CHAPTER 8 Determination of Intracellular Chloride ... · PDF fileDetermination of Intracellular Chloride Concentration in Dorsal Root ... of Ecl. C1--sensitive fluorescent dyes may

CHAPTER 8

Determination of Intracellular Chloride Concentration in Dorsal Root Ganglion Neurons by Fluorescence Lifetime Imaging

Hiroshi Kaneko, Ilva Putzier, Stephan Frings, and Thomas Gensch Institut fOr Biologische Informationsverarbeitung, Forschungszentrum Jtilich, 52425 Jttlich, Germany

I. Introduction II. Fluorescence Lifetime Microscopy with Two-Photon Excitation (TP-FLIM)

m. Fluorescence Lifetime Analysis of C1- Concentration IV. Determination of Intracellular C1- Concentration in Rat DRG Neurons V. Discussion

References

1. INTRODUCTION

Primary afferent neurons of the dorsal root ganglia (DRG) express Ca2+-activated C1- channels that have been studied in somata of isolated DRG neurons in various species (Bader et al., 1987; Scott et al., Currie et al., 1995; Kenyon and Goff, 1998). The channels open during a rise of the intracellular Ca 2+ concentration, [Ca2+]i, that follows Ca 2+ influx or Ca 2+ release (Crawford et al., 1997; Usachev and Thayer, 1997; Ayar and Scott, 1999) and substantially affect membrane properties by inducing a large anion conductance (roughly 5-30 ns). The channels thus constitute a link between [Ca2+]i and membrane excitability, but their role in sensory signal processing is still unclear. Critical information is missing about the cellular distribution of channels as well as about CI- homeostasis in the various functional compartments of the neurons, namely sensory endings, axon, soma, and synaptic terminals. Results obtained from acutely dissociated somata

Current Topics in Membranes, Volume 53 Copyright 2002, Elsevier Science (USA). All fights reserved. 1063-5823/02 $35.00

167

168 Kaneko et al.

of DRG neurons or from cultured sensory neurons cannot provide insight into the way Ca2+-activated C1- channels shape the sensory signal as it travels from the periphery to the synaptic ending in the spinal cord.

How Ca2+-activated C1- channels contribute to electrical signal processing, whether they hyperpolarize or depolarize the membrane, is determined by local levels of the C1- equilibrium potential, Ecl. Several researchers have found that E a in isolated somata of DRG neurons is in the range of -30 to - 4 0 mV and, thus, roughly 30 mV more positive than the resting membrane voltage of DRG neurons. This difference implies (1) that DRG neurons actively accumulate C1- against an electrochemical potential gradient, and (2) that opening of Ca2+-activated C1- channels induces C1- efflux and depolarization of the plasma membrane. Ecl values in these studies were obtained using Cl--selective microelectrodes (Alvarez-Leefmans et al., 1988; Alvarez-Leefmans, 1990) or by measuring the reversal voltage of GABAA receptors or Ca2+-activated C1- channels (Deschenes et al., 1976; Gallagher et al., 1978; Kenyon, 2001). These techniques are suited for experiments with cell somata but cannot be employed to study Ecl in cellular processes including sensory and synaptic endings of DRG neurons. Because the processes are thin (1-2/zm diameter) and cannot be dissected from their respec- tive tissues, a noninvasive optical method is required that can be applied to vital tissue slices and offers high spatial resolution for the examination of local levels of Ecl. C1--sensitive fluorescent dyes may be a solution to this problem, because they can be introduced into cellular processes and report changes in the local level of [C1-]i.

A variety of C1--sensitive dyes are used as optical probes for the intracellular C1- concentration, [C1-]i, (Verkman, 1990; Verkman and Biwersi, 1995). These probes report changes in [C1-]i because their fluorescence is physically quenched by C1- so that increasing [C1-]i causes a decrease of fluorescence intensity. To obtain absolute values for [C1-]i, the relation between fluorescence intensity and [C1-]i must be established. Calibration is necessary for each individual cell because the fluorescence intensity depends not only on [C1-]i but also on the amount of dye molecules in the excitation volume. Thus, the local probe concentration, the size of cells and cellular processes, as well as the sequestration of the probe by cellular organelles are factors that determine fluorescence intensity, but cannot be measured quantitatively. The calibration procedure involves selective permeabilization of the plasma membrane by ionophores, which allow equilibration of intracellular and extracellular [C1-] (Verkman, 1990). This procedure is difficult even in isolated cells (see below), but intracelluar calibration within processes of DRG neurons is probably not feasible. A solution for this problem may be fluorescence lifetime analysis of C1- probes. If the intracellular quenching efficiency is known, fluorescence lifetime, unlike fluorescence intensity, directly reports the C1- concentration and does not require knowledge about dye concentration or cell size. As a first step toward the application of fluorescence lifetime analysis to somatosensory neurons,

8. Intracellular Chloride Concentration in DRG 169

we examine the measurement of [C1-]i in isolated DRG somata. We test whether the Cl--sensitive fluorescent dye N-(6-methoxyquinolyl) acetoethyl ester (MQAE) is suited to be used as an intracellular C1- probe in fluorescence lifetime experiments with two-photon excitation. Two-photon optics combine several advantages for these experiments. The use of red or infrared light minimizes photodamage by the ultraviolet UV light that is necessary to excite MQAE fluorescence, because excitation of absorbing molecules by blue and UV light occurs only within the narrow focal plane of the objective lens. Red light also permits recording from cells within tissue slices because reduced light scattering increases the image quality and the penetration depth. Moreover, two-photon imaging provides excellent spatial resolution, and the femtosecond-pulse lasers used for two-photon excitation are a good light source for fluorescence lifetime analysis.

II. FLUORESCENCE LIFETIME MICROSCOPY WITH TWO-PHOTON EXCITATION (TP-FLIM)

In this section the basic principles of two-photon excitation, fluorescence lifetime measurements, fluorescence lifetime imaging, and the instrumentation needed for TP-FLIM will be presented.

Two-photon excitation is a nonlinear optical process that has certain advantages in microscopy applications compared to one-photon excitation [for more details see, e.g., Piston (1996) and references therein]. Before discussing those, the basics of two-photon excitation will be briefly explained. One-photon and two-photon excitation are schematically shown in Figure 1A. The first step in the interaction of a photon and a molecule is the so-called virtual absorption of the photon exciting an electron of the molecule. If the photon energy corresponds to an electronic transition, i.e., the energy difference between one of the occupied molecular orbitals and the unoccupied molecular orbitals (UMO), the electron transiently populates this UMO. In most fluorescence applications this UMO is the energetically lowest UMO and is named $1. This process is called one-photon excitation. If the photon energy is not in resonance with the electronic transition energies of the molecule the photon will be released eventually changing its energy and direction. These processes are called elastic and inelastic scattering.

The lifetime of the virtually excited state, however, is not infinitely short but has a duration of about 10-16 s. If within this time a second photon meets the molecule and the sum of the two photon energies is in resonance with the SI energy level, the electron will also end up in the $1 state. This process is called two-photon excitation. It requires very high temporal and spatial photon densities that are achievable by the use of a focused pulsed laser. Typically pulse widths of less than 5 ps are used and often the laser is focused via a high NA microscope objective to get higher spatial photon densities.

170 Kaneko et al.

A

B one-photon excitation two-photon excitation

J S1

So

It.

0.1

0.01

2*[Q] ~ [Q] ~0 _ ,, ,, r 1 E-,3 -

Q 0 10 20 30 40 50

x = 1 / (kf +knr+kqg:[Q]) Ume (ns)

FIGURE 1 Schematic representation of one- and two-photon excitation and the molecular origin of the fluorescence lifetime with consideration of physical quenching. (A) Schematic representation of one-photon (left) and two-photon (right) excitation and comparison of the localization of photodamage in microscopy applications. (B) Molecular origin of the fluorescence lifetime r in the absence and presence of physical quenching and the influence of quencher concentration on r. See text for description of the model.

There are three advantages of using two-photon over one-photon excitation in fluorescence microscopy: photodamage reduction, larger penetration depth, and higher light collection efficiency for high-resolution imaging.

