Calcium Green-1 Research Articles

Loss of calcium and increased apoptosis within the same neuron

Loss of neuronal calcium is associated with later apoptotic injury but observing reduced calcium and increased apoptosis in the same cell would provide more definitive proof of this apparent correlation. Thus, following exposure to vehicle or the calcium chelator, BAPTA (1–20 μM), primary cortical neurons were labeled with Calcium Green-1 which was then cross-linked with EDAC, prior to immuno-staining for various proteins. We found that BAPTA-induced changes in calcium were highly correlated with changes in expression of activated caspase-3 as well as the calcium binding proteins calbindin, calretinin, and parvalbumin. Additionally, in brain slices from P7 neonatal rats, BAPTA induced significant loss of calcium in a brain region we have previously shown to express only moderate levels of calcium binding proteins as well as display robust apoptosis following calcium entry blockade. In contrast, BAPTA had little influence on calcium levels in a brain region we have previously shown to express robust calcium binding proteins as well as display far less apoptosis following calcium entry blockade. These data suggest that the ability of developing neurons to buffer changes in calcium may be critical to their long-term survival.

Intracellular Zinc Elevation Measured with a “Calcium-Specific” Indicator during Ischemia and Reperfusion in Rat Hippocampus: A Question on Calcium Overload

Much of our current evidence concerning of the role of calcium (Ca2+) as a second messenger comes from its interaction with fluorescent probes; however, many Ca2+ probes also have a higher affinity for another divalent cation: zinc (Zn2+). In this study, using a selective Zn2+ probe (Newport Green), we investigated the accumulation of intracellular Zn2+ transients in acute rat hippocampal slices during ischemia, simulated by oxygen and glucose deprivation (OGD). Subsequent reperfusion with glucose-containing oxygenated medium resulted in an additional increase in intracellular Zn2+. Such observations compelled us to investigate the contribution of Zn2+ to the alleged intracellular Ca2+ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca2+ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn2+ chelator, at the peak of the fluorescence elevation (interpreted as Ca2+ overload), resulted in a significant drop in intensity, suggesting that rising Zn2+ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn2+ is responsible for the ischemic neuronal cell death, because Zn2+ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca2+-mediated excitotoxicity. Our results indicate that Zn2+ elevation contributes to conventionally recognized Ca2+ overload and also suggest that the role of Ca2+ in neurotoxicity described previously using Ca2+ probes may need to be re-examined to determine whether effect previously attributed to Ca2+ could, in part, be attributable to Zn2+.

Metabolic regulation of sodium–calcium exchange by intracellular acyl CoAs

The sodium-calcium exchanger (NCX) is a critical mediator of calcium homeostasis. In the heart, NCX1 predominantly operates in forward mode to extrude Ca(2+); however, reverse-mode NCX1 activity during ischemia/reperfusion (IR) contributes to Ca(2+) loading and electrical and contractile dysfunction. IR injury has also been associated with altered fat metabolism and accumulation of long-chain acyl CoA esters. Here, we show that acyl CoAs are novel, endogenous activators of reverse-mode NCX1 activity, exhibiting chain length and saturation dependence, with longer chain saturated acyl moieties being the most effective NCX1 activators. These results implicate dietary fat composition as a plausible determinant of IR injury. We further show that acyl CoAs may interact directly with the XIP (exchanger inhibitory peptide) sequence, a known region of anionic lipid modulation, to dynamically regulate NCX1 activity and Ca(2+) homeostasis. Additionally, our findings have broad implications for the coupling of Ca(2+) homeostasis to fat metabolism in a variety of tissues.

Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals

Investigations into the roles of Ca 2+ and Zn 2+ in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca 2+ or Zn 2+ in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a probe for the targeted ion. Here, we investigated metal-ion responses by screening nominally Zn 2+- or Ca 2+-selective fluorophores in solutions containing various concentrations of Ca 2+, as a potential interferent for Zn 2+, or Zn 2+, as a potential interferent for Ca 2+. The results suggested that Zn 2+-sensitive dyes were more specific for their targeted ion than dyes that targeted Ca 2+. Ca 2+-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn 2+, even responding to Zn 2+ in the presence of high concentrations of Ca 2+. We demonstrate that these Ca 2+ indicators can effectively measure dynamic changes of cytosolic Zn 2+. Our results appeal for a new generation of Ca 2+ fluorophores that are more specific for Ca 2+ over Zn 2+. One implication of these results is that data obtained using Ca 2+-sensitive dyes may need to be re-examined to determine if results previously attributed to Ca 2+ could, in part, be due to Zn 2+.

