Measurement of intracellular Ca2+ mobilization to study GPCR signal transduction.

Understanding G protein-coupled receptor (GPCR) structure-function relationship and its activation mechanism has been broadly explored using mutational strategy due to problems in GPCR crystallization. Probing into GPCR: effector (G protein/ß-arrestin) interactions and downstream signaling are important aspects of GPCR research. Among the G proteins, though there are some approaches to investigate Gq-mediated signaling, they involve the use of radioactivity and are qualitative in nature. Our method described here makes use of the cell permeable nature of fluorescent Ca2+ indicator dye, fura2AM, that binds with the Ca2+ released in response to GPCR: Gq interaction on ligand treatment. Using this spectrophotometric method, EC50 values of the GPCR: ligand binding can be calculated and the binding affinity can be analyzed.

Permeation of topically applied Magnesium ions through human skin is facilitated by hair follicles.

Magnesium is an important micronutrient essential for various biological processes and its deficiency has been linked to several inflammatory disorders in humans. Topical magnesium delivery is one of the oldest forms of therapy for skin diseases, for example Dead Sea therapy and Epsom salt baths. Some anecdotal evidence and a few published reports have attributed amelioration of inflammatory skin conditions to the topical application of magnesium. On the other hand, transport of magnesium ions across the protective barrier of skin, the stratum corneum, is contentious. Our primary aim in this study was to estimate the extent of magnesium ion permeation through human skin and the role of hair follicles in facilitating the permeation. Upon topical application of magnesium solution, we found that magnesium penetrates through human stratum corneum and it depends on concentration and time of exposure. We also found that hair follicles make a significant contribution to magnesium penetration.

Functional calcium imaging in developing cortical networks.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network. A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz & Shatz, 1996; Khaziphov & Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic & Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry. In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

A novel fluorescence imaging approach for comparative measurements of pancreatic islet function in vitro.

Pancreatic islet dysfunction is a key element in the development of type 2 diabetes. Determining possible early warning signs of dysfunction is thus important to determining the underlying causes of diabetes. We describe an improved fluorescent imaging approach to detect potential islet dysfunction. Using Cell Tracker Red (CTR, a mildly thiol-reactive fluorescent probe) to positively label particular islets, we measured intracellular free calcium with fura-2 AM in both CTR-labeled and unlabeled sets of pancreatic islets simultaneously in vitro. This approach enhances sensitivity by controlling for differences in background fluorescence, temperature, and perifusion dynamics. We confirmed that 200 nM CTR produced no spectral overlap with fura-2 and no significant physiological effects in selective tests of islet function. To demonstrate the utility of dual-labeling, we compared untreated islets with islets pretreated with low-dose pro-inflammatory cytokines (IL-6 + IL-1B) to induce mild dysfunction. We alternated CTR-labeling between control and test islets and identified consistent reductions in the amplitude and trajectory of glucose-stimulated calcium responses (GSCa) among cytokine-treated islets that were independent of labeling. Observations were verified using a MATLAB program specifically designed to identify key features in the GSCa. Our findings thus demonstrate the utility of CTR-labeling in identifying islet dysfunction and propose that this technique can be adapted for other cells and tissues.

Lipid-protein cargo transfer: a mode of direct cell-to-cell communication for lipids and their associated proteins.

Cells in tissues or in experimental cell colonies respond to stimuli in a co-ordinated manner when they are electrically and chemically coupled by gap junctions. These junctions permit the cell-to-cell passage of small molecules, such as inositol tris phosphate (IP(3)) within the colony and are important in co-ordinating tissue activity. This is the only recognised mechanism of direct chemical signalling that does not involve the release of an extracellular messenger between cells. However, the data in this article demonstrates a new mode of intercellular communication. Two potentially important signalling lipids, PIP(2) and ganglioside G-M1 were shown to move between cells in colonies by tracking (i) fluorescent lipids loaded into the plasma membranes of individual cells in a cell colony using a novel micropipette technique and (ii) movement of fluorescent lipids after localised photobleaching. Furthermore, a large protein molecule, cholera toxin B subunit bound to extracellularly facing ganglioside G-M1 was also shown to transfer between cells. The transfer was inhibited by pre-treatment with poly-L-lysine and polyethylenimine, suggesting a role for tight junctions, perhaps by permitting diffusion of lipids and their protein "cargo" across these cell-to-cell contact points. This is a hitherto unsuspected form of molecular signalling within cell colonies and tissues which may have implications for understanding co-ordinated cell colony behaviour.

