2 fluorescence imaging system. Preimplantation embryonic development was also evaluated; for this purpose the oocytes were incubated in TL-Hepes in the presence of 200 μM thimerosal for 30 minutes followed by a 20-min incubation in TL-Hepes with 8 mM dithiothreitol, a sulfhydryl reducing agent. Control oocytes were cultured in TL-Hepes for 50 minutes. After the treatments, the oocytes were held in 7.5 μg/ml cytochalasin B for 4 hours to stimulate the formation of diploid embryos then cultured in FOCM for 6 days. The total number of nuclei in the developing embryos was recorded by staining with Hoechst 33342. After the addition of thimerosal, a series of transient rises in the intracellular free calcium concentration were observed in 81.8% (9 out of 11) of the oocytes during the 30-min incubation period. Furthermore, thimerosal induced cleavage in 21.8% (14/64) of the activated oocytes; 4.7% (3/64) of the oocytes developed to morula or blastocyst stage. The average cell number of those morulae/blastocysts was 19.7±3.2. In the control group, no cleavage was observed (0/43). The results support the hypothesis that thimerosal can induce an oscillation in the intracytoplasmic free calcium levels in oocytes by oxidizing sulfhydryl groups on intracellular calcium release proteins. The series of calcium increases was sufficient to trigger the signal transduction pathway in cat oocytes that resulted in embryonic development up to the blastocyst stage. The results indicate the potential application of thimerosal for oocyte activation during cat nuclear transfer. Additional studies are needed for the optimization of the activation parameters. (poster)

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

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Little Strokes Fill Big Oaks: A Simple In Vivo Stain of Brain Cells

High-resolution functional imaging of neural activity in vivo relies on appropriate labeling methods. In this issue of Neuron, Nagayama et al. introduce a simple procedure for staining subsets of neurons with organic calcium indicator dyes via local electroporation. Neuronal populations are sparsely labeled, preserving the ability to resolve calcium signals in dendrites and synaptic structures.

Imaging synaptosomal calcium concentration microdomains and vesicle fusion by using total internal reflection fluorescent microscopy

Transmitter release at chemical synapses is triggered by high calcium concentration microprofiles at the presynaptic cytosol. Such microprofiles, generated by the opening of voltage-dependent calcium channels at the presynaptic plasma membrane, have been defined as calcium concentration microdomains. Using total internal reflection fluorescent microscopy in conjunction with calcium and vesicular release indicator dyes, we have directly visualized the close apposition of calcium concentration microdomains and synaptic release sites at single synaptic terminals from the CNS from rat cerebellar mossy fiber and squid optic lobe. These findings demonstrate the close apposition of calcium entry and release sites and the dynamics of such site locations over time. Kinetic analysis shows that vesicles can be released via two distinct mechanisms: full-fusion and kiss-and-run. Calcium triggers vesicular motion toward the membrane, and the speed of such movement is calcium concentration-dependent. Moreover, the immediately available vesicular pool represents molecularly trapped vesicles that can be located at a larger distance from the plasma membrane than the field illuminated by total internal reflection fluorescent microscopy.