The use of fluorescence microscopy with one-photon excitation in nonfixed cells and cell tissue is often limited by photodamage caused by the absorption of light by proteins with cofactors (ttavins, hemes, retinals, chlorophylls, etc.). Photodamage is a very complex phenomenon involving production of chemically reactive species and heating. It can hardly be understood and/or controlled. Rather, photodamage has to be avoided when working on living cells. One-photon excitation happens in all layers of the sample as schematically shown in Figure 1A. Two-photon excitation only occurs in the focal volume. The light used for two-photon imaging in living cells (red to near-infrared, 700-1100 nm) is not absorbed by proteins and most of the cofactors (exceptions are the photosynthetic complexes). Therefore photodamage with two-photon excitation is greatly reduced and is confined to the focal volume.

8. IntraceUular Chloride Concentration in DRG 171

Another general problem of light microscopy is the finite penetration depth. The scattering of light by matter limits the possibility for application of fluorescence microscopy in cell tissue. Scatter causes loss in resolution and detection efficiency. The scatter probability of light is inversely proportional to the fourth power of the wavelength (~,Z-4). This is a theoretical value; in biological tissue an exponent of 2.3-3.3 is found. Nevertheless, the penetration depth is much larger for microscopy using red light compared to blue light. Most of the known and established fluorophores for fluorescence microscopy, however, absorb in the blue and green spectral region. They can nicely be excited by two-photon excitation using light from 700 to 1000 nm. Therefore two-photon microscopy has a superior penetration depth compared to one-photon microscopy and is the method of choice for imaging tissue.

Laser scanning confocal microscopy with one-photon excitation has become the most widely used microscopy method for biological applications because of its very good three-dimensional spatial resolution (~200 nm in the x- and y- and ~600 nm in the z-direction using blue excitation light). The latter is achieved by using a pinhole in the imaging path, which allows only fluorescence light from the focal volume to reach the detector. As a drawback the amount of fluorescence photons detected from the focal volume by confocal imaging is also reduced. To account for that, as well as to counter the influence of scattering, more excitation power has to be used. This leads to increased photodamage and cell death, which is often not acceptable. Two-photon microscopy not only reduces the photodamage but has intrinsically a good spatial resolution without the use of confocal imaging. In this way it is possible to omit the pinhole and collect much more fluorescence light without losing the high-resolution image. As stated above, for practical reasons the same fluorophores are usually used in one- and two-photon microscopy. Because the spatial resolution is also proportional to the wavelength of the excitation light, two-photon resolution with red to near-infrared light excitation (~400 nm in the x- and y- and ~ 1/xm in the z-direction) is slightly less compared to one-photon excitation. But the detected light intensity is higher compared to confocal one-photon microscopy.

The parameter most often used as readout in fluorescence microscopy is the fluorescence intensity. It depends on many parameters and obviously on the concentration of the fluorophore. In applications to cells and tissue the fluorophore distribution is in general not homogeneous but highly heterogeneous, which makes comparisons beetween the fluorescence signals from different parts of one cell or between different cells difficult and may be misleading. More sophisticated fluorescence microscopy techniques use other fluorescence parameters such as the anisotropy, the spectrum, or the lifetime. Fluorescence lifetime has the advantage that it does not depend on the fluorophore concentration. Therefore, if a certain cell parameter can be read out via the fluorescence lifetime of a molecule, then different parts of the cell and different cells can be directly compared.

172 Kaneko et al.

The processes determining the fluorescence lifetime are shown in Fig. lB. The excited electron (here with two-photon excitation) can relax from the $1 state via nonradiative internal conversion with a rate k,r or via radiative relaxation (rate constant kF), i.e., fluorescence. Other photophysical processes such as inter- system crossing, charge separation, radical formation, and photochemical reactions are neglected for the sake of simplicity. In such a case the fluorescence lifetime is equal to the reciprocal value of the sum of the two rate constants for deactivation, knr + kF. It should be noted that the fluorescence behavior is in most cases independent of the excitation method. In other words, a fluorophore that is excited via one-photon absorption has very similar fluorescence properties compared to a fluorophore excited by two-photon absorption.

If an excited molecule is colliding with another molecule or ion in solution, an additional process may occur. The second particle--named the quencher (Q)-- may form a complex with the fluorophore. This complex can have a much larger k~r value, leading to nonradiative deactivation and hence decreasing the probability for fluorescence. The fluorescence intensity, but more importantly the fluorescence lifetime (see Fig. 1B) will decrease if the concentration of quencher molecules is high enough (usually millimolar) to ensure diffusional collision between fluorophore and quencher during the fluorophore excited state lifetime. In this way, an additional deactivation channel has been opened as shown in Fig. lB. If the lifetime at zero quencher concentration (r0) is known it is easy to calculate kq x [Q]. The concentration of the quencher can be calculated if kq is known (for more details see Section III) and a fluorophore highly specific for Q is used. In Fig. 1B (right panel) the change in fluorescence lifetime for increasing quencher concentrations is depicted.

Fluorescence lifetime imaging can be implemented in different ways (Lackowicz and Szmacinski, 1996; Wang et al., 1996). If frequency domain detection is used it is well suited for wide-field illumination and CCD camera detection. This allows very fast imaging rates. Time-domain detection is appropriate for point-scanning detection, which at present is still best suited for implementation with two-photon excitation. It requires longer acquisition times but offers a superior accuracy of lifetime determination. Time-domain detection has been used in the present study and will be explained below. In this case the excitation has to be performed with a pulsed light source, which has to be short compared to the fluorescence decay processes to be observed (that is < 1 ns).

It is not possible to detect the fluorescence decay curve in real time due to limita- tions in the speed of detectors and electronics. Therefore a repetitive measurement scheme [time-correlated single-photon counting (TCSPC)] is used in which one decay curve is obtained from many excitation cycles. This requires excitation sources with high repetition rates (typically MHz) to achieve faster acquisition times. The measurement is based on the exact determination of the temporal distance of two electronic pulses (see Fig. 2). The first pulse (start pulse) is formed by sending a small portion of the excitation light onto a fast photodiode. Every

0

0

1000,

100,

10,

I Nd:Vanadate-] laser ]

II, \

Ti:Sa-laser

Spectro- meter

cw-lamp ] n n a

.n_ ~ e e e o e e o o ~ , e

0 • v,,,,,l

I ~ bhoto-

/ ~iode !

. l

/ -

| : • o o e e e e o e o o e e o e o e e e e e e e e e o e o e o e o e o e o e ~

i ,; ~ i 17 time (ns)

FIGURE 2 TP-FLIM apparatus. Apparatus for fluorescence lifetime imaging with two-photon excitation (TP-FLIM) with explanation of the measurement principle for time-correlated single-photon counting (TCSPC). See text for details.

174 Kaneko et al.

excitation pulse will cause a start pulse. When the excitation pulse reaches the sample it will produce fluorescence photons, which are collected via the microscope and guided toward the detector. In our setup this is a special fast photomultiplier (PMT1 in Fig. 2). The first photon that reaches PMTI is detected and transformed into a second electronic pulse--the stop pulse. Both pulses are sent to an electronic device. When the start pulse arrives it starts a kind of electronic watch that is stopped when the stop pulse arrives. The time lag between the two pulses is dig- itized and registered in a histogram. This elementary cycle is repeated several ten thousand to million times (depending on the time resolution and signal-to-noise level needed) and in this way the fluorescence decay curve is obtained (see the left bottom comer in Fig. 2).

To prevent an artificially high detection probability for the early emitted photons, it is necessary to dim the excitation power, so that a photon is detected in only 1 of 50 to 100 excitation pulses. Doing so avoids the so-called pulse pileup effect that artificially shortens the detected fluorescence decay. The pulse pileup originates from the electronics, which allows detection of only the first arriving photon. If for every excitation pulse one photon is detected then the apparent decay time becomes shorter as the early emitted photons "close" the detector for the later ones. In this case the detection probability for the early photons is higher. By reducing the probability of having two photons produced in one excitation pulse arriving at the detector below 2% (that is 1 photon detected in 50 excitation pulses) pulse pileup becomes extremely unlikely and can be neglected.