Ensemble and Single-Molecule Fluorescence Spectroscopy of a Calcium-Ion Indicator Dye

The spectroscopic properties of Calcium Green 2 (CG-2), a dual-fluorophore Ca(2+) indicator dye, were characterized by a combination of steady state and time-resolved ensemble spectroscopic measurements, molecular mechanics calculations and single-molecule fluorescence spectroscopy. It was found that in Ca(2+) free solutions, CG-2 exists primarily as a highly quenched intramolecular dimer, but when bound to Ca(2+), the molecule adopts an extended, fluorescent conformation. The difference in emission properties of these two CG-2 conformations is explained in terms of simple exciton theory. Through single-molecule fluorescence measurements, we have shown that the bulk increase in ensemble fluorescence intensity correlates with a simple statistical increase in the number of fluorescent molecules in solution. In addition, we have also observed that the majority of CG-2 molecules photobleach in a single step, despite the molecule possessing two distinct fluorophores. A small fraction of molecules photobleach in multiple steps or show a series of transitions between emissive and nonemissive fluorescent states ("blinking"). We rationalize these photophysical phenomena using a simple model based on dipole-dipole Förster coupling between fluorophores in conjunction with irreversible photodamage to one of the constituent chromophores.

Correlative Analysis of [Ca2+]C and Apical Secretion during Pollen Tube Growth and Reorientation

The maintenance of a cytosolic free calcium gradient (Ca2+]c) and vesicle secretion in the apex of pollen tubes is essential for growth. It has been postulated that high [Ca2+]c levels promote and confine vesicle fusion with the apical plasma membrane and in this study we performed a correlative analysis of both events using specific fluorescent dyes and confocal scanning microscopy. [Ca2+]c was imaged with Calcium Green-1 10 kDa dextran (CG-1) while secretory events were followed with FM1–43 or FM4–64 in pollen tubes undergoing normal growth and reorientation events. During straight growth (no modification in direction), we found that changes in apical [Ca2+]c accompany changes in apical FM fluorescence indicating a tight coupling between [Ca2+]c and apical secretion. This coupling seems however to be perturbed during periods of reorientation of the pollen tube growth axis. Analysis of apical and sub-apical fluorescent signals during the reorientation events and subsequent re-entry in straight growth indicate that the increase in secretory events (higher fusion rate) precede the increases in [Ca2+]c that should be required for the transduction of the signal. Based on these findings, we discuss a model for membrane secretion and recycling which considers the apical and sub-apical region as a functional area containing all the elements required to promote and sustain growth.

A packed Cytodex microbead array for three-dimensional cell-based biosensing

A packed Cytodex 3 microbead array was fabricated as a simple three-dimensional (3-D) cell-based biosensing format. Resting membrane potentials and voltage-gated calcium channel (VGCC) function of SH-SY5Y human neuroblastoma cells cultured on the microbead array versus collagen-coated flat (2-D) substrates were evaluated by confocal microscopy with a potentiometric dye, tetramethylrhodamine methyl ester, and a calcium fluorescent indicator, Calcium Green-1. SH-SY5Y cells, differentiated with 1mM dibutyryl cAMP and 2.5 microM 5-bromodeoxyuridine, showed significant resting membrane potential establishment on the topographical scaffolds in a period of 13 days into differentiation, in contrast to the previously reported insignificant resting membrane potential establishment of the same cells within collagen hydrogels. On days 2, 8 and 13 into differentiation, cells on collagen-coated flat substrates developed resting membrane potentials of -6.0+/-19.5 mV (n=198), -30.5+/-19.9 mV (n=191) and -21.7+/-18.9 mV (n=308), in contrast to values for cells on 3-D scaffolds of -25.8+/-14.7 mV (n=112), -37.6+/-13.1 mV (n=120) and -28.7+/-12.2 mV (n=158), respectively. The development of VGCC function, as measured by percentage of cells responsive to 50 mM high K(+) depolarization, was significantly slower for cells on 3-D scaffolds (20.0% on day 13 into differentiation) than for cells on 2-D substrates (30.7% on day 8 into differentiation). The exaggerated 2-D cell calcium dynamics, in comparison with those of 3-D cells, is consistent with previous 2-D/3-D comparative studies. This study established the rationale and feasibility of the microbead array format for 3-D cell-based biosensing.

