Monitoring brain activity and behaviour in freely moving Drosophila larvae using bioluminescence.

We present a bioluminescence method, based on the calcium-reporter Aequorin (AEQ), that exploits targeted transgenic expression patterns to identify activity of specific neural groups in the larval Drosophila nervous system. We first refine, for intact but constrained larva, the choice of Aequorin transgene and method of delivery of the co-factor coelenterazine and assay the luminescence signal produced for different neural expression patterns and concentrations of co-factor, using standard photo-counting techniques. We then develop an apparatus that allows simultaneous measurement of this neural signal while video recording the crawling path of an unconstrained animal. The setup also enables delivery and measurement of an olfactory cue (CO2) and we demonstrate the ability to record synchronized changes in Kenyon cell activity and crawling speed caused by the stimulus. Our approach is thus shown to be an effective and affordable method for studying the neural basis of behavior in Drosophila larvae.

Exogenous oxidative stress induces Ca2+ release in the yeast Saccharomyces cerevisiae.

The Ca(2+) -dependent response to oxidative stress caused by H(2)O(2) or tert-butylhydroperoxide (tBOOH) was investigated in Saccharomyces cerevisiae cells expressing transgenic cytosolic aequorin, a Ca(2+) -dependent photoprotein. Both H(2)O(2) and tBOOH induced an immediate and short-duration cytosolic Ca(2+) increase that depended on the concentration of the stressors. Sublethal doses of H(2)O(2) induced Ca(2+) entry into the cytosol from both extracellular and vacuolar sources, whereas lethal H(2)O(2) shock mobilized predominantly the vacuolar Ca(2+). Sublethal and lethal tBOOH shocks induced mainly the influx of external Ca(2+), accompanied by a more modest vacuolar contribution. Ca(2+) transport across the plasma membrane did not necessarily involve the activity of the Cch1p/Mid1p channel, whereas the release of vacuolar Ca(2+) into the cytosol required the vacuolar channel Yvc1p. In mutants lacking the Ca(2+) transporters, H(2)O(2) or tBOOH sensitivity correlated with cytosolic Ca(2+) overload. Thus, it appears that under H(2)O(2)-induced or tBOOH-induced oxidative stress, Ca(2+) mediates the cytotoxic effect of the stressors and not the adaptation process.

Monitoring neural activity with bioluminescence during natural behavior.

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of approximately 20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.

Measurements of mitochondrial calcium in vivo.

Mitochondria play a pivotal role in intracellular Ca(2+) signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca(2+) handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca(2+) transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca(2+) sensors, respectively. Both methods revealed a consistent picture of Ca(2+) uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca(2+) levels. The big future challenge is to understand the functional impact of such Ca(2+) signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory.

Calcium-sensitive photoproteins.

The recombinant Ca2+ sensitive photoprotein aequorin was the first probe used to measure specifically the Ca2+ concentration, [Ca2+], inside the intracellular organelles of intact cells. Aequorin-based methods offer several advantages: (i) targeting of the probe is extremely precise, thus permitting a selective intracellular distribution; (ii) the use of wild-type and low Ca2+-affinity aequorins allows covering a large dynamic range of [Ca2+], from 10(-7) to 10(-3)M; (iii) aequorin has a low Ca2+ buffering effect and it is nearly insensitive to changes in Mg2+ or pH; (iv) it has a high signal-to-noise ratio; (v) calibration of the results in [Ca2+] is made straightforward using a simple algorithm; and (vi) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine is necessary to generate the functional photoprotein and this procedure requires at least 1h; (iii) in the case of aequorin targeted to high Ca2+ compartments, because of the high rate of aequorin consumption at steady-state, only relatively brief experiments can be performed and, because of the steepness of the Ca2+-response curve, the calibrated [Ca2+] values may not reflect the real mean in cells or compartments with dyshomogeneous behavior; and (iv) expression of targeted aequorins requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones.

Mechanisms of electrically mediated cytosolic Ca2+ transients in aequorin-transformed tobacco cells.

Cytosolic Ca(2+) changes induced by electric field pulses of 50-micros duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca(2+)-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca(2+) concentration reaching a peak value within <100-200 ms. Peaking of [Ca(2+)](cyt) was followed by a biphasic decay due to removal of Ca(2+) (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of approximately 0.5 and 3-5 s, respectively. Experiments with various external Ca(2+) concentrations and conductivities showed that the fast decay arises from Ca(2+) fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca(2+) fluxes through the tonoplast. The amplitude of the [Ca(2+)](cyt) transients increased with increasing field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical field strength of approximately 450 V/cm, whereas breakdown of the tonoplast required approximately 580 V/cm. The above findings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca(2+) and presumably other vacuolar ingredients.