In our setup (Fig. 2) we use the frequency-doubled output (532 nm) of a Nd: Vanadate Laser (Verdi 5.5 W, Coherent) laser that pumps a mode-locked Titan- Sapphire laser (Mira 900, Coherent), tunable from 700 to 1000 nm with a 150-fs pulse width, a repetition rate of 75.6 MHz, and an output power of >500 mW. The power on the sample in the microscope after passing all the optical components is about half of that. For typical measurements we need 1% or less of the maximum power, i.e., less than 2.5 roW. A small portion of the light is sent to an optical fiber, which guides it to a spectrophotometer. Here the excitation pulse spectrum and mode locking can be monitored. The latter is reflected in the spectral bandwidth of the pulse. The shorter a pulse the broader its spectrum as a consequence of the Heisenberg uncertainty relationship for energy and time. In the experiments presented here we have used light of 750 nm and 150-fs pulses. A small portion of the excitation light is guided onto the fast photodiode to produce the start pulse for the TCSPC measurement. The excitation light is fed into a beam scanner (T.I.L.L. Photonics, Miinchen, Germany), which allows movement of the light spot in the x- and y-direction in the sample plane. The scanner is mounted on an upright fluorescence microscope (BX50WI, Olympus) with epifluorescence detection. We use a water-immersion microscope objective with long working distance (NA = 0.9). This gives us a spatial resolution of better than 500 nm in the x- and y-direction and better than 1.5/zm in the z-direction. When measuring in the


fluorescence lifetime imaging mode, the fluorescence light was guided onto PMT 1, which worked in the single-photon counting mode. The time resolution of the apparatus is characterized by the instrument response function, which is limited by the time response of PMT1 to about 400 ps full width at half maximum (FWHM). If only two-photon imaging was performed, we used the more sensitive PMT2. The electronics (SPC730) and software for TCSPC and fluorescence lifetime analysis and imaging are from Becker & Hickl (Berlin, Germany). The whole setup, which has many more features such as a UV/blue light source for uncaging experiments, was designed together with T.I.L.L. Photonics.

The fluorescence decay measurements of the cell-free in vitro characterization of the fluorophore were performed in a stand-alone time-resolved fluorescence spectrometer (Fluotime 200, Picoquant). The excitation source was a pulsed light-emitting diode (LED) with the following specifications: Lexc = 380 + 10 nm, 590-ps pulse width, 40 MHz maximum repetition rate, 12/zW average power. The fluorescence is collected in a rectangular configuration with a lens and guided through a monochromator onto a red-enhanced PMT with a time response similar to the one used in the TP-FLIM apparatus. The time resolution of the spectrometer is determined by both LED and PMT resulting in an instrument response function with an FWHM of 700-750 ps.

Figure 3A and B illustrates the analysis of the fluorescence lifetime data as performed for TP-FLIM (Fig. 3A) and in vitro experiments (Fig. 3B). In time-resolved fluorescence spectroscopy, the time response of the measurement apparatus has to be considered, as it is in the same time range (subpicosecond to 1 ns) as the fluorescence lifetime--typically in the nanosecond time range, but often also faster. The time response is characterized by the instrument response function (IRF), which is usually measured by detecting the light scattered by a sample that does not absorb and fluoresce. The IRF of the Fluotime 200 is depicted in Fig. 3B. It is a nonsymmetric pulse function due to the characteristics of the PMT and the LED. The IRF of the TP-FLIM apparatus is more difficult to measure as a consequence of the two-photon excitation: only red to near-infrared photons are scattered. To improve the signal-to-noise ratio, however, a low pass filter blocks photons from the red and near-infrared spectral region so that only visible photons (i.e., fluorescence photons) can reach the detector. Therefore the IRF has to be measured in a different way by using a fluorophore with a fast fluorescence lifetime compared to the instrument response function. We have done this and estimated an IRF with an FWHM of 400 ps.

The measured fluorescence signal can be mathematically described as the convolution of the apparatus IRF and the exponential function describing the fluorescence decay. If the fluorescence decay time is in the order of or longer than the FWHM of the IRF, it is in principle possible to analyze the data by simulating an exponential model function. The deviation of the model function (the fit) and the measured curw~ (the g 2, that is the sum of the squared differences of measured data and fit

176 Kaneko et al.

A

100-

I : = 0 U

10

I I I I I I I i i i

" t "~ 2

~- .2 t V'l',~-~,'v'v'y'y"~rT"v"'" 0 2 4 6 8

time (ns)

B

0 20 40 60 time (ns)

FIGURE 3 Fluorescence lifetime determination. (A) Fluorescence decay from one pixel in the TP-FLIM image shown in Fig. 7A, the fitted monoexponential model function (r --- 5.3 ns, X 2 = 1.08) and the residuals (bottom panel). Fluorescence was excited with 750 nm light (150 fs, 75.6 MHz repetition rate, 2 mW) and recorded in the range from 400 to 680 nm. (B) Fluorescence decay from an in vitro time-resolved fluorescence experiment of MQAE (100/zM) in aqueous solution containing 5 mM KC1 fitted monoexponentially (3 = 14.9 ns, X 2 = 1.12), the instrument response function (IRF), and the residuals (bottom panel). Excitation was at 381 nm (780 ps, 10 MHz repetition rate, 1/zW) and recorded at 460 nm.

at all t ime points) serves as the estimator for the quality of the simulation. The function parameters are iteratively changed by a nonlinear least-square algorithm (e.g., the Marquard-algorithm) until X 2 reaches a minimum. Another parameter to evaluate the quality of a fit is the plot of the residuals, that is, the difference between

measured and simulated values at each time point [Fig. 3 (bottom panels)]. The residuals have to be randomly distributed around zero without structures or wings. If the residuals do look different the model function was not properly chosen to describe the data.

A more accurate analysis, however, does include a separate measurement of the IRF and more complicated fitting procedure called deconvolution. Instead of a simple exponential model function a convolution of the IRF with the exponential model function is used to simulate the data. The iterative minimization of X 2 occurs in a way similar to the simple exponential model function. The IRF has to be measured accurately and in close temporal distance to the sample to account for long-term changes of the time response of the apparatus. Al l in vitro


measurements for the characterization of the fluorescence C1- probe MQAE (see next section) have been analyzed with deconvolution. With the exception of experiments at very high MQAE concentrations a monoexponential model function was sufficient to obtain a good description of the data (nonstructured residuals, X 2 < 1.2). The experiments at high MQAE concentrations (>1 raM) have to be extended to obtain a full picture of the MQAE photophysics.

As mentioned above, it is more difficult to measure the correct IRF at the TP-FLIM apparatus. Because the FWHM of the IRF of the TP-FLIM apparatus is in the order of 400 ps and the measured fluorescence lifetimes in the intracellular experiments were between 4 and 10 ns we could simplify the measurements. Instead of measuring the correct IRF, the IRF was mathematically extracted from the rising part of the fluorescence signal assuming a symmetric pulse function. Although this is not correct, it serves as a good approximation for the IRF for this long fluorescence lifetimes.

For every pixel in the image (typically 128 times 128) we recorded the total fluorescence intensity and the fluorescence decay curve, which was obtained as explained above. In the present study we obtained satisfactory fits with a monoexponential model function (nonstructured residuals, X 2 < 1.3). No indications for biexponential fluorescence behavior was found in the intracellular FLIM measurements. We accumulated for 1-3 min per image to achieve 100-5000 counts in the maximum of each fluorescence decay curve. This corresponds to total count numbers of 30,000 to 1,500,000 per pixel histogram. It has recently been shown that for such fluorescence decay curves the least-square estimator gives reliable results (Maus et al., 2001). To speed up the imaging and keep the low excitation power (we did not observe any photodamage!) it is necessary to obtain decays with much smaller numbers of total counts. In this case a different estimator (the maximum likelihood estimator) has to be used that works properly for total count numbers of 1000 and less (Maus et al., 2001).

ii!. FLUORESCENCE LIFETIME ANALYSIS OF CI- CONCENTRATION

To be useful for fluorescence lifetime analysis of [Cl-]i, a fluorescent probe should ideally have the following properties: (1) loading cells with the probe should be efficient and noninvasive; (2) the probe should have low toxicity; (3) the probe should be resistant to metabolic enzymes in the cytosol, and leakage from cells should be slow; (4) fluorescence quenching by cellular components should be minimal; (5) the fluorescence signal should be resistant to pH changes in the physiological range; (6) absorption coefficient, fluorescence quantum yield, and stability of the probe should be high enough to permit fluorescence detection in small cellular structures; (7) the CI- sensitivity should be sufficient to detect changes of [Cl-]i in the range of a few millimolar; and (8) compounds commonly used to examine C1- homeostasis (niflumic acid, disulfonic acid stilbenes,

178 Kaneko et al.

u )

c

8 ¢ -

9. 0

. I c C I .