Calcium‐fluorescence lifetime imaging in ex‐vivo skin

Calcium controls several key events in keratinocytes and epidermis: epidermal barrier homeostasis and repair, neonatal barrier establishment, differentiation, signaling, cell adhesion, and various pathologic states. Yet, tissue- and cellular Ca2+ concentrations in physiologic and diseased states are only partially known. The very barrier properties of epidermis pose a major obstacle in determinations of the Ca2+ concentrations in intact skin. Methodology to measure and localize Ca2+ in intact and unfixed tissue has been lacking, while so far Ca2+-precipitation electron microscopy, or proton-induced x-ray emission were used. Both techniques are limited in that they can determine Ca2+ in only very small sample volumes, at or below light microscopic resolution levels, require fixed tissue and a chemical precipitation, or determine only total Calcium, irrespective of ionization or binding. So far, neither cellular and/or subcellular localization can be determined through these approaches. In cells, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca2+ concentrations, and in recent publications, we described Fluorescence Lifetime Imaging Microscopy (FLIM) to assess pH in intact epidermis. Here we report a method to measure and visualize Ca2+ in ex-vivo biopsies of unfixed epidermis, via FLIM. Using Calcium Green as the calcium sensor and the phasor-plot approach to separate raw lifetime components, we could establish a protocol to assess dynamic changes of Calcium throughout epidermis. We believe that this method will contribute to elucidating basic physiology as well as various pathologic situations in dermatology.

Calcium permeability and flux through osmosensory transduction channels of isolated rat supraoptic nucleus neurons

Hypertonic stimuli delivered into the supraoptic nucleus provoke neuropeptide release from the somata of magnocellular neurosecretory cells (MNCs) in the presence of tetrodotoxin, suggesting that such stimuli can increase intracellular calcium concentration ([Ca2+]i) in the absence of action potentials. We therefore examined whether the stretch-inhibited cation (SIC) channels of MNCs can mediate calcium influx. Whole-cell recordings were made in MNCs isolated from the supraoptic nuclei of adult rats. Measurements of reversal potentials in different solutions revealed that the current induced by a suction-evoked decrease in cell volume (ISIC) displays a selectivity sequence for monovalent cations of K+>Cs+>Na+>NMDG+. The permeability of SIC channels to Ca2+, relative to Na+, was approximately 5. In the presence of physiological concentrations of external Na+ and K+, the amplitude of inward ISIC was reduced dose-dependently by external Ca2+ with an IC50 of 4.9 mM. This was not due to reduced suction-evoked volume changes or to an accumulation of [Ca2+]i. Confocal imaging of cytoplasmic Calcium Green-1 fluorescence revealed that activation of ISIC significantly increases [Ca2+]i in physiological solutions. This effect is absent in Ca2+-free solution, or when Gd3+ (300 microM) is added to Ca2+-containing solution. Part of this effect is inhibited in the presence of dantrolene (10 microM) and heparin (4 mg/mL), suggesting that release from intracellular Ca2+ stores participates in suction-evoked Ca2+ signalling. These observations indicate that SIC channels are highly permeable to Ca2+, mediate significant Ca2+ entry and release of Ca2+ from internal stores under conditions when the volume of MNCs is decreased.

Long‐term in vivo modulation of synaptic efficacy at the neuromuscular junction of Rana pipiens frogs

