Olfactory coding in the antennal lobe of the bumble bee Bombus terrestris.

Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honey bees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe of the bumble bee Bombus terrestris. Our results show that odorants induce reproducible neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecule's chemical structure, i.e. its carbon chain length and functional group. In addition, pairwise similarity among odor representations are conserved in bumble bees and honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Quantitative fluorescence spectroscopy in turbid media: a practical solution to the problem of scattering and absorption.

The presence of practically unavoidable scatterers and background absorbers in turbid media such as biological tissue or cell suspensions can significantly distort the shape and intensity of fluorescence spectra of fluorophores and, hence, greatly hinder the in situ quantitative determination of fluorophores in turbid media. In this contribution, a quantitative fluorescence model (QFM) was proposed to explicitly model the effects of the scattering and absorption on fluorescence measurements. On the basis of the proposed model, a calibration strategy was developed to remove the detrimental effects of scattering and absorption and, hence, realize accurate quantitative analysis of fluorophores in turbid media. A proof-of-concept model system, the determination of free Ca(2+) in turbid media using Fura-2, was utilized to evaluate the performance of the proposed method. Experimental results showed that QFM can provide quite precise concentration predictions for free Ca(2+) in turbid media with an average relative error of about 7%, probably the best results ever achieved for turbid media without the use of advanced optical technologies. QFM has not only good performance but also simplicity of implementation. It does not require characterization of the light scattering properties of turbid media, provided that the light scattering and absorption properties of the test samples are reasonably close to those of the calibration samples. QFM can be developed and extended in many application areas such as ratiometric fluorescent sensors for quantitative live cell imaging.

Paying the piper: the cost of Ca2+ pumping during the mating call of toadfish.

Superfast fibres of toadfish swimbladder muscle generate a series of superfast Ca(2+) transients, a necessity for high-frequency calling. How is this accomplished with a relatively low rate of Ca(2+) pumping by the sarcoplasmic reticulum (SR)? We hypothesized that there may not be complete Ca(2+) saturation and desaturation of the troponin Ca(2+) regulatory sites with each twitch during calling. To test this, we determined the number of regulatory sites by measuring the concentration of troponin C (TNC) molecules, 33.8 µmol per kg wet weight. We then estimated how much SR Ca(2+) is released per twitch by measuring the recovery oxygen consumption in the presence of a crossbridge blocker, N-benzyl-p-toluene sulphonamide (BTS). The results agreed closely with SR release estimates obtained with a kinetic model used to analyse Ca(2+) transient measurements. We found that 235 µmol of Ca(2+) per kg muscle is released with the first twitch of an 80 Hz stimulus (15(o)C). Release per twitch declines dramatically thereafter such that by the 10th twitch release is only 48 µmol kg(-1) (well below the concentration of TNC Ca(2+) regulatory sites, 67.6 µmol kg(-1)). The ATP usage per twitch by the myosin crossbridges remains essentially constant at ∼25 µmol kg(-1) throughout the stimulus period. Hence, for the first twitch, ∼80% of the energy goes into pumping Ca(2+) (which uses 1 ATP per 2 Ca(2+) ions pumped), but by the 10th and subsequent twitches the proportion is ∼50%. Even though by the 10th stimulus the Ca(2+) release per twitch has dropped 5-fold, the Ca(2+) remaining in the SR has declined by only ∼18%; hence dwindling SR Ca(2+) content is not responsible for the drop. Rather, inactivation of the Ca(2+) release channel by myoplasmic Ca(2+) likely explains this reduction. If inactivation did not occur, the SR would run out of Ca(2+) well before the end of even a 40-twitch call. Hence, inactivation of the Ca(2+) release channel plays a critical role in swimbladder muscle during normal in vivo function.

Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging.

Using optical imaging of retinal ganglion cell (RGC) calcium dynamics in living intact retinal wholemount preparations, we tested whether RGCs in an experimental rat glaucoma model were more sensitive to exogenously applied glutamate as a result of deficient glutamate clearance mechanisms. In contrast to post-natal rat RGCs in purified cultures, in which the calcium influx induced by 200 microm NMDA and 10 microm glutamate was approximately equivalent, application of up to 500 microm glutamate did not affect calcium levels in RGCs in retinal wholemounts, even though the RGCs responded to 200 microm NMDA. Glutamate (500 microm) did elicit a RGC calcium response in retinal wholemounts when glutamate transporters were inhibited pharmacologically with DL-threo-beta-benzyloxyaspartate, confirming the presence of glutamate clearance mechanisms in this intact retina preparation. The effect of glutamate was then assessed on retinas from rats with chronically elevated intraocular pressure in one eye, produced by the injection of hypertonic saline into an episcleral vein. Application of up to 500 microm glutamate had no effect on RGC calcium levels, while millimolar concentrations of glutamate induced a calcium signal in RGCs that was indistinguishable from that in fellow control retinas. Therefore, there was no evidence for a global defect in glutamate uptake in this rat model of experimental glaucoma. Imaging glutamatergic calcium dynamics of RGCs in retinal wholemounts represents a novel methodology to probe glutamate transporter function and dysfunction in an intact CNS tissue system.

Optical assessment of motoneuron function in a "twenty-four-hour" acute spinal cord slice model from fetal rats.