Early perception responses of Nicotiana tabacum cells in response to lipopolysaccharides from Burkholderia cepacia.

Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe-/pathogen-associated molecular patterns, have diverse roles in plant-microbe interactions, e.g. LPS are able to promote plant disease tolerance through activation of induced or acquired resistance. However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans. The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. The purified LPS(B.cep.) was found to trigger a rapid influx of Ca2+ into the cytoplasm of aequorin-transformed tobacco cells. An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence. These early perception responses were accompanied by K+/H+ exchange and alkalinization of the extracellular medium. Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS(B.cep.) elicitation was dissected, elucidated and compared to that induced by a yeast elicitor. These results suggest that LPS(B.cep.) interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells.

The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.

Activation of the oxidative burst in aequorin-transformed Nicotiana tabacum cells is mediated by protein kinase- and anion channel-dependent release of Ca2+ from internal stores.

The source of Ca2+ involved in transducing an oxidative-burst defense signal was examined in aequorin-transformed tobacco (Nicotiana tabacum L.) cells using modulators of Ca2+ entry. Treatments that either increased or decreased the influx of Ca2+ from external stores were found to have little effect on the magnitude or kinetics of an osmotically stimulated oxidative burst. In contrast, treatments that reduced the discharge of Ca2+ from internal stores inhibited dilution-activated H2O2 production. Curiously, most of the modulators commonly employed in animal studies as internal Ca(2+)-release inhibitors were neither effective in blocking discharge of intracellular Ca2+ nor in preventing the oxidative burst. When three different biochemical elicitors of the oxidative burst were similarly examined, both the H2O2 production and Ca2+ fluxes stimulated were found to be sensitive to modulators of internal Ca2+ release, but neither was impacted by alterations in externally derived Ca2+ influx. We hypothesize, therefore, that the oxidative burst does not depend on the influx of external Ca2+, but instead may generally be mediated by the release of internal Ca2+ in a manner that depends on the proper function of kinases and anion channels. These Ca2+ pulses trigger downstream signaling events that include the activation of Ca(2+)-regulated protein kinases, which are required for stimulation of the oxidative burst.

Switching from simple to complex oscillations in calcium signaling.

We present a new model for calcium oscillations based on experiments in hepatocytes. The model considers feedback inhibition on the initial agonist receptor complex by calcium and activated phospholipase C, as well as receptor type-dependent self-enhanced behavior of the activated G(alpha) subunit. It is able to show simple periodic oscillations and periodic bursting, and it is the first model to display chaotic bursting in response to agonist stimulations. Moreover, our model offers a possible explanation for the differences in dynamic behavior observed in response to different agonists in hepatocytes.

Mastoparan induces an increase in cytosolic calcium ion concentration and subsequent activation of protein kinases in tobacco suspension culture cells.

Mastoparan induced a transient elevation of cytosolic free Ca2+ concentration ([Ca2+]cyt) in tobacco suspension culture cells. The mastoparan-induced [Ca2+]cyt elevation was inhibited by 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl and neomycin but not by depletion of extracellular Ca2+, suggesting that the elevation was the result of Ca2+ release from the intracellular stores caused by stimulation of phosphoinositide turnover. Hydrogen peroxide which has been shown to induce an oxidative burst in soybean cells by mastoparan treatment [L. Legendre, P.F. Heinstein, P.S. Low, Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells, J. Biol. Chem., 267 (1992) 20140-20147], also induced a transient [Ca2+]cyt elevation in the tobacco cells. However, mastoparan did not induce an oxidative burst in the tobacco cells. Activation of a 50, a 75 and a 80 kDa protein kinases after the mastoparan-induced [Ca2+]cyt elevation was shown by an in-gel protein kinase assay. This activation was inhibited by neomycin, suggesting that the [Ca2+]cyt elevation is necessary for the mastoparan-induced activation of the protein kinases. The activation was inhibited also by pretreatment with staurosporine and was sustained by pretreatment with calyculin A, suggesting that the protein kinase activity is regulated by protein phosphorylation/dephosphorylation. The present report shows that mastoparan induces an increase in [Ca2+]cyt without oxidative burst and subsequent activation of protein kinases in tobacco cells.

A history of stress alters drought calcium signalling pathways in Arabidopsis.