Prolonged changes in motor neurone activity can result in long-term changes in synaptic transmission. We investigated whether mechanisms commonly thought to be involved in determining synaptic efficacy of vertebrate motor neurones are involved in these long-term changes. The nerve supplying the cutaneous pectoris muscle was chronically stimulated via skin surface electrodes in freely moving frogs for 5-7 days. Chronic stimulation induced a 50% reduction in evoked endplate potential (EPP) amplitude at stimulated neuromuscular junctions (NMJs). These changes appear to be presynaptic since miniature EPP (mEPP) amplitude was unchanged while mEPP frequency was decreased by 46% and paired-pulse facilitation was increased by 26%. High frequency facilitation (40 Hz, 2 s) was also increased by 89%. Moreover, stimulated NMJs presented a 92% decrease in synaptic depression (40 Hz, 2 s). An increase in mitochondrial metabolism was observed as indicated by a more pronounced labelling of active mitochondria (Mitotracker) in stimulated nerve terminals, which could account for their greater resistance to synaptic depression. NMJ length visualized by alpha-bungarotoxin staining of nAChRs was not affected. Presynaptic calcium signals measured with Calcium Green-1 were larger in stimulated NMJs at low frequency (0.2 Hz) and not different from control NMJs at higher frequency (40 Hz, 2 s and 30 s). These results suggest that some mechanisms downstream of calcium entry are responsible for the determination of synaptic output, such as a down-regulation of some calcium-binding proteins, which could explain the observed results. The possibility of a change in frequenin expression, a calcium-binding protein that is more prominently expressed in phasic synapses, was, however, refuted by our results.

Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement

We have investigated the fluorescence lifetime properties of 8 calcium ion probes, calcium-green-1, calcium green-2, calcium green-5N, calcium orange, oregon green 488 BAPTA-6F, fluo-3, fluo-4, and fluo-5N. We found that the decay time of calcium green-5N varied more sensitively with calcium concentration than calcium green-1 which was known to be a highly sensitive probe. We have also found that the center of observable range of calcium concentration by fluorescence lifetime measurement is lower than that by fluorescence intensity measurement.

1250 流れ場における培養内皮細胞の細胞間Ca^<2+>伝達(J03-1 細胞の構造と流れのメカニクス(1),J03 細胞の構造と流れのメカニクス)

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in human body, and shear stress affects the intracellular calcium ([Ca^<2+>]_i), which initiates cellular responses. Thus, we studied the intercellular communication of cultured ECs under shear stress. We used cultured bovine aortic cells and a parallel plate flow chamber for shear stress loading. We measured [Ca^<2+>]_i responses in cultured ECs by using a fluorescent microscope and calcium indicator (Calcium Green-1/AM), and measured [Ca^<2+>]_i, responses under shear stress in the presence of various inhibitors. The [Ca^<2+>]_i responses induced by shear stress in the presence of suramin (100μM), apyrase (10 units/ml), and FCCP (100μM) decreased to 40%, 25%, and 20%, respectively. However, the response in the presence of 18α-GA (gap junction inhibitor) did not change compared to no-treatment sample. Furthermore, after loading caged IP_3 into ECs, caged IP_3 was photoliberated in ECs. IP_3-triggered [Ca^<2+>]_i response propagated to adjacent cells downstream. These results have indicated the intercellular communication of ECs under shear stress is paracrine manner.

Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts inAplysia kurodai

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.

Activation of MAPK Kinase pathway by Gal/GalNAc adherence lectin of E. histolytica: Gateway to host response

Amoebiasis caused by the protozoan parasite Entamoeba histolytica is one of the leading parasitic causes of morbidity and mortality in the developing countries. Among the variety of virulence factors, an adherence lectin (Gal/GalNAc, 260 kDa) has been known to mediate colonization and subsequent host responses. It is a major cell surface antigen which is universally recognized by the immune sera of patients with amoebic liver abscess (ALA). The role of this lectin in cytolysis and phagocytosis of human colonic mucin glycoproteins has also been established. The objective of the present study was to elucidate the signal transduction events induced in response to Entamoeba histolytica derived Gal/GalNAc lectin in the target epithelial cells. We have attempted to define a pathway in target cells that could link this immunodominant antigen to a known biological pathway for target cell activation and triggering of subsequent disease pathology/parasite survival. Lectin stimulated cells showed immediate rise in (Ca2+)i concentration corresponding to 1517.31+/-16.3 nM (approximately) at 0-2 min. The intracellular calcium also extruded from the cells as was measured by increase in calcium green-1 fluorescence. Expression of several protein kinases was checked by western blotting to delineate the signaling pathway. Results showed that the expression of PLA2, PI3K, Ras p21, Ras GAP, ERK-MAPK, p38MAPK and PKC was significantly increased. Expression of Raf-1 and MEK-1 was also found to be significant, as determined by intensity analysis. Overall, it indicated activation of MAPKinase pathway which is implicated in a variety of cellular functions. On the basis of our observations it can be stated that there is a calcium mediated activation of PKC in target cells, by lectin, which inturn activates cyclic nucleotides and other protein kinases. These protein kinases further phosphorylated downstream signals in a sequential manner, thus leading to the activation of MAPKinase cascade. Activation of MAPK cascade, in our studies, is implicated in a variety of physiological cellular functions including apoptosis, proliferation, cytoskeleton rearrangements and permeability changes. However, future screening of the genes responsible for the transcription and translation of new proteins and their biological functions in response to lectin stimulation will prove useful in understanding this host-parasite relationship.