Differences in mitochondrial movement and morphology in young and mature primary cortical neurons in culture.

Mitochondria have many roles critical to the function of neurons including the generation of ATP and regulation of intracellular Ca2+. Mitochondrial movement is highly dynamic in neurons and is thought to direct mitochondria to specific cellular regions of increased need and to transport damaged or old mitochondria to autophagosomes. Morphology also varies between individual mitochondria and is modulated by fusion and fission proteins such as mitofusin-1 and dynamin-related protein-1, respectively. Although mitochondrial movement and morphology are thought to be modulated to best meet cellular demands, few regulatory signals have been identified. In this study, we examined how the different cellular environments of synaptically immature and mature rat cortical neurons affect mitochondrial movement, morphology, distribution and function. In younger cells, mitochondria were more mobile, were shorter, occupied a smaller percentage of neuronal processes, and expressed greater mitofusin-1 and lower dynamin-related protein-1 protein levels compared with older cells. However, the number of mitochondria per mum of neuronal process, mitochondrial membrane potential and the amount of basally sequestered mitochondrial Ca2+ were similar. Our results suggest that while mitochondria in young neurons are functionally similar to mature neurons, their enhanced motility may permit faster energy dispersal for cellular demands, such as synaptogenesis. As cells mature, mitochondria in the processes may then elongate and reduce their motility for long-term support of synaptic structures.

Tributyltin-induced cell death is mediated by calpain in PC12 cells.

Tributyltin, an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide and component of antifouling paints. In this study, we investigated whether calpain is involved in tributyltin toxicity in undifferentiated PC12 cells. Tributyltin (2 microM) induced an increase of lactate dehydrogenase release, a marker of cytotoxicity, in PC12 cells in a time-dependent manner. It also induced calpain activation in a dose-dependent manner, and a calpain inhibitor, MDL28170 (40 microM), decreased the cellular toxicity, suggesting that calpain is involved in tributyltin toxicity in PC12 cells. Because calpain is a calcium-dependent protease, we examined the effect of EGTA, an extracellular Ca(2+) chelator and BAPTA-AM, an intracellular Ca(2+) chelator. Calpain activation induced by tributyltin was decreased by BAPTA-AM (50 microM), but not by EGTA (1 mM), suggesting that calpain activation is associated with calcium release from intracellular Ca(2+) stores. Further, we investigated the relationship between caspase-3 and calpain. Inhibition of caspase-3 reduced calpain activity induced by tributyltin. In conclusion, we have demonstrated that tributyltin induced cell death through calpain activation, and that intracellular Ca(2+) increase and caspase-3 activation are required for calpain activation by tributyltin.

Pharmacologically induced calcium oscillations protect neurons from increases in cytosolic calcium after trauma.

Increases in cytosolic calcium ([Ca(2+)](i)) following mechanical injury are often considered a major contributing factor to the cellular sequelae in traumatic brain injury (TBI). However, very little is known on how developmental changes may affect the calcium signaling in mechanically injured neurons. One key feature in the developing brain that may directly impact its sensitivity to stretch is the reduced inhibition which results in spontaneous [Ca(2+)](i) oscillations. In this study, we examined the mechanism of stretch-induced [Ca(2+)](i) transients in 18-days in vitro (DIV) neurons exhibiting bicuculline-induced [Ca(2+)](i) oscillations. We used an in vitro model of mechanical trauma to apply a defined uniaxial strain to cultured cortical neurons and used increases in [Ca(2+)](i) as a measure of the neuronal response to the stretch insult. We found that stretch-induced increases in [Ca(2+)](i) in 18-DIV neurons were inhibited by pretreatment with either the NMDA receptor antagonist, APV [D(-)-2-Amino-5-phosphonopentanoic acid], or by depolymerizing the actin cytoskeleton prior to stretch. Blocking synaptic NMDA receptors prior to stretch significantly attenuated most of the [Ca(2+)](i) transient. In comparison, cultures with pharmacologically induced [Ca(2+)](i) oscillations showed a substantially reduced [Ca(2+)](i) peak after stretch. We provide evidence showing that a contributing factor to this mechanical desensitization from induced [Ca(2+)](i) oscillations is the PKC-mediated uncoupling of NMDA receptors (NMDARs) from spectrin, an actin-associated protein, thereby rendering neurons insensitive to stretch. These results provide novel insights into how the [Ca(2+)](i) response to stretch is initiated, and how reduced inhibition - a feature of the developing brain - may affect the sensitivity of the immature brain to trauma.