A

1000

100

10

B

25 B

t--

I.J

lO

C

I I I I I I i . . . . . . . . i . . . . . . . . i

1 10 100 0 100 time (ns) CI (mM)

20

o

10

5 '

0 | , = | |

0 .

CI (mM)

FIGURE 4 Fluorescence lifetime analysis of MQAE in solution containing C1-. (A) Fluorescence decays obtained by time-correlated single-photon counting using aqueous solutions containing 500/zM MQAE and (in mM): 94.5, 67.5, 54, 40.5, 27, 13.5, and 0 KC1 (from left to right). Fluorescence was excited at 10 MHz with ~.ex ----- 380 nm using a time-resolved spectrometer and recorded at lem = 460 nm. The time course of the intensity decay depends on C1- concentration. (B) Dependence of time constant r on [C1-]. r was derived from the time course of fluorescence decay l(t) = I(0) exp(-t/r), where I(0) is the fluorescence intensity at the end of the excitation light pulse. (C) Stern-Volmer analysis of the same data. A Stern-Volmer constant Ksv of 185 M -1 was obtained from the slope of the plot according to z0/r = 1 + Ksv [C1-], where 30 is the decay time constant in Cl--free solution.

100

furosemide, bumetanide) should not interfer with fluorescence lifetime analysis. One of the most widely used C1- probes is N-(6-methoxyquinolyl) acetoethyl ester (MQAE) (Verkman, 1990). M Q A E was sucessfully employed for studies of [C1-]i by fluorescence intensity measurements in various cell types, including hippocampal neurons (Inoue et aL, 1991), smooth muscle cells (Koncz and Daugirdas, 1994), and olfactory sensory neurons (Kaneko et aL, 2001). We therefore tested the use of this probe for the examination of [C1-]i in DRG neurons by fluorescence lifetime analysis.

We first examined properties of M Q A E in a cell-free assay using a t ime-resolved spectrometer (Fluo Time 200, PicoQuant, Berlin, Germany) at 3~exc = 380 nm, )~em --- 460 nm. C1- reduces the fluorescence lifetime of M Q A E by physical quenching, and the decay of fluorescence intensity after a brief light pulse is, therefore, accelerated in the presence of C1-. Figure 4 shows that the time course of fluorescence decay depends on [CI-]. Fluorescence decay is well fitted by a monoexponential function, indicating a homogeneous population of fluorophores. The decay time constant r decreases from 25.1 ns at 0 C1- to 1.4 ns at 94.5 m M C1- (Fig. 4B). These data were analyzed by the Stern-Volmer equation, r o / r = 1 + Ksv [C1-], where r0 and r are, respectively, the decay time constants in the absence and presence of CI- , and the Stem-Volmer-constant , Ksv = kqr0, is a measure of the C I - sensitivity of the probe. Plotting r 0 / r versus [CI-] (Fig. 4C) yields a Ksv value


A

5 • HEPES 0 HP04 z /

• HCO 3" /

Zk S042"

• H~.PO 4 V NO3 /

| | | | | |

0 anion (mM) 100

B

30

10

;o

0 5 M~E (raM)

I I I 0 100

MQAE (IJM) FIGURE 5 Quenching of MQAE fluorescence by nonchloride anions and self-quenching.

(A) Anions that are present in the cytosol, HPO 2-, HCO~-, SO42-, display only weak or no quenching (note scaling of ordinate) with Ksv values of 7, 3, and 0 M -l , respectively. The pH buffer HEPES quenches fluorescence with Ksv = 40M- 1, while NO~, which is used in Cl--substitution experiments, has no measurable affect on fluorescence lifetimes. Solutions contained 100/xM MQAE and the indicated concentrations of either Na2HPO4, NaH2PO4, NaHCO3, Na2SO4, KNO3, or HEPES at pH 7.4. (B) Decline of fluorescence lifetime by self-quenching of MQAE. At dye concentrations > 100/zM, decay times are strongly reduced, characterized by a Ksv value for self-quenching of 330 M -1 (inset).

I

10000

of 185 M -1, consistent with results from fluorescence intensity measurements (155-200 M - l ) (Verkman, 1990; Koncz and Daugirdas, 1994). Thus, MQAE displays similar C1- sensitivity in fluorescence lifetime and intensity measurements.

To find out whether intracellular anions affect decay times of MQAE, we determined Ksv values for HPO 2-, SO42-, and HCO 3, which are present in the cytosol at concentrations of roughly 100, 20, and 10 mM, respectively. Only HPO 2- and HCO 3 slightly accelerated the fluorescence decay, displaying Ksv values of 7 and 3 M -1, respectively (Fig. 5A). N-(2-hydroxyethyl)piperazine-N ~- (2-ethanesulfonic acid) (HEPES), which is often used as a pH buffer in physiological experiments, had a substantial quenching effect (Ksv = 40M-l) . H2PO 4 did not change decay times, indicating that phosphate-buffered solutions are suited for fluorescence lifetime experiments with MQAE. In contrast to probes for the detection of [Ca2+]i or pHi, which are used at concentration of about 10/zM, CI- probes are often used in the millimolar range. Because the interaction between fluorophores can lead to self-quenching at such concentrations, we tested whether the fluorescence lifetime signals change with MQAE concentration. Figure 5B illustrates that decay times did not change up to about 100/zM MQAE, but strongly decreased at higher concentrations. The Stern-Volmer plot of decay times in the millimolar range reveals pronounced self-quenching of MQAE with a Ksv of

180 Kaneko et al.

330 M -1 (inset in Fig. 5B). Thus, for the interpretation of MQAE signals, the propensity of the probe for self-quenching in the millimolar range has to be taken into account. The reduced C1- sensitivity of MQAE by self-quenching (Ksv values for C1- at 0.1, 0.5, and 5 mM MQAE are 200, 185, and 67 M -1, respectively) would lead to underestimation of [C1-]i at high probe concentrations. Thus, self- quenching of MQAE at concentrations exceeding 1 mM represents a major contri- bution to the decrease of C1- sensitivity inside cells. The photophysical mechanism of this process has still to be determined.

Intracellular proteins carry negative charges that originate mainly from ionized carboxylate groups and may also contribute to quenching of MQAE fluorescence (Koncz and Daugirdas, 1994). To examine this point, we measured the Ksv value for MQAE quenching by C1- in the presence of bovine serum albumin (BSA). MQAE quenching by C1- was attenuated by BSA: 1% BSA (0.75 mM) reduced r0 from 28 to 22 ns, but its effect on Ksv for C1- was not significant (Fig. 6A). How- ever, a 1% BSA solution is a poor model for the cell interior with its multitude

A

30

m c

10

0

/

0 Cl-(mM)40

+BSA

0 CI- (rnM) 40

B

In" CH~(~OCH2CH~

500 . ' " ' .