A procedure to label inner ear afferent nerve endings for calcium imaging

Characterization of synaptic transmission between the inner ear sensory cells and primary neuron dendrites has been hampered by the limited access to the postsynaptic terminals. Because direct physiological recording of postsynaptic currents are difficult to achieve, no information regarding the synaptic and dendritic events are available. This is due to the small size of the postsynaptic afferent nerve endings that do not allow a clear identification, and thus compromise direct electrophysiological recordings of the buttons. To study the physiology of afferent nerve endings, we have developed a two-photon imaging technique in cochlear and vestibular slice preparations from neonatal rats and turtles. This technique is based on a retrograde labeling of afferent nerve endings with high-affinity calcium-sensitive dyes. Dye filling was achieved by 6 h application of the dextran–amine conjugate of calcium green-1. Calcium changes were measured in afferent nerve endings in line scan and time lap mode. To address recording in a near-physiological situation, iontophoretic application of K + was performed in the area of the stereocilia whereas glutamate was applied at the basal pole of sensory hair cells. Both types of application cause a reversible and sustained increase of Ca 2+ in the button of afferent nerve fibers. Typical recordings are presented and potential interests for pharmacological studies of inner ear sensory cell synapses are discussed.

Spatiotemporal pattern of calmodulin and [Ca2+]i is related to resumption of meiosis in mouse oocytes.

During meiotic maturation, mammalian oocytes undergo a series of morphological and physiological changes that prepare them for fertilization. Calcium-initiated signaling is thought to trigger these processes. In this study, we examine the spatio-temporal pattern of calcium and calmodulin (CaM), its downstream receptor, in order to investigate their association with meiotic maturation. Intracellular free calcium and activated CaM levels were measured using the fluorescent probes Calcium Green-1 and TA-CaM, respectively. The distribution patterns were examined using confocal microscopy. Both calcium and activated CaM showed a dynamic spatiotemporal distribution during meiotic maturation. After release from IBMX buffer, calcium was found to periodically translocate from the perinuclear region to the germinal vesicle (GV) in 90 s intervals. After 90 min, calcium stopped oscillating and became concentrated within the GV. After a further 60 min, the GV broke down and calcium dispersed into the ooplasm, but calcium levels were slightly lower here than in the original nuclear region. Activated CaM also showed a dynamic patterning process similar to calcium. Taking the data from calcium chelation and CaM inhibition together, our results suggest that the dynamic distribution patterns of calcium and activated CaM are crucial for oocyte maturation.

Ciliary reorientation is evoked by a rise in calcium level over the entire cilium.

Internal Ca2+ levels control the pattern of ciliary and flagellar beating in eukaryotes. In ciliates, ciliary reversal is induced by a rise in intra-ciliary Ca2+, but the mechanism by which Ca2+ induces reversal is not known. We injected the fluorescent Ca2+ indicator Calcium Green into a ciliate Didinium nasutum and observed the intra-ciliary Ca2+ level during the initial reversed stroke preceding spontaneous cyclic reversed beating. In D. nasutum, Ca2+ rose throughout the length of the cilia undergoing initial reversed stroke. Electron microscopy with a combined oxalate-pyroantimonate method showed Ca2+ deposits distributed throughout the reversed cilia. We injected caged Ca2+ into D. nasutum and irradiated the base or mid region of the cilia with UV to locally increase Ca2+ concentration. Uncaging Ca2+ in the middle of the cilia produced reversal distally, but not proximally to the site of Ca2+ release. These results strongly suggest that not only Ca2+ influx sites, but also Ca2+ binding sites and vectoral bending machineries for ciliary reversal, are distributed throughout the cilium.