Cannabidiol-induced intracellular Ca2+ elevations in hippocampal cells.

The phytocannabinoid cannabidiol (CBD) is at the forefront of therapeutic cannabinoid research due to its non-psychotropic properties. Research supports its use in a variety of disorders, yet the cellular mechanisms of its action remain unclear. In this study, the effect of CBD upon Ca2+ homeostasis in hippocampal cells was characterised. CBD (1 microM) elevated intracellular Ca2+ ([Ca2+]i) by approximately +45% of basal Ca2+ levels in both glia (77% responders) and neurones (51% responders). Responses to CBD were reduced in high excitability HEPES buffered solution (HBS), but not affected in low excitability/low Ca2+ HBS. CBD responses were also significantly reduced (by 50%) by the universal Ca2+ channel blocker cadmium (50 microM) and the L-type specific Ca2+ channel blocker nifedipine (20 microM). Interestingly, intracellular store depletion with thapsigargin (2 microM) had the most dramatic effect on CBD responses, leading on average to a full block of the response. Elevated CBD-induced [Ca2+]i responses (>+100%) were observed in the presence of the CB1 receptor antagonist, AM281 (1 microM), and the vanilloid receptor antagonist, capsazepine (CPZ, 1 microM). Overall, our data suggest that CBD modulates hippocampal [Ca2+]i homeostasis via intracellular Ca2+ stores and L-type VGCC-mediated Ca2+ entry, with tonic cannabinoid and vanilloid receptor signalling being negatively coupled to this pathway.

Mg2+-malate co-transport, a mechanism for Na+-independent Mg2+ transport in neurons of the leech Hirudo medicinalis.

Mg2+-extrusion from Mg2+-loaded neurons of the leech, Hirudo medicinalis, is mediated mainly by Na+/Mg2+ antiport. However, in a number of leech neurons, Mg2+ is extruded in the nominal absence of extracellular Na+, indicating the existence of an additional, Na+-independent Mg2+ transport mechanism. This mechanism was investigated using electrophysiological and microfluorimetrical techniques. The rate of Na+-independent Mg2+ extrusion from Mg2+-loaded leech neurons was found to be independent of extracellular Ca2+, K+, NO3-, HCO3-, SO4(2-), HPO4(2-), and of intra- and extracellular pH. Na+-independent Mg2+ extrusion was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), furosemide, ouabain, vanadate, iodoacetate, 4-amino-hippurate, or alpha-cyano-4-hydroxycinnamate and was not influenced by changes in the membrane potential in voltage-clamp experiments. Na+-independent Mg2+ extrusion was, however, inhibited by the application of 2 mM probenecid, a blocker of organic anion transporters, suggesting that Mg2+ might be co-transported with organic anions. Extracellularly, of all organic anions tested (malate, citrate, lactate, alpha-ketoglutarate, and 4-amino-hippurate) only high, but physiological, concentrations of malate (30 mM) had a significant inhibitory effect on Na+-independent Mg2+ extrusion. Intracellularly, iontophoretically injected malate, citrate, or fura-2, but not Cl-, alpha-ketoglutarate, glutamate, succinate, or urate, were stimulating Na+-independent Mg2+ extrusion from those neurons that initially did not extrude Mg2+ in Na+-free solutions. Our data indicate that Mg2+ is co-transported with organic anions, preferably with malate, the predominant extracellular anion in the leech. The proposed model implies that, under experimental conditions, malate drives Mg2+ extrusion, whereas under physiological conditions, malate is actively taken up, driven by Mg2+, so that malate can be metabolized.

GABAA-receptor-mediated increase in intracellular Ca2+ concentration in the regenerating retina of adult newt.

