Ca2+- and Metabolism-Related Changes of Mitochondrial Potential in Voltage-Clamped CA1 Pyramidal Neurons In Situ

S. Schuchmann,2 M. Lückermann,1 A. Kulik,1 U. Heinemann,2 and K. Ballanyi1

 1II. Physiologisches Institut, Universität Göttingen, D-37073 Göttingen; and  2Institut für Physiologie, Humboldt-Universität Berlin, Universitätsklinikum Charité, D-10117 Berlin, Germany


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Schuchmann, S., M. Lückermann, A. Kulik, U. Heinemann, and K. Ballanyi. Ca2+- and Metabolism-Related Changes of Mitochondrial Potential in Voltage-Clamped CA1 Pyramidal Neurons In Situ. J. Neurophysiol. 83: 1710-1721, 2000. In hippocampal slices from rats, dialysis with rhodamine-123 (Rh-123) and/or fura-2 via the patch electrode allowed monitoring of mitochondrial potential (Delta Psi ) changes and intracellular Ca2+ ([Ca2+]i) of CA1 pyramidal neurons. Plasmalemmal depolarization to 0 mV caused a mean [Ca2+]i rise of 300 nM and increased Rh-123 fluorescence signal (RFS) by <= 50% of control. The evoked RFS, indicating depolarization of Delta Psi , and the [Ca2+]i transient were abolished by Ca2+-free superfusate or exposure of Ni2+/Cd2+. Simultaneous measurements of RFS and [Ca2+]i showed that the kinetics of both the Ca2+ rise and recovery were considerably faster than those of the Delta Psi depolarization. The plasmalemmal Ca2+/H+ pump blocker eosin-B potentiated the peak of the depolarization-induced RFS and delayed recovery of both the RFS and [Ca2+]i transient. Thus the Delta Psi depolarization due to plasmalemmal depolarization is related to mitochondrial Ca2+ sequestration secondary to Ca2+ influx through voltage-gated Ca2+ channels. CN- elevated [Ca2+]i by <50 nM but increased RFS by 221% as a result of extensive depolarization of Delta Psi . Oligomycin decreased RFS by 52% without affecting [Ca2+]i. In the presence of oligomycin, CN- and p-trifluoromethoxy-phenylhydrazone (FCCP) elevated [Ca2+]i by <50 nM and increased RFS by 285 and 290%, respectively. Accordingly, the metabolism-related Delta Psi changes are independent of [Ca2+]i. Imaging techniques revealed that evoked [Ca2+]i rises are distributed uniformly over the soma and primary dendrites, whereas corresponding changes in RFS occur more localized in subregions within the soma. The results show that microfluorometric measurement of the relation between mitochondrial function and intracellular Ca2+ is feasible in whole cell recorded mammalian neurons in situ.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

In central neurons, particularly in hippocampal CA1 neurons, pharmacological approaches established that synaptically evoked local changes of the free concentration of intracellular Ca2+ ([Ca2+]i) play a key role in excitability and synaptic plasticity (Bliss and Collingridge 1993; Edwards 1995; Ito et al. 1995). For a decade, whole cell recording techniques have been used in combination with microfluorometric measurements of [Ca2+]i (Neher 1989) to analyze cellular mechanisms of synaptic processes in neurons of functionally intact slice preparations (Eilers et al. 1995; Regehr et al. 1989). Recently it was proposed that activity-evoked changes of energy metabolism may contribute to adaptive neuronal processes by redox modulation of ion channels (Kohr et al. 1994; Tang and Zucker 1997). A full test of this hypothesis requires simultaneous monitoring of metabolic activity with membrane properties and [Ca2+]i in neurons that are embedded in their natural environment.

Metabolic parameters such as uptake (Hubel et al. 1978) or catabolism (Sibson et al. 1998) of glucose or oxygen saturation of hemoglobin (Bonhoeffer et al. 1995) have been used to visualize neuronal activity in vivo. Mitochondrial membrane potential (Delta Psi ) is a further measure of metabolism with a high temporal resolution (Duchen 1992, 1999; Gunter et al. 1994; McCormack et al. 1990). So far, microfluorometric measurements of relative changes in Delta Psi were done in acutely isolated or cultured neurons that were bulk-loaded with rhodamine-123 (Rh-123) (Duchen 1992, 1999; Duchen and Biscoe 1992; Nowicky and Duchen 1998; Schinder et al. 1996; Schuchmann et al. 1998; White and Reynolds 1996). However, synaptic integrity as maintained within brain slices is necessary to study the interaction of activity-related changes in metabolism and neuronal excitability. In this regard, it recently was demonstrated that mitochondrial function can be analyzed in Rh-123 bulk-loaded hippocampal slices (Bindokas et al. 1998). Under these conditions, the dye distributes nonselectively in diverse compartments of various cellular elements within the slice. Discrimination between pre- and postsynaptic processes as well as discrimination between neurons and different types of glial cells is rather difficult under these conditions. The latter technique does also not provide information on the temporal relation between synaptic or intrinsic membrane currents and Delta Psi and thus metabolic changes in single hippocampal neurons within the network of the slice.

