Departments of 1Medicine, 2Neurobiology, Pharmacology and Physiology, and 3Pediatrics, Division of Biological Sciences, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois
Submitted 5 January 2005 ; accepted in final form 3 March 2005
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ABSTRACT |
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pancreatic -cell; optical electrophysiology; islet electrical coupling
Cellular voltage changes include slow, prolonged shifts in Vm that may last from a few seconds to several minutes, as well as varying numbers and patterns of spikes lasting on the order of milliseconds. Optical voltage dyes are classified as fast or slow, depending on the speed of signal response to voltage changes (26). Optical recording with fast dyes has been used in various excitable cells, but these measurements suffer from small changes in signal, typically 0.1%/mV. In contrast, slow dyes report larger responses but cannot resolve action potentials. However, newer formulations have used dye pairs to enhance the magnitude and temporal responses by 10-fold (26).
Herein we report the use of fluorescence (Förster) resonance energy transfer (FRET) between two voltage reporting dyes in combination with laser scanning confocal microscopy (LSCM) to record changes in Vm throughout optical sections of whole pancreatic islets and the combination of this FRET-based Vm reporter with fluorophores reporting [Ca2+]i. The amount of FRET is profoundly affected by distance (inverse sixth power of distance) between the donor and acceptor dye molecules and typically falls to nearly zero at >10 nm separation of the two. The voltage sensor probes (VSPs; Invitrogen), first described by González and Tsien (9, 10) and initially developed by Aurora Biosciences, were studied in our experiments. The FRET ratio pair that we used is able to report changes in Vm with relatively large signal changes (>1% ratio value per mV) with the time constant of 200 ms. N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine (CC2-DMPE; coumarin fluorophore linked to a phospholipid) functions as the FRET donor, remaining embedded in the outer leaflet of the cell membrane. The bis-(1,3-diethylthiobarbiturate) trimethine oxonol component [DiSBAC2(3), a slow, voltage-sensitive dye of the oxonol class] moves from the outer side of the membrane in polarized cells to the cytoplasmic face with depolarization, thus decreasing the amount of FRET. The DMPE-CC2 and DiSBAC2(3) pair has been used previously to measure changes in membrane potential in keratinocytes (4) and population activity in cortical neurons (14, 25).
In the present study, we used the VSPs in conjunction with LSCM to scan, for the first time, simultaneous voltage changes from multiple cells within an islet. This method revealed characteristic synchronous electrical activity within islets. By combining this method with confocal imaging of changes in [Ca2+]i in the peripheral islet cells, we also simultaneously assessed the relationship between [Ca2+]i and Vm oscillations in multiple individual islet cells in response to glucose and tetraethylammonium (TEA). The use of noninvasive confocal imaging techniques with VSPs dramatically expands the ability to study excitation-secretion coupling and cell-to-cell communication within relatively intact tissue specimens, such as pancreatic islets.
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METHODS |
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Confocal microscopy. Experiments were performed using the Leica TCS SP2 AOBS spectral laser scanning confocal microscopy system (Leica Microsystems, Mannheim, Germany). Coverslips containing islets were placed into an open perfusion chamber (Medical Systems International, Greenvale, NY) on a stage of an inverted Leica DMIRE2 microscope and maintained at 37°C. Structural data acquisition was performed in the sequential line mode for the best spatiotemporal reliability. CC2-DMPE was excited with a 405-nm diode laser, and DiSBAC2(3) was excited with the 476-nm line of an argon laser. The resulting fluorescence was recorded in two channels set up to detect emitted light correspondingly in the ranges 430490 nm and 545625 nm. For FRET experiments only, the donor dye CC2-DMPE was excited using the 405-nm diode laser. Donor and acceptor dye fluorescence signals were recorded in line mode simultaneously in two independent channels set to detect light in the ranges 430490 nm and 545625 nm, respectively.