We used optical recording with the Ca(2+)-sensitive dye, fura-2, in living slice preparations from the newt retina at different stages of regeneration. gamma-Aminobutyric acid (GABA) induced pronounced [Ca(2+)](i) rise in progenitor cells and differentiating ganglion cells in the 'intermediate' stage of retinal regeneration. This [Ca(2+)](i) rise became less pronounced at the beginning of synapse formation in the late regenerating retina. At the late period of the late regenerating retina with the IPL thickness comparable to that of the control retina, GABA-induced [Ca(2+)](i) rise became undetectable or sometimes a small decrease in [Ca(2+)](i) was observed in regenerated ganglion cells. In contrast, N-methyl-d-aspartate (NMDA)-induced [Ca(2+)](i) rise appeared in premature ganglion cells and became prominent gradually as the regeneration proceeded. The [Ca(2+)](i) rise to GABA was mediated by GABA(A) receptors. This was shown by inhibition of GABA-induced Ca(2+) response with the preincubation of the GABA(A) receptor antagonist, bicuculline. The [Ca(2+)](i) rise due to GABA was suppressed in the absence of extracellular Ca(2+) or in the presence of the L-type voltage-gated Ca(2+) channel blocker, verapamil, suggesting that Ca(2+) may be entered through L-type Ca(2+) channels. Transient appearance of [Ca(2+)](i) rise to GABA during regeneration and origin of GABA-induced [Ca(2+)](i) rise were similar to those in the developing retina [J. Neurobiol. 24 (1993) 1600]. These similarities may suggest that common mechanisms may control neurogenesis and/or synaptogenesis during development and regeneration.

Excitatory effects of ATP on rat dorsomedial hypothalamic neurons.

We investigated P2X purinoceptors in rat dorsomedial hypothalamic (DMH) neurons using nystatin-perforated patch-clamp recordings and fura-2 microfluorometry. Adenosine triphosphate (ATP) concentration-dependently evoked an inward current and increased cytosolic Ca(2+) ([Ca](i)). The rise in [Ca](i) was dependent on external Ca(2+) and Na(+), was blocked by Ca(2+) channel antagonists and had pharmacological properties consistent with P2X2 receptors. These results suggest that P2X receptor-mediated depolarization activates voltage-gated Ca(2+) channels, resulting in an increase in [Ca](i).

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis.

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell.

We wrote a program that runs as a Microsoft Excel spreadsheet to calculate the diffusion of Ca2+ in a spherical cell in the presence of a fixed Ca2+ buffer and two diffusible Ca2+ buffers, one of which is considered to be a fluorescent Ca2+ indicator. We modeled Ca2+ diffusion during and after Ca2+ influx across the plasma membrane with parameters chosen to approximate amphibian sympathetic neurons, mammalian adrenal chromaffin cells, and rat dorsal root ganglion neurons. In each of these cell types, the model predicts that spatially averaged intracellular Ca2+ activity ([Ca2+]avg) rises to a high peak and starts to decline promptly on the termination of Ca2+ influx. We compared [Ca2+]avg with predictions of ratiometric Ca2+ measurements analyzed in two ways. Method 1 sums the fluorescence at each of the two excitation or emission wavelengths over the N compartments of the model, calculates the ratio of the summed signals, and converts this ratio to Ca2+ ([Ca2+]avg,M1). Method 2 sums the measured number of moles of Ca2+ in each of the N compartments and divides by the volume of the cell ([Ca2+]avg,M2). [Ca2+]avg,M1 peaks well after the termination of Ca2+ influx at a value substantially less than [Ca2+]avg because the summed signals do not reflect the averaged free Ca2+ if the signals come from compartments containing gradients in free Ca2+ spanning nonlinear regions of the relationship between free Ca2+ and the fluorescence signals. In contrast, [Ca2+]avg,M2 follows [Ca2+]avg closely.

Hydrolysis of Ca2+-sensitive fluorescent probes by perfused rat heart.

Rat hearts were loaded with the fluorescent calcium indicators fura 2, indo 1, rhod 2, or fluo 3 to determine cytosolic calcium levels in the perfused rat heart. With fura 2, however, basal tissue fluorescence increased above anticipated levels, suggesting accumulation of intermediates of fura 2-AM deesterification. To examine this process, we separated the intermediates of the deesterification process using HPLC after incubation of fura 2-AM with tissue homogenates and after loading in the rat heart. Loading of hearts with fura 2-AM resulted in tissue levels of fura 2 free acid that were only 5% of the total heart dye content of all fura 2 species. The parent fura 2-AM form accumulated without accumulation of intermediate products. Similar results were obtained with indo 1-AM. Fluo 3 loaded very poorly in perfused hearts. Unlike other indictors, rhod 2 rapidly loaded in perfused hearts and was completely converted to the free acid form. To determine the subcellular localization of the free acid form of these indictors, mitochondria from indicator-loaded hearts were assayed for the free acid form. Approximately 75% of the total amount of rhod 2 in hearts could be recovered in isolated mitochondria. Subcellular localization of indo 1 and fura 2 was more evenly distributed between mitochondria and nonmitochondrial compartments. We conclude that measurement of calcium in the perfused rat heart using surface fluorescence with either indo 1 or fura 2 is complicated by an inconsistent accumulation of the parent ester and that the resulting signal cannot be easily calibrated using "in situ" methods using the free acid form. Rhod 2 does not display this shortcoming, but like other indicators, it also loads into the mitochondrial matrix.

