Journal of Neuroscience Methods 240 (2015) 48–60

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Basic Neuroscience

Improving AM ester calcium dye loading efficiency Mohammad I.K. Hamad a,∗ , Martin Krause b , Petra Wahle a a b

AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr Universität Bochum, D-44780 Bochum, Germany Lehrstuhl für Allgemeine Zoologie und Neurobiologie, Ruhr Universität Bochum, D-44780 Bochum, Germany

h i g h l i g h t s • • • • •

The efficiency of AM dye loading depends on the concentration of PF127. Both, PF127 and DMSO are required for successful dye loading. The labeled cells display spontaneous and evoked calcium transients. This method allows repetitive measurements for up to 24 h. This method is optimal for calcium imaging in slice cultures.

a r t i c l e

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Article history: Received 18 September 2014 Received in revised form 11 November 2014 Accepted 12 November 2014 Available online 20 November 2014 Keywords: Calcium imaging AM ester dye Organotypic cultures Osmolality DMSO Pluronic PF127

a b s t r a c t Background: Calcium imaging has unraveled the calcium-dependent mechanisms underlying neuronal function. Acetoxymethyl ester (AM) dyes are widely employed for calcium imaging. Pluronic F127 (PF127) as a surfactant and dimethyl sulfoxide (DMSO) as a solvent are used to dissolve the dyes, but concentrations vary between protocols. How these substances affect loading efficiency is not well characterized. New method: We aimed to characterize dye loading in slice cultures. We determined minimum concentrations of surfactant, solvent and dye. The current study shows that the efficiency of AM dye loading depends on the initial stock concentration of PF127. Lowering the PF127 and DMSO concentrations can improve the loading efficiency. Results: Both, pluronic and DMSO are required for successful dye loading. However, dissolving the dyes in lower concentrations of PF127 yielded better staining efficiency. Moreover, lowering the DMSO concentration to ∼0.25% improves the efficiency. The strategy allows standard two-photon or confocal microscope monitoring of neuronal activity. The labeled cells display spontaneous and evoked calcium transients, and repetitive measurements for up to 24 h after loading indicate that the method is not deleterious to neuronal function. Comparison with existing method(s): Dissolving the AM dyes in lower concentrations of PF127 combines the advantages of high loading efficiency, preserves cell viability and functional integrity, and allows repetitive measures over hours and days. Moreover, we found that the dye itself can be diluted to a final concentration of 1 ␮M which reduces the experimental costs. Conclusion: The method is optimal for calcium imaging in slice cultures. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: AM, acetoxymethyl ester; PF127, pluronic F127; DMSO, dimethyl sulfoxide; OTCs, organotypic cultures; APV, dl-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; AMPA, ␣-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid; NMDA, N-methyl-d-aspartate; OGB-1, Oregon Green 488 BAPTA-1; ACSF, artificial cerebrospinal fluid; PI, propidium iodide; arb. units, arbitrary units; DIV, days in vitro; TTX, tetrodotoxin. ∗ Corresponding author. Tel.: +49 02343224344. E-mail address: [email protected] (M.I.K. Hamad). 0165-0270/© 2014 Elsevier B.V. All rights reserved.

Calcium imaging became a powerful tool for analyzing single neuron and neuronal network activity in acute slices, in cultures as well as in vivo. AM ester dyes have proven useful for the analysis of network activity. Several methods are commonly used for loading the indicator dye. Filling with a patch pipette (Goldberg et al., 2003; Goussakov et al., 2010; Jia et al., 2010; Neher, 2013; Svoboda et al., 1996) targets a single neuron with high spatial resolution and permits calibration of the dye, but over time will dialyze the cell. Labeling of individual cells is also achieved by transfecting calcium indicator-coated particles using the gene gun (Kettunen et al., 2002;