In acute slice preparations of most brain regions, neuronal functions are preserved for only few hours. Since the effects of growth factors or neurotoxic agents are often manifested beyond this time scale, corresponding studies are typically performed on cultured cells. However, cell cultures are generated and maintained under vastly different conditions that can grossly alter neuronal properties. For example, glutamate application to motoneuronal cultures has been reported to modulate neurite formation in some studies while in others it has been reported to kill cells. Here, we have examined whether acute spinal cord slices from rat fetuses can be used within a time window of 24 h for assessment of long-term effects of neuromodulators. In these slices, we have studied the action of glutamate on lumbar motoneurons loaded with fura-2 and rhodamine-123 to monitor intracellular Ca2+ ([Ca2+]i) and mitochondrial potential (Deltapsi), respectively. Further, loading with fura-2 or propidium iodide allowed for morphological assessment of cell viability and death, respectively. Pulses (15 s) or 1 h application of glutamate (300 microM) evoked a moderate (approximately 500 nM) [Ca2+]i rise, but no change of Deltapsi. Even after 24 h, no glutamate-induced cell death was observed and glutamate pulse-evoked [Ca2+]i transients were comparable to controls. The data demonstrate that glutamate does not deregulate [Ca2+]i homeostasis in fetal motoneurons in situ. We propose that acute spinal cord slices from perinatal rodents are a robust model that allows for analysis of neuronal properties and cell viability within a time window of at least 24 h.

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader.

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.

GABAB receptor-mediated presynaptic potentiation of ATP ionotropic receptors in rat midbrain synaptosomes.

Nucleotides can activate ionotropic P2X receptors that induce calcium-responses in rat midbrain synaptosomes. In this report, we show that ATP elicits Ca(2+) responses producing a monophasic dose-response curve with an EC(50) value of 24.24+/-1.42 micro M. In the presence of gamma-aminobutyric acid (GABA), the ATP dose-response curve becomes biphasic with EC(50) values of 3.69+/-0.44 nM and 59.65+/-8.32 micro M. Moreover, the maximal calcium response induced by ATP is 52.1% higher than the control. This effect is mimicked or blocked by the specific GABA(B) receptor agonist and antagonist, baclofen and saclofen, respectively. Presynaptic GABA(B) receptors, identified by immunocytochemistry are present in 62% of the total synaptosomal population. Adenylate cyclase and protein kinase A cascades are involved in the potentiatory effects mediated by baclofen and their activation or inhibition modifies calcium signalling and synaptosomal cAMP levels. The potentiatory action of baclofen was confirmed by microfluorimetry performed on single synaptic terminals. In its presence, 86% of the terminals responding to 100 micro M ATP, are also able to respond to nanomolar concentrations (100 nM) of this nucleotide. This potentiatory effect is reduced to 32% in the presence of pertussis toxin. Our data suggest that the activity of P2X receptors is modulated by GABA(B) receptors in midbrain synaptosomes.

Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis.

Normal and malignant epithelial cells are induced to undergo apoptosis by a large variety of mechanistically diverse agents. Regardless of inducing agent, apoptosis characteristically occurs asynchronously within a population of epithelial cells over a period of 12-96 h and is associated with permeability and enzymatic perturbations. Pre-loading of cells with acetoxymethyl esters (AM) derivatives of fluorescent Ca2+ indicators (i.e. fura-2, indo-1, fluo-3) by passive diffusion allows longitudinal kinetic analysis of acute [Ca2+] changes subsequent to exposure to apoptosis inducing agents. Using prostate cancer cell lines, however, it is demonstrated that dye leakage and compartmentalization into organelles limit such passive loading to longitudinal [Ca2+]i measurements of < 2 h. Post-loading of cells exposed to the apoptosis inducing agent for several hours is also inaccurate owing to decreased loading efficiency and de-esterification of the probes resulting in increased production of fluorescent Ca(2+)-insensitive dye species. To accurately measure kinetics of [Ca2+]i changes longitudinally in individual cells undergoing apoptosis, cells were microinjected with fura dextran and maintained in a physiologic environment. [Ca2+] and morphological changes characteristic of apoptosis were then followed simultaneously in individual cells over several days following exposure to the apoptosis inducing agent.

Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes.

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca2+ mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+]i) level and the mechanisms of Ca2+ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca2+ mobilization as well as the mechanisms of changes in [Ca2+]i in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrated by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobilization. The increase in intracellular Ca2+ in cardiomyocytes by PA, unlike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca2+ movements.

Assessment of platelet cytosolic concentration of free magnesium in healthy subjects.

We measured basal cytosolic concentration of free Mg2+ ([Mg2+]i) in platelets from 30 healthy volunteers by using mag-fura-2, a new fluorescent Mg2+ indicator. The mean [Mg2+]i was 381 +/- 22 mumol/L, with values ranging from 226 to 771 mumol/L. The day-to-day intrasubject coefficient of variation was relatively small (3.6%). [Mg2+]i was significantly higher in men than in women (430 +/- 38 vs 332 +/- 17 mumol/L, p < 0.03), and was not correlated with age. There was no significant relation between platelet [Mg2+]i and serum total Mg concentration. The results indicate that the gender but not age may decide intracellular Mg2+ status. In addition, the mechanisms that regulate [Mg2+]i may be independent of those that influence the extracellular concentration of Mg.

Assessment of Fura-2 for measurements of cytosolic free calcium.

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement.

The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.