Environmental stresses commonly encountered by plants lead to rapid transient elevations in cytosolic free calcium concentration ([Ca2+]cyt) (Bush, 1995; Knight et al., 1991). These cellular calcium (Ca2+) signals lead ultimately to the increased expression of stress-responsive genes, including those encoding proteins of protective function (Knight et al., 1996; Knight et al., 1997). The kinetics and magnitude of the Ca2+ signal, or 'calcium signature', differ between different stimuli and are thought to contribute to the specificity of the end response (Dolmetsch et al., 1997; McAinsh and Hetherington, 1998). We measured [Ca2+]cyt changes during treatment with mannitol (to mimic drought stress) in whole intact seedlings of Arabidopsis thaliana. The responses of plants which were previously exposed to osmotic and oxidative stresses were compared to those of control plants. We show here that osmotic stress-induced Ca2+ responses can be markedly altered by previous encounters with either osmotic or oxidative stress. The nature of the alterations in Ca2+ response depends on the identity and severity of the previous stress: oxidative stress pre-treatment reduced the mannitol-induced [Ca2+]cyt response whereas osmotic stress pretreatment increased the [Ca2+]cyt response. Therefore, our data show that different combinations of environmental stress can produce novel Ca2+ signal outputs. These alterations are accompanied by corresponding changes in the patterns of osmotic stress-induced gene expression and, in the case of osmotic stress pre-treatment, the acquisition of stress-tolerance. This suggests that altered Ca2+ responses encode a 'memory' of previous stress encounters and thus may perhaps be involved in acclimation to environmental stresses.

Ca2+ homeostasis in the endoplasmic reticulum: coexistence of high and low [Ca2+] subcompartments in intact HeLa cells.

Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).

External Ca2+-dependent Ca2+ release in directly stimulated diaphragm muscles of mice.

Intracellular Ca2+ release in directly stimulated diaphragm muscles of mice was found to be dependent on external Ca2+, using the intracellular Ca2+ aequorin luminescence transient technique. The Ca2+ mobilization into the cells may operate via a voltage- and external Ca2+-dependent mechanism, and via a caffeine- and external Ca2+-independent one, from the Ca2+ pools in plasma membranes or in the terminal cisternae of sarcoplasmic reticulum.

Bioluminescence in the sea: photoprotein systems.

Photoproteins are the primary reactants of the light-emitting reactions of various bioluminescent organisms. A photoprotein emits light in proportion to its amount, like a luciferin, but its light-emitting reaction does not require a luciferase. There are about two dozen types of bioluminescent organisms for which substantial biochemical knowledge is presently available, and about one third of them involve photoproteins. Most photoproteins are found in marine organisms. There are various types of photoproteins: the photoproteins of coelenterates, ctenophores and radiolarians require Ca2+ to trigger their luminescence; the photoproteins of the bivalve Pholas and of the scale worm appear to involve superoxide radicals and O2 in their light-emitting reactions; the photoprotein of euphausiid shrimps emits light only in the presence of a special fluorescent compound; the photoprotein of the millipede Luminodesmus, the only known example of terrestrial origin, requires ATP and Mg2+ to emit light. The Ca2+-sensitive photoproteins of coelenterates have been most frequently studied and most widely used. Therefore, they are overwhelmingly popular compared with other types. All coelenterate photoproteins, including aequorin, halistaurin, obelin and phialidin, have relative molecular masses close to 20 000, contain an identical functional group, and emit blue light in aqueous solution when a trace of Ca2+ is added, in the presence or absence of molecular oxygen. Aequorin contains an oxygenated form of coelenterazine in its functional group. When Ca2+ is added, aequorin decomposes into three parts, i.e., apo-aequorin, coelenteramide and CO2, accompanied by the emission of light. Apo-aequorin can be reconstituted into active aequorin indistinguishable from the original sample, by incubation with an excess of coelenterazine in a buffer containing 5 mM-EDTA and a trace of 2-mercaptoethanol, even at 0 degree C. Thus, aequorin and other coelenterate photoproteins can be luminesced and recharged repeatedly. The regeneration of coelenterate photoproteins in this manner probably takes place in vivo, utilizing stored coelenterazine. The photoproteins of coelenterates, and their chemically modified forms, are useful in measuring and monitoring calcium ions in biological systems, especially in single cells.

Voltage-dependent facilitation of Ca2+ entry in voltage-clamped, aequorin-injected molluscan neurons.

Voltage-clamp experiments were performed on giant neurons of the nudibranch Anisodoris nobilis injected with the Ca-sensitive photoprotein, aequorin. Depolarization beyond -10 to +5 m V produced an aequorin signal, the amplitude of which depended on the extracellular Ca2+ concentration, the amplitude of the depolarization, and its duration. In paired pulse experiments, the amplitude of the aequorin signal produced in response to the second of two identical depolarizing pulses was larger than that produced during the first, resulting from an increased entry of Ca2+ during the second pulse. The increment in Ca conductance inferred from the augmented signal during the second pulse was independent of Ca2+ influx during the first pulse but, instead, was related to the amplitude and duration of the first pulse.