To simultaneously record [Ca2+]i, fluo-4 was excited with the 488-nm line of the argon laser, and a 25-nm-wide band (505530 nm) of fluorescence was acquired with a third detector. Data were collected using a x63 magnification, 1.40 numerical aperture (NA) oil-immersion UV or a x20 0.70 NA air objective using 1-kHz unidirectional or 2-kHz bidirectional scan rates.
Simultaneous electrophysiological and optical recordings.
Electrophysiological recordings were performed in dissociated islet cells using a L/M-EPC7 patch-clamp amplifier (List Medical Electronics, Darmstadt, Germany) controlled using Clampex 8 acquisition software (Axon Instruments, Union City, CA) via a DigiData 1200A interface (Axon Instruments). Cells were loaded with the VSPs and placed in the Leica confocal system for FRET recordings as described above. KRB supplemented with 16 mM glucose was used as the bath solution. In a typical experiment, a single cell exhibiting satisfactory voltage dye membrane loading was approached with a borosilicate recording pipette (glass type 8250, 1.5/0.9 mm diameter, pipette resistance 46 M; Garner Glass, Claremont, CA) filled with the intracellular solution (in mM: 130 KCl, 5 NaCl, 2 CaCl2, 2 MgCl2, 10 EGTA, 5 MgATP, and 10 HEPES, pH 7.3). Pipette movements were controlled using the Water Robot Micromanipulator WR-88 (Narishige Scientific Instrument Lab, Tokyo, Japan). After the formation of a gigaohm seal was observed, a short negative pressure pulse was used to establish a whole cell configuration and the cell was held at 60 mV until the beginning of recordings. Electrophysiological and optical recordings were triggered simultaneously. For the electrophysiological component, the following voltage-step protocol was used. The cell was held at 60 mV, and seven steps with 20-mV increments were applied, starting from 70 mV and increasing to +50 mV at the final step. Each voltage step was 5 s long and was separated from the next one by a 5-s intermediate step at 60 mV. Electrical signals were recorded at a 10-kHz rate. The optical component was recorded as described above with the acquisition rate of 5 Hz.
Data analysis. The images were analyzed using Leica confocal software (Leica Microsystems), MetaMorph software (Universal Imaging, Downingtown, PA) and NIH ImageJ software (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). The electrophysiological data were processed and analyzed using Clampfit software (Axon Instruments). Data were typically processed with a Kalman filter (0.5 gain factor) or three-dimensional (3-D) hybrid median filters (NIH ImageJ software) to minimize noise. Pixel-by-pixel ratios were calculated using MetaMorph or ImageJ software with donor/acceptor [CC2-DMPE/DiSBAC2(3)] (F460/F580). 3-D reconstructions were created with ImageJ, MetaMorph, or Voxx2 software (5). Microsoft Excel, Word, and PowerPoint software were used for preparation of the figures.
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RESULTS |
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With optimal loading established, we used LSCM to scan islet cells in three fluorescent and differential interference contrast (DIC) optical channels to assess the spatial distribution of the VSPs and the degree of FRET (Fig. 1). In individual dissociated -cells (Fig. 1A), CC2-DMPE was accumulated mostly in plasmalemma, while DiSBAC2(3) was distributed throughout the entire cell and the FRET signal was clearly observed in the cell membrane region. In small islets or islet fragments (Fig. 1B), the intracellular VSP distribution was quite similar and the FRET signal could be detected readily in the cell membranes of essentially all cells in the islet, creating a lattice-like effect. While the degree of labeling of interior cells is not uniform, useful signals can be extracted from most cells of the islet even at relatively fast acquisition times for islet responses. In the islet cells, where glucose affects the Vm, the DiSBAC2(3) distribution between the plasma membrane and the cytoplasm was dependent on glucose concentration, while in presumably more stably hyperpolarized cardiomyocytes and neurons, both VSPs were localized primarily to the plasmalemma (data not shown).