M.I.K. Hamad et al. / Journal of Neuroscience Methods 240 (2015) 48–60

Lang et al., 2006). It results in small numbers of fully intact transfectants which can be analyzed at high spatial resolution. Pressure injection of the dye solution (Ch’ng and Reid, 2010; Dombeck et al., 2007; Dzhala et al., 2010; Garaschuk et al., 2006; Golshani et al., 2009; Grewe et al., 2010; Komiyama et al., 2010; Lillis et al., 2008; Schultz et al., 2009; Stosiek et al., 2003; Wilson et al., 2007) is usually done in vivo; the dye is taken up by subsets of nearby cells, and neurons and glial cells are differentiated by spike patterns or sulforhodamin 101 co-labeling. The injection creates a small lesion, and requires specialized instruments and practice; the advantage is that larger populations of neurons can be analyzed, and further, it seems to label cells in adult brain efficiently. In vitro, cultures or acute slices are usually incubated with the indicator dye, and the time required for efficient loading is positively correlated to the age of the tissue (Cossart et al., 2003; Hamad et al., 2011, 2014; Nikolenko et al., 2007; Tyzio et al., 2011). AM ester dyes are non-fluorescent in solution. After cellular uptake and intracellular de-esterification, fluorescence is emitted upon chelating cytosolic calcium. Common to all AM ester protocols the dye loading solution consists of PF127 and DMSO. Yet concentrations of dye and of the two major components vary between protocols and the exact loading requirements are not well known. It is thus of interest to characterize the role of the components in order to increase dye loading efficiency at lowest possible AM dye concentrations. In the present study we used 5–20 days in vitro (DIV) organotypic cultures (OTCs) from visual cortex which are electrically active. We analyzed dye loading efficiency by systematically varying the concentrations of the two components with a loading protocol which does not cause lesion to the tissue. Our results suggest that both components are essential, and the loading efficiency can be increased by using initially low concentrations of PF127 dissolved in DMSO. 2. Materials and methods 2.1. Organotypic cultures OTCs from newborn P0/1 rat visual cortex were prepared as described (Klostermann and Wahle, 1999). Tissues were chopped in 350 ␮m slices and fixed on a coverslip with a plasma/thrombin coagulate. Cultures were kept in a plastic tube with 0.7 ml of semiartificial medium containing 25% horse serum and cultivated in a non-gas roller incubator at 37 ◦ C. Medium was changed every third day.


at 50 ␮M and the competitive AMPA receptor antagonist CNQX as used at 10 ␮M. To induce cell damage, a mixture of 100 ␮M AMPA, 25 ␮M kainate and 10 ␮M NMDA (all from Tocris, Wiesbaden, Germany) were applied for 15 min. To test whether activity deprivation affects the loading we pharmacologically altered or blocked the activity by adding 10 ␮M nifedipine, or 10 ␮M bumetanide (Sigma–Aldrich, Deisenhofen, Germany), or 3 ␮M gabazine (Tocris, Wiesbaden, Germany), or 50 ␮M APV, or 10 ␮M CNQX, or all agents mixed to the loading solution. To block spontaneous activity in OTCs we applied 500 nM of tetrodotoxin (TTX; Sigma-Aldrich, Deisenhofen, Germany) to the recording solution. 2.3. AM ester calcium dye loading and osmolality measurement Various stock solutions of PF127 (Sigma, Steinheim, Germany) were prepared by initially dissolving PF127 at 1, 5, 10, 20% in 100% DMSO (w/v) (J.T. Baker, Center Valley, USA) by heating to 70 ◦ C. 50 ␮g of Oregon Green 488 BAPTA-1 AM (OGB-1), or Calcium Orange AM, or Fluo-3 AM, or Fluo-4 AM, or Indo-1 AM (all from Molecular Probes, Eugene, OR, USA) was dissolved in 100 ␮l of each of the PF127/DMSO stock solutions mentioned above and vortexed vigorously. To prepare the loading solution, 2.5 ␮l of the various dye/PF127/DMSO mixtures were diluted to 1 ml with carbogenegassed artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1 mM MgSO4 , 25 mM NaHCO3 , 1.25 mM NaH2 PO4 , 25 mM glucose, pH 7.4). This loading solution had the following final concentrations: OGB-1 was 1 ␮M and the PF127 (taken from the 1, 5, 10 and 20% stocks) was 0.0025, 0.0125, 0.025, and 0.05% (v/v) respectively, and DMSO was 0.25% (v/v). OTCs were washed 3 times with carbogene-gassed ACSF to remove the culture medium. The ACSF was discarded and 1 ml of the loading solution was applied to the culture and incubated for 45–60 min at 37 ◦ C (stationary, not in the roller drum). In this way, a 100 ␮l dye/PF127/DMSO stock mixture is enough to load 40 cultures. The stock solution is stable for 2 months at 4–8 ◦ C. Upon usage, it has to be warmed up to 37 ◦ C for 15 min and vortexed vigorously. Afterwards, the OTCs were washed several times to remove excess dye and placed into the roller incubator to recover for at least 1 h to ensure complete deesterification. The OTCs were then transferred to the recording chamber mounted on a fixed stage of an upright microscope, and perfused with carbogene-gassed ACSF (3–5 ml/min at 32 ± 2 ◦ C). The osmolality was 295 ± 5 mOsm as determined with a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany). For some experiments, the ACSF was made hypoosmotic by diluting the ACSF with water or hyper-osmotic either by adding DMSO or mannitol (Sigma, Steinheim, Germany).