234 PARTHENOGENETIC ACTIVATION OF DOMESTIC CAT OOCYTES USING STRONTIUM

Cloning domestic cats is useful in comparative medicine programs as it may provide insight into unique disease mechanisms and facilitate investigation of new therapeutic options. It is also believed to be beneficial for the conservation of precious animal models. However, as in many species, low birth rates after nuclear transfer remain a formidable challenge. One potential reason for the low efficiency is poor embryo development following activation of the reconstructed oocytes. The number of methods available to induce a transient increase in the oocytes' cytosolic free calcium level to stimulate development is rather limited. Although strontium has been reported to successfully activate the developmental program of mature mouse and rat oocytes, it was without effect in all other species studied. Here we investigated the effect of strontium on mature cat oocytes. Oocytes collected from the cat ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mm cysteine, 0.1 mm cysteamine, 1 IU mL–1 eCG, 2 IU mL–1 hCG, 25 ng mL–1 epidermal growth factor (EGF) for 24 h. For intracellular calcium measurements, mature oocytes were incubated in the presence of 2 µ m fura-2Am, a calcium indicator dye, and 0.02% pluronic F-127 for 40 min. Individual oocytes were transferred into calcium-free HEPES, and SrCl2 was added to the medium at a final concentration of 20 mm. Changes in the intracellular free calcium levels were then monitored using an InCyt Im2™ fluorescence imaging system (Intracellular Imaging, Cincinnati, OH, USA). Preimplantation embryonic development was also evaluated by incubating the oocytes with 20 mm SrCl2 in calcium-free HEPES medium supplemented with 7.5 µg mL–1 cytochalasin B for 6 h. Control oocytes were activated by two 20-µs-long, 100 kV cm–1 direct current pulses and incubated in the presence of 7.5 µg mL–1 cytochalasin B for 6 h. After activation, the oocytes were cultured in FOCM for 6 days. At the end of the culture period, embryonic development was recorded; the nuclear number of the embryos was also determined after staining with Hoechst 33342. Data were subjected to one-way ANOVA, and differences between treatments were analyzed using the Tukey test. We found that strontium triggered a transient rise in the intracellular free calcium concentration in all oocytes tested (N = 20). Strontium treatment also induced cleavage in 49.7% (92/185) of the oocytes, while 4.9% (9/185) of the activated oocytes developed to the blastocyst stage. In the electroporated group, cleavage frequency was 57.1% (104/182) and blastocyst formation was 8.8% (16/182). Data analysis showed that there was no significant difference between the two groups in terms of cleavage frequency and blastocyst formation. This is the first study to demonstrate that strontium can induce cytoplasmic calcium increase in cat oocytes and trigger development up to the blastocyst stage. The results also indicate that SrCl2 may be useful for oocyte activation during cat nuclear transfer. Additional studies are needed to determine whether SrCl2 can trigger development more effectively than current activation techniques.

Calcium uptake and membrane trafficking in response to PTH or 25(OH)D3 in polarized intestinal epithelial cells

Cell culture techniques providing retention of the polarized enterocyte morphology has allowed, for the first time, comparison of parathyroid hormone (PTH)- and 25-hydroxyvitamin D3 [25(OH)D3]-induced 45Ca uptake with membrane trafficking events discerned using confocal microscopy. Treatment of cells with 65pM bPTH(1-34) promoted enhanced 45Ca uptake between 1 and 10min after peptide. The protein kinase A (PKA) antagonist, RpcAMP inhibited hormone-mediated uptake. At the microscopic level, cells labeled with the endocytic tracking dye FM1-43 revealed increased punctate staining 50–550s after hormone. Pretreatment of cells with RpcAMP abolished this pattern of staining. The calcium indicator dye fluo-3 AM revealed faint punctate labeling in controls, with increased bands of punctate labeling in the apical region of the cells after peptide hormone, and ultimately the basal region. Parallel studies conducted with the metabolite 25(OH)D3 resulted in a slower stimulation of 45Ca uptake 5–10min after steroid, which was also inhibited by preincubation with RpcAMP. Cells labeled with FM1-43 and then treated with steroid showed no change in distribution of fluorescence during the 10min incubation period. Confocal microscopy with fluo-3 revealed intense apical fluorescence – that after steroid – streamed to a perinuclear position, and ultimately the basal area. Uniformly diffuse staining, which would indicate cytoplasmic calcium transport, was observed only in controls. Membrane trafficking and compartmentalized calcium appear to be integral to agonist mediated cation transport.

A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca 2+ flux

Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca 2+ flux measurements. With advances in technology and the availability of low-power air-cooled lasers, there is interest in alternative protocols. Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. The reagents were examined in parallel to detect Ca 2+ flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Ca 2+ flux was detected with a FACSVantage SE equipped with an Omnichrome Series 74 Helium–Cadmium, or a Spectra Physics 177-G1202 Argon ion air-cooled laser. Following determination of optimal loading conditions, Ca 2+ flux was examined in response to membrane receptor stimulation or intracellular Ca 2+ mobilization. Dose dependent Ca 2+ flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca 2+ flux detected by Indo-1 or with fluo-3 and Fura Red appeared similar, with the combination of fluo-3 and Fura Red more sensitive under the particular test conditions. The results clearly demonstrated that Indo-1 could be usefully excited with a low-power air-cooled laser. The alternative use of fluo-3 and Fura Red does not require the availability of a UV capable laser and produced equivalent data.