.c

30O

. . /

f2: O~.T . . . . . 0 100

." .. Ct" (raM)

. : : . i ~ . . ~

i l | m | | | i i •

410 430 480 500 ~m (nrn)

FIGURE 6 Protein effects on MQAE fluorescence and hydrolysis of MQAE. (A) Lifetime is reduced by bovine serum albumin (BSA). Fluorescence lifetimes were determined with 100/xMMQAE without BSA (closed circles) and with 1% (0.7 raM) BSA (open circles). The quenching constant for C1- is almost unaffected by BSA: Ksv decreases from 171 to 165 M - I (inset). (B) Hydrolysis of an ester bond in the MQAE molecule (arrow) leads to a blue shift of the fluorescence emission spectrum, combined with an increase of maximal fluorescence intensity. Spectra were recorded in 100/zM MQAE (buffered to pH 7.4 with 1.8 mM NaH2PO4F/.8 mM Na2HPO4) before (solid line, Nil) and after hydrolysis (dotted line, H). Hydrolysis was induced by exposing the dye to alkaline solution (buffered to pH 9.2 with 1.6 mM Na2CO3/18.4 mM NaHCO3). Hydrolysis strongly decreased CI- sensitivity: Ksv was reduced from 178 to 90 M -1.


of soluble and fixed anions, which may all add to quenching of MQAE inside cells. Moreover, the concentration of soluble protein in the cytosol is much higher (roughly 10-15 mM) than the BSA concentration we used, suggesting that the interaction of MQAE with proteins contributes significantly to intracellular quenching of the probe.

Koncz and Daugirdas (1994) found that an ester bond in the MQAE molecule is partially hydrolyzed inside smooth muscle cells by endogeneous esterases, and that hydrolysis causes a blue shift of the emission spectrum, combined with an increase of fluorescence intensity and the complete loss of a subtle pH sensitivity observed with the nonhydrolyzed probe. We used fluorescence lifetime analysis to compare the C1- sensitivity of nonhydrolyzed MQAE to the sensitivity of the probe after hydrolysis by mild alkaline treatment. Hydrolysis reduced the Ksv for CI- (measured in phosphate-buffered solution) by a factor of 2 to 90 M-1 (Fig. 6B).

Various compounds are used for the examination of [C1-]i and C1- transport processes. These include the ionophores tributyltin, nigericin, and valinomycin, which are used for the calibration of MQAE signals (see below) as well as blockers of Cl-channels and inhibitors of C1- transporters and pumps. We found that the ionophores (20/zM each) did not have measurable autofluorescence at the wave- lengths used for MQAE recording, and that only valinomycin reduced decay times of MQAE (data not shown). Because the valinomycin concentration in calibration experiments is much lower (1-10/xM) than [Cl-]i, its effect on the time course of fluorescence is negligible. The C1- channel blocker niflumic acid (500/xM) did not show autofluorescence and had no effect on decay times. In contrast, two commonly used inhibitiors of C1- transport, bumetanide and furosemide, displayed autofluorescence and introduced a second, fast component to the fluorescence decay kinet- ics (data not shown). This may create a problem for the fluorescence lifetime analysis of C1- homeostasis and must be examined in appropriate control experiments.

Taken together, our data show that the fluorescence lifetime signal originating from intracellular MQAE can be influenced by various factors including HPO 2-, proteins, self-quenching, and hydrolsis of the probe. All factors reduce the C1- sensitivity of MQAE, making it necessary to calibrate the relation between [C1-]i and decay time with MQAE inside the cell.

IV. DETERMINATION OF INTRACELLULAR CI- CONCENTRATION IN RAT DRG NEURONS

Dorsal root ganglia were prepared from adult rats and cut into small pieces. Neurons from 20 to 30 ganglia were isolated by enzymatic treatment. The tissue was first incubated in Dulbecco's modified Eagle's medium (DMEM) containing 0.3% collagenase (Sigma, C-9891) for 1 h at 37°C. Following centrifugation (5 min

182 Kaneko et al.

at 200g), the tissue was resuspended in minimum essential medium containing 0.25% trypsin (Sigma, T-1426), gently triturated, and incubated for 30 min at 37°C. Cells were then sedimented (5 min at 200g), resuspended in 2 ml DMEM, and triturated to obtain a homogeneous suspension of isolated cells. Cells were plated on concanavalin A-coated coverslips (250/zl cell suspension per cover- slip) and kept at 37°C until the experiment. For fluorescence lifetime imaging, cells were incubated with 5 mM MQAE (Molecular Probes) in a Ringer's solution (see legend to Fig. 7) for 30 min at 37°C. Extracellular MQAE was then washed away and coverslips were transferred to the FLIM microscope. Although MQAE readily diffuses into DRG neurons, leakage from the neurons is very slow, allowing stable fluorescence recordings for several hours. This trapping of the probe inside the cell is probably due to a reduced membrane permeability of the hydrolyzed MQAE molecule. The stable fluorescence intensity over periods exceeding 30 min suggests that all MQAE in DRG neurons is hydrolyzed and, therefore, trapped. The fluorescence lifetime of intracellular MQAE was recorded in Ringer's solution using a 60x water-immersion lens (NA = 0.9). Two-photon excitation of MQAE was achieved by applying 150-fs pulses of 750 nm light at intervals of 13 ns. Fluorescence photons emitted by MQAE were collected and the fluorescence lifetime of each photon was determined by TCSPC as described above.

Figure 7A shows two images of the same DRG neuron, one depicting fluorescence intensity and the other a color-coded representation of fluorescence lifetimes. The cytoplasm displays a largely uniform fluorescence intensity and a lifetime near 5 ns. The MQAE signal originating from the nucleus has lower intensity compared to the cytosol and distinctly longer lifetimes near 6 ns. The histogram in Fig. 7B illustrates the distribution of lifetimes in this cell: the mean lifetime of the cytosolic region is 5.1 q- 0.1 ns, whereas values obtained from the nuclei range from 5.5 to 6.5 ns (mean: 5.85 -4- 0.22 ns). Collected results from 28 DRG neurons yield a mean lifetime near 5.3 ns for the cytosol (Fig. 7C). In- terpretation of the difference between signals obtained from cytosol and nucleus is difficult. If C1- exchanges freely between nucleus and cytoplasm, so that nuclear and cytosolic C1- concentrations are similar, the different lifetime values may indicate a difference in quenching properties between the two compartments. Alternatively, if MQAE-quenching efficiency is similar, our result suggests that the concentrations of both MQAE and CI- in the nucleus are lower than in the cytoplasm. Previous measurements of the C1 content in sympathetic neurons by energy-dispersive X-ray microanalysis (Galvan e ta l . , 1984) showed no difference between nuclei and cytosol. This finding favors the notion that no CI- gradient exists across the nuclear envelope, and that the different lifetime signals indicate that nucleoplasm and cytosol constitute significantly different environments for MQAE fluorescence.

To determine the relation between [C1-]i and fluorescence lifetime for cytosol and nuclei of DRG neurons, [Cl-]i levels must be adjusted to defined values. Exposing DRG neurons to extracellular C1- concentrations in the range of

8. Intracel lular Chlor ide Concent ra t ion in D R G 183

A B oot I C

2000

1000

0 5 6 4.5 s.o 8.0 6.5 4.5 5.0 6.0 6.5

1; (ns) 1; (ns) 1; (ns)

D E

[Cf]~ 0 10 mM 20 mM satellite 1

mmm 1.2

1.0 DRG = = i = =

5.5 I; (ns) 7.0 0 Cr(mM) 20

FIGURE 7 MQAE fluorescence-lifetime analysis in rat DRG neurons. (A) Two-photon images of a freshly isolated rat DRG neuron. The neuron was loaded with MQAE by incubation for 30 rain at 37°C in a solution containing (mM) 140 NaC1, 5 KC1, 2.5 CaC12, 1 MgC12, 10 HEPES, I0 glucose, 5 MQAE (pH 7.4, 340 mOsM). Upper panel: Fluorescence-intensity image after washing off external MQAE shows a largely homogeneous fluorescence signal in the cytosol and a weaker signal originating from the nucleus. Lower panel: Fluorescence-lifetime image of the same cell with color-coded lifetime values. (B) Histogram of lifetimes obtained from all pixels of the cell depicted in (A) Lifetime values originating from the cytosol have a mean of 5.1 4- 0.1 ns (red curve). Values from the nuclei range from 5.5 to 6.5 ns (mean: 5.85 4- 0.22 ns; green curve). (C) Collected FLIM results from 28 DRG neurons. Differences in lifetime signals between individual neurons broaden the distribution of the collected results, yielding an all-pixel histogram with a mean ~ value near 5.3 ns. (D) Fluurescence-lifetime images of three DRG neurons at different C1- concentrations. Cells are held in solutions containing KC1 at the indicated concentrations, together with 1 m M EGTA, 8.1 m M Na2Ht~4, 1.9 mM NaH2PO4, as well as KNO3 to give a total K + concentration of 150 mM. The pH was 7.2. Tributyltin (40/~M) and