2.2. Expression plasmids, biolistic transfection and pharmacological treatment

2.4. Two-photon calcium imaging

OTCs were transfected using a Helios Gene Gun (BioRad, München, Germany). Cartridges were prepared by coating 10 mg gold particles (1 ␮m diameter; BioRad) with 10 ␮g endotoxinfree plasmid encoding mCherry (pmCherry; Clontech, Heidelberg, Germany) using 50 ␮l of 0.05 M spermidine. The suspension was briefly vortexed and precipitated with 50 ␮l of 1 M CaCl2 solution. After 10 min, the samples were centrifuged and the supernatant was discarded. The gold precipitation was then washed 3 times with 100% ethanol to dehydrate the gold precipitates. The handheld Helios gene gun was used to blast gold particles into the cultures using helium gas at pressure between 180 and 200 psi. To prevent excitotoxicity during transfection at DIV 5–6, the culture medium was supplemented with a cocktail of glutamate receptor antagonists: 3 mM kynurenic acid (Sigma–Aldrich, Deisenhofen, Germany) and 50 ␮M APV (Sigma–Aldrich, Deisenhofen, Germany), and after 2 h cultures were switched to fresh medium. Two-photon calcium recordings were done at DIV 10 and older. APV was used

Fluorometric recordings were made using a custom-built twophoton laser scanning microscope equipped with a Ti:sapphire laser system (Spectra-Physics, Mountain View, CA, USA) for modelocked laser light (pulse width less than 100 femtoseconds; repetition rate, 80 MHz), a laser scanning system attached to a movable objective system on an upright microscope (Sutter Instruments, Lambrecht/Pfalz, Germany), and a water-immersion objective (20×, 0.8 NA; Olympus). To monitor calcium changes, the excitation wavelength was set to 820 nm and the laser power was reduced (5 h) of cortical neurons with very low concentrations of DMSO (0.05%) can induce a decreased action potential output upon stimulation with depolarizing currents (Tamagnini et al., 2014). This may be related to the published neuroprotective actions of DMSO; usually, dosages between 0.5 and 1.5% (v/v) are well tolerated (Lu and Mattson, 2001). Concentrations of DMSO of 10% (v/v) or higher in vitro induce pore formation in the plasma membrane and promote cell death (Ménorval et al., 2012). However, DMSO has also been reported to be toxic. For instance, exposure to 2–4% DMSO for 24 h induces apoptosis in retinal neurons in vivo and in vitro by depressing mitochondrial respiration and increasing calcium fluctuations (Galvao et al., 2014). In early postnatal dissociated hippocampal neurons, 8 h exposure to 0.5% and 1.0% of DMSO has been reported to be lethal because DMSO depresses the excitability of the cells; indeed, KCl-mediated depolarization rescues the young neurons in the presence of DMSO (Hanslick et al., 2009). In the current study, we tested the toxicity of the DMSO and PF127 using PI-staining as a readout for plasma membrane permeabilization. We showed that the short-term treatment of PF127 and DMSO for around 45–60 min does not increase cell death rates. Further, we showed that the short-term treatment of these agents did not alter the electrophysiological properties of membrane potential, input resistance and spiking threshold. The dye-loaded OTCs display an early network activity comparable to that previously published with imaging or multielectrode arrays in acute cortical slices (Dupont et al., 2006) and neocortical OTCs (Stewart and Plenz, 2008). Further, by recording OTC on the following day we showed that the OTCs were spontaneously active indicating that the dye loading does not evoke cell death. A final argument is the financial aspect. Garaschuk et al. (2006) already reported that the loading can also be accomplished with less dye in the injection pipet (100 ␮M instead of 1 mM dye; injection volume ∼400 femtoliter; DMSO concentration 1–10%). We observed efficient loading at even lower concentrations. We now routinely use the dye at 1 ␮M final concentration such that 50 ␮g OGB-1 (one commercial tube) will be good for imaging 40 cultures. This can save substantially on the budget. The dye/PF127/DMSO mixture can be kept in the refrigerator for up to 2 months. A daily fresh preparation of the loading solution is not necessary, and this saves time for the experimenter.

Acknowledgements This study was funded by Deutsche Forschungsgemeinschaft (DFG) (German Research Council) [DFG WA 541/9-2 to P.W. and DFG WA 541/10-1 to P.W.]. We also thank Dr. Melanie Mark for critically reading the manuscript.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at 2014.11.010.


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Improving AM ester calcium dye loading efficiency.

Calcium imaging has unraveled the calcium-dependent mechanisms underlying neuronal function. Acetoxymethyl ester (AM) dyes are widely employed for cal...
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