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DISCUSSION |
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It is therefore of considerable interest to record simultaneous electrical activity throughout the islet. One approach has been the use of optical probes that change their emission on the basis of electrical changes such as the membrane potential. Previous methods, such as those based on oxonol dyes alone, were very slow and suffered from a low signal-to-noise ratio or did not return quickly to a basal state.
Recently, several new approaches using pairs of molecules, including CC2-DMPE and DiSBAC2(3), that couple by FRET have been reported (17). With cell depolarization, FRET becomes less efficient; correspondingly, the CC2-DMPE signal increases and the DiSBAC2(3) signal decreases. The FRET signal, calculated as the ratio of CC2-DMPE to DiSBAC2(3) signal values in our experiments, matches the known electrical behavior of islet cells. We have also verified the efficacy of these dyes using isolated chick cardiomyocytes and mouse hippocampal neurons in culture (Kuznetsov A, Bindokas VP, Philipson LH, and Marks JD, manuscript in preparation), supporting its application to cardiac and cortical imaging (11). These and similar dyes have been used primarily as tools to report relative changes in membrane potential using individual cells in multiwell plates (17) as, for example, in high-throughput screening assays of drugs (26) that might affect ion channels or ion transporters.
With the availability of appropriate lasers and optics, we have used these dyes with live cell LSCM at frame rates of 0.230 Hz. The higher frame rates are possible using subregions, and even higher ones can be used in conjunction with line-scanning modes (>1,000 Hz; data not shown). The duration of image acquisition in the time-lapse mode is essentially unlimited, provided that illumination is set low to prevent photobleaching. Recently introduced fast line scan confocal imaging technologies (e.g., the Carl Zeiss "5 live" system) will further increase full-resolution x-y capture rates to >120 Hz as well as provide faster x-y-z capabilities.
With a time constant of 200 ms for 100-mV depolarization at 20°C (10), these dyes are well suited for pancreatic islet and
-cell physiology. Moreover, the VSPs makes it possible to conduct studies of many different biological processes, from very fast ones, such as subsecond-scale individual Vm spikes in neurons (25), to much slower events, such as changes in the Vm of stimulated cells of islets of Langerhans occurring during a period of minutes or tens of minutes.
We also found that the VSPs give an essentially linear response to graded membrane potential. With the appropriate loading conditions, isolated cells and intact islets could be loaded, showing unambiguous localization of the dye to the plasma membrane compartment. In contradistinction to such widely used dyes as fura-2 AM and fluo-4 AM, which allow imaging of only peripherally located cells, CC2-DMPE and DiSBAC2(3) demonstrate excellent penetration throughout the islet, allowing measurement of Vm in multiple cells deep inside the islet. Glucose challenge resulted in membrane depolarization and repolarization cycles that temporally clearly resembled oscillations detected with microelectrode perforated patch recordings. We also increased the frequency of glucose-induced oscillations using TEA and found that the dye provided excellent fidelity, with the FRET signal continuing to report membrane oscillations.
When cells are loaded with both the Vm and [Ca2+]i indicators, the corresponding fluorescent signals can be recorded and quantified with high fidelity because of the excellent spatial separation of the cytoplasmic and plasmalemmal areas of the resulting images. We added fluo-4 AM to the islet bath during the loading conditions and found that the Ca2+ signals in the outer cells, the only ones that are capable of being loaded by acetoxymethyl ester dyes in intact islets, matched the electrical changes reported by the FRET signal extremely well.
In conclusion, this first use of the FRET pair system to report Vm changes simultaneously within multiple cells of a pancreatic islet represents a significant advance in the ability to understand integrated islet physiology. Continued advances, such as the use of faster DiSBAC derivatives [e.g., DiSBAC4(2), also available from Invitrogen] should continue to improve this approach to measuring Vm in islets. The faster dyes should provide better temporal resolution even with the currently used LSCM system, in which time resolution of 30 ms can be achieved. It is important to apply these tools to islets of other species, models of diabetes mellitus, and other experimental models of islet dysfunction (2, 22).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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