2+ nigericin (10/zM) were added to equilibrate intracellular and extracellular C1- concentrations. Ca - free solution was used to prevent Ca 2+ overload and lysis of cells that can be induced by tributyltin (Viviani et al., 1995). The red arrows point to perineuronal satellite ceils attached to the DRG neurons. (E) Stern-Volmer plots describing the dependence of MQAE fluorescence lifetime on [C1-]i. Collected results yield Ksv values of 9.1 M -1 (31 cells) for DRG neurons and 29.8 M -1 (29 cells) for perineuronal satellite cells. (See Color Plate)

/ / m / /

184 Kaneko et al.

0-100 mM did not affect [C1-]i, indicating a low C1- permeability of the plasma membrane. To clamp [C1-]i to known values we used a double ionophore technique (Chao et aL, 1989). Cells were incubated with 40 #M tributyltin, a CI-/OH- exchanger that dissipates C1- gradients across the plasma membrane, as well as with 10/zM nigericin, a K+/H + exchanger that attenuates changes of intracellular pH resulting from the OH- transport mediated by tributyltin. At lower concentrations of tributyltin (10-30/zM), permeabilization of DRG neurons to C1- was not achieved reliably. To obtain r0, the fluorescence lifetime at [C1-]i = 0, cells were first washed in a solution in which all Cl-was replaced by NO 3 (see legend to Fig. 7D). Cells were loaded with MQAE (5 mM, 30 min, 37°C) in C1--free solution and, subsequently, incubated for 30 min with ionophores in C1--free solution. After this procedure, cells displayed a homogeneous lifetime distribution that we assume represents r0. The mean r0 values from 13 DRG neurons were 6.8 + 0.3 ns in the cytosol and 7.0 + 0.5 ns in the nucleus. Increasing C1- concentration to 20 mM reduced lifetimes (Fig. 7D), and the mean steady-state values obtained from 8-13 different cells per C1- concentration are presented as a Stern-Volmer plot in Fig. 7E. The results show considerable differences between individual cells at the same C1- concentration (illustrated by standard deviations in Fig. 7E). Linear regression fits to the measured points yield a Ksv value of 9.1 M -1 for the cytosol. At C1- concentrations exceeding 20 mM, lifetimes did not change in a reproducible and consistent manner. Thus, calibration of lifetime signals with tributyltin-permeabilized DRG neurons was possible only within a very limited range of low [C1-]i. Lifetime values recorded from the nuclei of the same cells displayed even larger scatter, which, in part, may be attributed to the smaller number of pixels recorded from nuclei compared to cytosol. The estimated quenching constant was 6 M -1 (data not shown). However, the variability of the data precludes analysis of differences between cytosol and nucleus with respect to quenching efficiency and C1- concentration. Perineuronal satellite cells, which are tightly associated with the somata of somatosensory neurons within the DRGs, and, like the DRG neurons, have been shown to express Ca2+-activated C1- channels (England et al., 2001), can often be seen attached to isolated DRG neurons (Fig 7D, red arrows). The C1- dependence of lifetime signals from satellite cells is characterized by a Ksv of 29.8 M -1 (Fig. 7E).

The data we obtained at 0-20 mM [C1-]i indicate that the sensitivity of MQAE to cytosolic C1- in DRG neurons is much lower than the sensitivity to C1- in aqueous solution (Ksv values of 8.8 vs. 185 M-l) , consistent with results from a variety of cell types where Ksv values for MQAE are in the range of 5-25 M - 1 (Kaneko et al., 2001; Lau et al., 1994; Bevensee et aL, 1997; Eberhardson et al., 2000; Maglova et al., 1998). This profoundly reduced C1- sensitivity inside cells is thought to reflect quenching of MQAE fluorescence by nonchloride anions in the cytoplasm, including proteins and soluble anions (Chao et aL, 1989), an interpretation supported by our observation that r0 is reduced from 28 ns in aqueous

B

BB 5

x (ns)

A C

8

2000

1000

D

I

6 4.5 5.0 (ns)

[Cf~ 0 10 mM 20 mM

I

6.0 6.5

/ / s.s x (ns) 7.0

0 4.5 5.0 6.0 6.5 x (ns)

E 181 1.2

1.0 DRG = = i | i

0 CI-(mM) 20

FIGURE 7 MQAE fluorescence-lifetime analysis in rat DRG neurons. (A) Two-photon images of a freshly isolated rat DRG neuron. The neuron was loaded with MQAE by incubation for 30 min at 37°(; in a solution containing (mM) 140 NaC1, 5 KC1, 2.5 CaC12, 1 MgC1 v 10 HEPES, 10 glucose, 5 MQAE (pH 7.4, 340 mOsM). Upper panel: Fluorescence-intensity image after washing off external MQAE shows a largely homogeneous fluorescence signal in the cytosol and a weaker signal originating from the nucleus. Lower panel: Fluorescence-lifetime image of the same cell with color- coded lifetime values. (B) Histogram of lifetimes obtained from all pixels of the cell depicted in (A). Lifetime values originating from the cytosol have a mean of 5.1 _+0.1 ns (red curve). Values from the nuclei range from 5.5 to 6.5 ns (mean: 5.85_+0.22 ns; green curve). (C) Collected FLIM results from 28 DRG neurons. Differences in lifetime signals between individual neurons broaden the distribution of the collected results, yielding an all-pixel histogram with a mean "r value near 5.3 ns. (D) Fluorescence-lifetime images of three DRG neurons at different C1- concentrations. Cells are held in solutions containing KC1 at the indicated concentrations, together with 1 mM EGTA, 8.1 mM Na2HPO 4, 1.9 mM NaH2PO 4, as well as KNO3 to give a total K ÷ concentration of 150 mM. The pH was 7.2. Tributyltin (40 WkI) and nigericin (10 ~ were added to equilibrate intracellular and extracellular C1- concentrations. Ca2÷-free solution was used to prevent Ca 2÷ overload and lysis of cells that can be induced by tributyltin (Viviani et al., 1995). The red arrows point to perineuronal satellite cells attached to the DRG neurons. (E) Stern-Volmer plots describing the dependence of MQAE fluorescence lifetime on [C1-]i. Collected results yield Ksv values of 9.1 M -1 (31 ceils) for DRG neurons and 29.8 M -1 (29 cells) for perineuronal satellite ceils.


solution (Fig. 4B) to 6.7 ns inside DRG neurons. Using the lifetime parameters determined for the cytosol (r0 = 6.8 ns, Ksv = 9.1M -1) and the mean lifetime measured in intact DRG neurons (r = 5.3 ns), we obtain from the Stern-Volmer relation [C1-]i = ('r0/lr - 1)/Ksv, an estimate for [C1-]i of 31 mM. Our data for perineuronal satellite cells (~0 ---- 9.1 ns, Ksv = 29.8M -1, and r = 5.4 ns in intact cells) yields an estimate of 23 mM for [C1-]i.

V. DISCUSSION

Previous results and the experiments shown here indicate that MQAE is a mod- erately useful probe for FLIM studies of [C1-]i in DRG neurons. Points in favor of MQAE include the following: (1) loading of cells with MQAE is noninvasive and, in contrast to C1- probes that have to be loaded by hypotonic shock (e.g., SPQ) (Chao et al., 1989), can be achieved without altering [C1-]i; (2) MQAE appears to have low toxicity for DRG neurons; (3) hydrolysis of an ester bond inside the cell renders MQAE less membrane permeant, strongly reducing probe leakage from the cell; (4) the fluorescence signal of the hydrolyzed form of MQAE is in- sensitive to pH changes in the physiological range (Koncz and Daugirdas, 1994); (5) the photochemical properties of MQAE permit fluorescence lifetime measurements in small cellular structures. The main critical points include the low residual C1- sensitivity of MQAE inside cells, characterized by Stern-Volmer constants of 6-30 M -1, as well as the pronounced self-quenching at MQAE concentrations exceeding 100/zM.