Nerve growth factor and chronic ethanol treatment alter calcium homeostasis in developing rat septal neurons.

Chronic ethanol treatment (CET) during development produces cellular adaptations resulting in tolerance to the acute effects of ethanol (EtOH). The objectives of this study were to determine whether CET during the prenatal period (PCET) followed by a period of in vitro CET (PCET-CET) altered intracellular calcium [Ca(2+)](i) and produced tolerance to acute EtOH treatment (AET), and whether nerve growth factor (NGF) modulated the effects of PCET-CET in cultured developing rat septal neurons. Fetuses were obtained from EtOH-fed and sucrose-fed (diet-control) female rats. Neurons from PCET fetuses were cultured in the presence of NGF (+NGF) and 200 mg/dl (mg %) EtOH and diet-control cultures received NGF and no EtOH. PCET and diet-control cultures were then divided into two groups, +NGF and -NGF (withdrawn from NGF), and exposed acutely to one of five doses of EtOH during stimulation with potassium (K(+)) chloride. [Ca(2+)](i) was measured using fura-2. PCET-CET did not affect resting [Ca(2+)](i). PCET-CET decreased and acute EtOH withdrawal increased overall K(+)-stimulated changes in [Ca(2+)](i), but only in +NGF PCET neurons. Reducing the level of EtOH from 200 to 100 mg % decreased overall K(+)-stimulated [Ca(2+)](i) in -NGF PCET neurons. The effects of PCET-CET or PCET-CET combined with NGF on overall K(+)-stimulated changes in [Ca(2+)](i) occurred mostly in the early and middle phases of the K(+)-response. NGF reduced overall K(+)-stimulated changes in [Ca(2+)](i) in PCET neurons during EtOH withdrawal and during AET with 200 mg % EtOH and increased overall K(+)-stimulated changes in [Ca(2+)](i) during AET with 400 and 800 mg % EtOH. There was no effect of NGF on overall K(+)-stimulated changes in [Ca(2+)](i) in diet-control neurons with the exception that NGF-treatment decreased overall K(+)-stimulated changes in [Ca(2+)](i) during AET with 400 mg % EtOH. The effects of AET on overall K(+)-stimulated changes in [Ca(2+)](i) mostly occurred in +NGF PCET neurons. In conclusion, CET during development of the brain could adversely affect Ca(2+)-dependent functions such as neuronal migration, neurite outgrowth, and synaptogenesis in neurons even in the presence of neurotrophin support.

Two-photon excitation imaging of pancreatic islets with various fluorescent probes.

Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca(2+)-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca(2+) measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.

Intracellular calcium signals in the surround of rat visual cortex lesions.

Focal lesions of the visual cortex induce deafferentiation, excitotoxic cell death as well as functional reorganization in the surrounding tissue. The intracellular second messenger calcium is involved in a wide range of cellular responses including excitotoxicity and functional reorganization following cortical injuries. We investigated the intracellular calcium concentration [Ca2+]i in neurons of the visual cortex using fluorescence imaging of fura-2 signals in a slice preparation obtained from lesioned and sham-operated cortices. We observed an increase in resting and stimulus evoked [Ca2+]i in the surround of the lesion, which were mediated by NMDA and non-NMDA ionotropic glutamate receptors. This increase in [Ca2+]i might be an important factor for lesion-induced functional reorganization in the rat visual cortex.

Role of kainate receptor activation and desensitization on the [Ca(2+)](i) changes in cultured rat hippocampal neurons.

We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the [Ca(2+)](i) caused by kainate showed cell-to-cell variability. The [Ca(2+)](i) increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe [Ca(2+)](i) changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the [Ca(2+)](i) and toxicity.

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells.

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.