Functional separation of deep cytoplasmic calcium from subplasmalemmal space calcium in cultured human uterine smooth muscle cells

For smooth muscle, two important functions of free intracellular calcium (Ca 2+ i) are modulation of plasma membrane excitability properties and modulation of the contractile apparatus. As proposed by van Breemen, Ca 2+ i can be divided into the subplasmalemmal space (Ca 2+ sps) and the deep cytosol (Ca 2+ d) by the superficial calcium buffer barrier. Using these distinctions, Ca 2+ sps activates the large conductance calcium-activated potassium channel (BK), and Ca 2+ d binds calcium-dependent fluorescent probes in the cytoplasm. We present here combined fluorescence—patch clamp experiments designed to simultaneously assess Ca 2+ d and Ca 2+ sps in cultured human uterine smooth muscle cells. Open probabilities ( P o) of the BK channel were measured using the cell-attached patch clamp technique. P o was used to approximate changes of [Ca 2+ sps]. Relative concentrations of Ca 2+ d were approximated by observing fluorescence of Calcium green-1 ( F). Under control conditions, we found similar time courses for rises of P o and F following 10 nM oxytocin (OT) addition. In parallel experiments, but with lanthanum (La 3+) added to the bath to block transmembrane calcium flux, P o was only slightly affected, but F increases were delayed and blunted. These data paradoxically indicate that following OT stimulation, the primary source of calcium for Ca 2+ sps is internal stores, and calcium entry from the extracellular space is required to raise Ca 2+ d. When cells were exposed to cyclopiazonic acid (CPA) to release SR calcium stores, P o increased slowly, then persisted at large values. The persistence of P o rises suggests that removal of calcium from the subplasmalemmal space is primarily via reuptake into the SR. In the presence of La 3+, OT-induced rises of F were slightly prolonged, suggesting that transmembrane calcium flux contributes to decreasing Ca 2+ d, but is not the primary mechanism. In summary, these data demonstrate that Ca 2+ d and Ca 2+ sps are not always intimately linked, but indicate a functional separation of the deep cytosol and the subplasmalemmal space that is consistent with the existence of a barrier to calcium diffusion between these two regions.

Two-photon imaging of brain pericytes in vivo using dextran-conjugated dyes.

Pericytes in the central nervous system (CNS) are hypothesized to be involved in important circulatory functions, including local blood flow regulation, angiogenesis, immune reaction, and regulation of blood-brain barrier. Despite these putative functions, functional correlates of pericytes in vivo are scarce. We have labeled CNS pericytes using the dextran-conjugated fluorescent calcium indicator Calcium Green I and imaged them in somatosensory cortex of the mouse in vivo. Intracellular calcium concentration in pericytes showed spontaneous surges lasting for several seconds. Furthermore, population bursts of neuronal activity were associated with increased Ca(2+) signal in a portion of the pericytes. Selective in vivo labeling of pericytes with functional markers may help reveal their physiological function in neuronal activity-associated regulation of local cerebral blood flow.

Characterization of 3-D collagen hydrogels for functional cell-based biosensing

To address the growing demand for functional cell-based assay technologies with accelerated drug discovery applications, we have proposed the use of human neuroblastoma cells (IMR-32) immobilized in three-dimensional (3-D) collagen hydrogel matrices. The gel protects weakly adherent cells from fluid mechanical forces while providing a more physiologically relevant 3-D environment. Hydrogels made up of collagen, between 0.5 and 1.0 mg/ml, exhibited mechanical stability adequate to withstand fluid mechanical forces (<0.11 mN) typical of automated commercial fluid transfer equipment. Collagen-entrapped cells visualized with the aid of confocal microscopy and a potentiometric-sensitive dye, TMRM, exhibited round morphology in comparison to flat morphology typical of cells in two-dimensional (2-D) monolayer cultures. Morphological differentiation characterized by neurite extension and cell aggregation was observed for both 2-D and 3-D cultures. Differentiated IMR-32 cells failed to develop a resting membrane potential typical of excitable cells. Free intracellular calcium was monitored with Calcium Green-1. Depolarization-induced Ca 2+ influx was only observed with differentiated 3-D cells unlike 2-D cells, where calcium flux was observed in both differentiated and undifferentiated cells. Taken together, the results revealed that collagen hydrogels (0.5 mg/ml collagen) were suitable structural supports for weakly adherent cells. However, for voltage-dependent calcium channel function applications, further investigations are needed to explain the difference between 2-D monolayer and 3-D collagen-entrapped cells.