Fluorescence lifetime measurements take advantage of the favorable properties of MQAE and partly compensate for the critical points. The high accuracy of lifetime measurements (At of 0.1 ns can be resolved) permits recording of C1- signals in the physiological range (5-50 raM) with sufficient precision, despite the low intracellular C1- sensitvity of MQAE. Combination of FLIM with two- photon excitation reduces the problem of UV damage to cells and allows the application of lifetime measurements to tissue slices. Utilizing the high spatial resolution of two-photon imaging, the combination of two-photon microscopy and FLIM analysis can be used to generate optical sections of cells and tissues, yielding information about three-dimensional distribution of [C1-]i. Optical sectioning and three-dimensional reconstruction will be a valuable tool for the examination of ion concentrations in sensory and synaptic endings of DRG neurons. The temporal resolution of FLIM experiments is limited by the time necessary to accumulate sufficient photons for each individual point of the image. For the image shown in Fig. 7A (128 × 128 pixels), fluorescence was recorded for 60 s. For studies of [Cl-]i homeostasis in parts of axons or dendrites, imaging can be restricted to fewer pixels and lifetime images can be recorded at intervals of a few seconds. This temporal resolution is sufficient to probe C1- transport mechanisms in DRG

186 Kaneko et al.

neurons using specific transport blockers, because changes in [C1-]i typically de- velop within about 2-5 minutes after application of the blockers (Kettenmann, 1999; Hara et al., 1992; Inagaki et aL, 1996).

The most problematic aspect of [C1-]i determination is the difficulty associated with calibration of intracellular fluorescence signals (intensity and lifetime) by selective permeabilization of the plasma membrane to CI- ions. Tributyltin, which is widely used for that purpose, is highly toxic to cells, disrupting within minutes various physiological functions and inducing apoptosis (e.g., Viviani et aL, 1995; Nishikimi et aL, 2001). Accordingly, we frequently observed DRG neurons damaged by 15-30 min exposure to 40/zM tributyltin, which had to be excluded from analysis. Lower concentrations of the ionophore did not result in permeabilization and, even with 40/zM tributyltin, calibration was limited to very low C1- concentrations. Thus, the double ionophore technique seems to be suited for DRG neurons only within a narrow range of [C1-]i. At present, triorganotins are the only available C1- ionophores, but other compounds may also operate as C1-/OH- exchangers or C1-/H + symporters [see Sato et al. (1998) and references therein] and may be used for C1- calibration in the future. Methods for calibration without C1- ionophores cannot be applied with confidence to DRG neurons. Such noninvasive methods for the determination of [C1-]i rely on the function of endogenous C1- channels and transporters. In some cell types, [C1-]i can be clamped without using tributyltin because C1- is readily exchanged between cytosol and the extracellular solution (Chao et al., 1989; Mansoura et al., 1999). The C1- permeability of DRG neurons is, however, too low for this method. An alternative method, filling cells with solutions of known CI- concentration through the patch pipette (Kuner and Augustine, 2000), may allow endogenous soluble quenchers to diffuse from the cell. This would change r0 and Ksv and would compromise the valid- ity of the calibration. Thus, in the absence of an efficient and reliable method of C1- calibration in tissue slices, the absolute values of [C1-]i in sensory and synaptic endings of DRG neurons cannot be determined using FLIM experiments. Calibra- tions obtained from isolated somata cannot serve to calculate [C1-]i in cellular processes because r0 and Ksv can change substantially not only between individual cells but also between different locations within the same cell. This was recently demonstrated for olfactory sensory neurons, where Ksv changes more than 4-fold between dendrite and soma (Kaneko et al., 2001). Nevertheless, two-photon-based FLIM can be used to examine with high sensitivity changes of [C1-]i that occur in the cell processes of DRG neurons due to sensory signaling or pharmacological manipulations. Extending technical possibilities from studies of isolated somata to the examination of cellular processes in intact tissue, FLIM studies of [Cl-]i may thus help to elucidate the role Ca2+-activated C1- channels in somatosensory signal processing.

Two independent methods have been used to determine [C1-]i in DRG neurons: Alvarez-Leefmans et al. (1988) used C1--sensitive microelectrodes to directly


measure [C1-]i in freshly isolated DRG neurons of adult frogs and found a mean value of 23.6 4- 1 mM. The most frequently used method to obtain an estimate for [C1-]i is to determine EGABA, the reversal voltage of GABAA receptor-mediated currents, and to calculate [C1-]i under the assumption that the C1- equilibrium potential, Eca, equals F-~ABA (Kryozis and Reichling, 1995; Ebihara et al., 1995). Applying this technique to freshly dissociated DRG neurons of adult cats and to 10-day-old chick embryos, estimates of, respectively, 53 4- 12 mM (Gallagher et al., 1978) and 31 4- 2.5 mM (Kenyon, 2001) were obtained. Consistent with these results, our estimate of 31 mM demonstrates that freshly isolated DRG neurons have higher levels of [C1-]i than expected for passive distribution across the plasma membrane. Intracellular C1- accumulation is supported by an inwardly directed uphill C1- transport, the molecular nature of which is still unclear. It has been suggested that Na +, K +, 2C1- cotransporters and C1-/HCO 3 exchangers mediate C1- accumulation in DRG neurons (Alvarez-Leefmans et al., 1990, 2001). With [C1-]i above equilibrium, opening of Ca2+-activated C1- channels in the cell bodies of DRG neurons mediates C1- efflux and depolarization. Whether similar C1- gradients exist in sensory and synaptic endings and how Ca2+-dependent C1- currents contribute to somatosensory signal processing are still open questions.

Like other methods of [C1-]i determination, TP-FLIM relies on the accuracy of intracellular calibration procedures. Nevertheless, TP-FLIM offers several advantages over other experimental approaches. (1) In contrast to microelectrode-based techniques, TP-FLIM can be used to explore neuronal dendrites, axons, and probably even presynaptic terminals in intact tissue. (2) Fluorescence lifetime signals do not depend on cell size, a considerable advantage over fluorescence intensity imaging. (3) Intensity-based fluorescence analysis requires each individual cell to survive a calibration procedure. In contrast, fluorescence lifetime experiments allow determination of the mean fluorescence lifetime in intact cells and allow the calibration procedure to be performed in a separate group of the same type of cells. Only with FLIM is it possible to use the calibration parameters obtained in this way to calculate the native [C1-]i in intact cells. This is a decisive advantage for studies on cells that do not survive calibration techniques involving the application of toxic ionophores such as tributlytin.

References Alvarez-Leefrnans, E J. (1990). Intracellular C1- regulation and synaptic inhibition in vertebrate and

invertebrate neurons. In "Chloride Channels and Carders in Nerve, Muscle, and Glial Cells" (F. J. Alvarez-Leefmans and J. M. Russell, Eds.), pp. 109-158. Plenum Press, New York.

Alvarez-Leefmans, F. J., Gamino, S. M., Giraldez, F., and Nogueron, I. (1988). Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-sensitive microelectrodes. J. Physiol. 406, 225-246.

Alvarez-Leefrnans, F. J., I.e,6n-Olea, M., Mendoza-Sotelo, Alvarez, F. J., Ant6n, B., and Garduno, R. (2001). Immunolocalization of the Na+-K+-2C1 - cotransporters in peripheral nervous tissue of vertebrates. Neurosci. 104, 569-582.

1 8 8 Kaneko et al.

Ayar, A., and Scott, R. H. (1999). The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca 2+ release. Naunyn Schmiedeberg's Arch. Pharmacol. 359, 81-91.

Bader, C. R., Bertrand, D., and Schlichter, R. (1987). Calcium-activated chloride current in cultured sensory and parasympathetic quail neurones. Z Physiol. (Lond.) 394, 125-148.

Bevensee, M. O., Apkon, M., and Boron, W. E (1997). Intracellular pH regulation in cultured astrocytes from rat hippocampus. J. Gen. Physiol. 110, 467-483.

Chao, A. C., Dix, J. A., Sellers, M. C., and Verkman, A. S. (1989). Fluorescence measurement of chloride transport in monolayer cultured cells. Biophys. J. 56, 1071-1081.

R 2+ Crawford, J. H., Wootton, J• F., Seabrook, G. R., and Scott, . H. (1997). Activation of Ca -dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP- ribose precursors. J. Neurophysiol. 77, 2573-2584.

Currie, K. P. M., Wootton, J. E, and Scott, R. H. (1995). Activation of Ca2+-depend~nt C1- currents in cultured rat sensory neurones by flash-photolysis of DM-nitrophen. J. Physiol. (Lond.) 482, 291-307.

Deschenes, M., Feltz, P., and Lamour, Y. (1976). A model for an estimate in vivo of the ionic basis of presynaptic inhibition: An intracellular analysis of the GABA-induced depolarization in rat dorsal root ganglia. Brain Res. 118, 486-493.

Eberhardson, M., Patterson, S., and Grapengiesser, E. (2000). Microfluorimetric analysis of C1- permeability and its relation to oscillatory Ca 2+ signalling in glucose-stimulated pancreatic/3-cells. Cell. Signal. 12, 781-786.

Ebihara, S., Shirato, K., Harata, N., and Akaike, N. (1995). Gramicidin-pefforated patch recording: GABA response in mammalian neurones with intact intracellular chloride. J. Physiol. 484, 77-86.

England, S., Heblich, E, James, I. J., Robbins, J., and Docherty, R. J. (2001). Bradykinin evokes an Ca2+-activated chloride current in non-neuronal cells isolated from neonatal rat dorsal root ganglia. J. Physiol. 530, 395-403.

Gallagher, J. P., Hihashi, H., and Nishi, S. (1978). Characterization and ionic basis of GABA-induced depolarizations recorded in vitro from cat primary afferent neurons. J. Physiol. 275, 263-282.

Galvan, M., DOrge, M., Beck, E, and Rick, R. (1984). Intracellular electrolyte concentrations in rat sympathetic neurones measured with an electron microprobe. Pfliigers Arch. 400, 274-279.

Hara, M., Inoue, M., Yasukara, T., Olmishi, S., Mikami, Y., and Inagaki, C. (1992). Uneven distribution of intracellular C1- in rat hippocampus. Neurosci. Lett. 143, 135-138.

Inagaki, C., Hara, M., and Zeng, X.-T. (1996). A C1- pump in rat brain neurons. J. Exp. Zool. 275, 262-268.

Inoue, M., Hara, M., Zeng, X. T., Hirose, T., Ohnishi, S., Yasukura, T., Uriu, T., Omori, K., Minato, A., and Inagaki, C. (1991). An ATP-driven C1- pump regulates C1- concentration in rat hippocampal neurons• Neurosci. Lett. 134, 75-78•

Kaneko, H., Nakamura, T., and Lindemann, B. (2001). Noninvasive measurement of chloride concentration in rat olfactory receptor cells with use of a fluorescent dye. Am~ J. Physiol. 280, C1387-C1393.

Kenyon, 2+ J. L. (2001). The reversal potential of Ca -activated C1- currents indicates that chick sensory neurons accumulate intracellular C1-. Neurosci. Lett. 296, 9-12.

• 2 + 2 + • - K e n y o n , J. L., and Goff, H. R. (1998). Temperature dependencies of Ca current, Ca -acavated CI

current and Ca 2+ transients in sensory neurones. Cell Calcium 24, 35-48. Kettenmann, H. (1990). Chloride channels and carriers in cultured glial cells. In "Chloride Channels

and Carriers in Nerve, Muscle, and Glial Cells" (F. J. Alvarez-Leefmans and J. M. Russell, Eds.), pp. 193-208. Plenum Press, New York.

Koncz, C., and Dangirdas, J. T. (1994). Use of MQAE for measurement ofintracellular [C1-] in cultured aortic smooth muscle cells. Am. J. Physiol. 267, H2114-H2123.

8. IntraceUular Chloride Concentration in DRG 189

Kryozis, A., and Reichling, D. B. (1995). Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concenlration. J. Neurosci. Methods 57, 27-35.

Kuner, T., and Augustine, G. J. (2000). A genetically encoded ratiometric indicator for chloride: Capturing chloride transients in cultured hippocampal neurons. Neuron 27, 447-459.

Lackowicz, J. R., and Szmacinski, H. (1996). Imaging applications of time-resolved fluorescence spectroscopy. In "Chemical Analysis, Vol. 137, Fluorescence Imaging Spectroscopy and Microscopy" (X. F. Wang and B. Herman, Eds.), pp. 272-311. J. Wiley & Sons, New York.

Lau, K. R., Evans, R. L., and Case, R. M. (1994). Intracellular C1- concentration in striated intralobular ducts from rabbit mandibular salivary glands. Pfliigers Arch. 427, 24-32.

Maglova, L. M., Crowe, W. E., Smith, P. R., Altamirano, A. A., and Russel, J. M. (1998). Na+-K+-C1- cotransport in human fibroblasts is inhibited by cytomegalovirus infection. Am. J. Physiol. 275, C1330-C1341.

Mansoura, M. K., Biwersi, J., Ashlock, M. A., and Verkman, A. S. (1999). Fluorescent chloride indicators to assess the efficacy of CFI'R cDNA delivery. Hum. Gene Ther. 10, 861-875.

Maus, M., Cotlet, M., Gensch, T., Hofkens, J., De Schryver, E C., Schaffer, A., and Seidel, C. A. M. (2001). Comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules. An experimental approach. Anal. Chem. 73, 2078- 2086.

Nishikimi, A., Kira, Y., Kasahara, E., Sato, E. F., Kanno, T., Utsumu, K., and Inoue, M. (2001). Tributyltin interacts with mitochondria and induces cytochrome C release. Biochem. J. 356, 621- 626.

Piston, D.W. (1996). Two-photon excitation microscopy. In "Chemical Analysis, Vol. 137, Fluorescence Imaging Spectroscopy and Microscopy" (X. E Wang and B. Herman, Eds.), pp. 253-272. J. Wiley & Sons, New York.

Sato, T., Konno, H., Tanaka, Y., Kataoka, T., Nagai, K., Wassermann, H. H., and Ohkuma, S. (1998). Prodigiosins as a new group of H+/C1 - symporters that uncouple proton translocators. J. Biol. Chem. 273, 21455-21462.

Scott, R. H., Sutton, K. G., Griffin, A., Stapleton, S. R., and Cuttle, K. P. M. (1995). Aspects of calcium-activated chloride currents: A neuronal perspective. Pharmacol. Ther. 66, 535-565.

2+ fro r Usachev, Y. M., and Thayer, S. A. (1997). All-or-none Ca release m intracellular sto es triggered by Ca 2+ influx through voltage-gated Ca 2+ channels in rat sensory neurons. J. Neurosci. 17, 7404-7414.

Verkman, A. S. (1990). Development and biological application of chloride-sensitive fluorescent indicators. Am. J. Physiol. 256, C375-C388.

Verkman, A. S., and Biwersi, J. (1995). Chloride-sensitive fluorescent indicators. Methods Neurosci. 27, 328-339.

Viviani, B., Rossi, A. D., Chow, S. C., and Nicotera, P. (1995). Organotin compounds induce calcium overload and apoptosis in PC12 cells. NeuroToxicology 16, 19-26.

Wang, XC. E, Periasamy, A., Wodnicki, P., Gordon, G. W., and Herman, B. (1996). Time-resolved fluorescence lifetime imaging microscopy: Instrumentation and biomedical applications. In "Chem- ical Analysis, Vol. 137, Fluorescence Imaging Spectroscopy and Microscopy" (X. E Wang and B. Herman, Eds.), pp. 313-350. J. Wiley & Sons, New York.

Documents

CHAPTER 8 Determination of Intracellular Chloride ... · PDF fileDetermination of Intracellular Chloride Concentration in Dorsal Root ... of Ecl. C1--sensitive fluorescent dyes may