In the present study, we have used photomultiplier-based optical techniques in hippocampal slices to investigate whether long-term recording of Delta Psi is feasible in individual CA1 pyramidal neurons that are dialyzed with Rh-123 via the patch electrode. It is known from measurements on isolated neurons or mitochondria that a rise of [Ca2+]i produces a robust depolarization of Delta Psi (Duchen 1992; Duchen and Biscoe 1992; Gunter et al. 1994; Loew et al. 1994; McCormack et al. 1990). Accordingly, we have studied the extent to which a rise of [Ca2+]i, evoked by depolarization of the plasma membrane, affects Delta Psi in whole cell recorded hippocampal neurons in situ. For comparison of the effects of plasma membrane depolarization with Delta Psi responses due to direct modulation of energy metabolism, we have analyzed the effects on [Ca2+]i and Delta Psi of block of aerobic metabolism by CN- (Ballanyi and Kulik 1998; Biscoe and Duchen 1990; Duchen 1992). We furthermore have used digital imaging techniques to study putative spatial differences of activity- and metabolism-related changes of Delta Psi and [Ca2+]i. The results show that this novel technique is an appropriate tool for monitoring the temporal and causal relation between membrane excitability and metabolism of individual neurons in a functional network.


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Preparation and solutions

The experiments were performed on hippocampal slices from 9- to 14-day-old Wistar rats of either sex. The animals were anesthetized with ether and decapitated. The forebrain with the hippocampus was isolated and kept for 5 min in ice-cold artificial cerebrospinal fluid (standard solution; composition see following text; Ca2+ concentration reduced to 0.5 mM). Eight to 10 longitudinal slices (200 µm) were cut from the ventral side in ice-cold low Ca2+ solution. Before transfer to the recording chamber, the slices were stored at 30°C in standard solution. The recording chamber (volume, 3 ml) was superfused with oxygenated standard solution (flow rate, 5 ml/min, 30°C) of the following composition (in mM): 118 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 25 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose. The pH was adjusted to 7.4 by gassing with 95% O2-5% CO2. Chemical anoxia was induced by addition of 1 mM NaCN to the standard solution (Ballanyi and Kulik 1998). In the Ca2+-free solution, which also contained 1 mM EGTA as a Ca2+ buffer, the Mg2+ concentration was elevated to 5 mM. Drugs were purchased from Sigma (München, Germany), Biomol (Köln, Germany), or Tocris Cookson (Bristol, UK).

Intracellular recording

Patch pipettes were produced from borosilicate glass capillaries (GC 150TF, Clark Electromedical Instruments, Pangbourne, UK) using a horizontal electrode puller (Zeitz, München, Germany). The standard patch pipette solution (osmolarity, 270-285 mosmol) contained (in mM) 140 potassium gluconate, 1 MgCl2, 0.5 CaCl2, 10 HEPES, 1 K4-BAPTA, and 1 Na2-ATP, pH 7.3-7.4. For measurements of [Ca2+]i, the patch solution contained neither Ca2+ nor BAPTA because of the Ca2+-buffering properties of fura-2 (Neher 1989; Tsien 1990). The DC resistance of the electrodes ranged from 4 to 6 MOmega . Fura-2 (100 µM; Molecular Probes, Eugene, OR) or 1-10 µg/ml Rh-123 (Sigma) was added to the pipette solution before the experiment. In one series of experiments, 100 µM eosin-B also was added to the patch electrode solution. Whole cell recordings were performed on superficial CA1 pyramidal neurons under visual control using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany), driven by Pulse/Pulsefit software (HEKA) on a PowerPC (Apple Computer, Cupertino, CA). Seal resistance ranged from 1 to 3 GOmega , and series resistance was between 10 and 25 MOmega . Membrane conductance (gm) was measured by application of hyperpolarizing voltage pulses (-20 mV) with a duration of 500 ms. Holding potential in voltage clamp was -60 mV.

Fluorescence measurements

Fluorescence measurements were done with either a photomultiplier (Luigs and Neumann, Ratingen, Germany) or an imaging system using a 12-bit CCD camera (T.I.L.L. Photonics, Planegg, Germany) that was fixed to an upright microscope (Standard-16 or Axioskop, Zeiss, Oberkochen, Germany). The microscope was equipped with epifluorescence optics and a monochromator (Polychrome II, T.I.L.L. Photonics) to allow for fluorescence excitation at both 360 and 390 nm ([Ca2+]i measurements) or 485 nm (Delta Psi measurements). Emission was measured at 510 nm ([Ca2+]i measurements) or 530 nm (Delta Psi measurements). In one set of experiments, both fura-2 and Rh-123 were added to the patch electrode solution for simultaneous measurements of [Ca2+]i and Delta Psi . In this case, alternating excitation was done at 390 and 485 nM, and emission was measured at 530 nm. For the photomultiplier system, a pinhole diaphragm was used to avoid disturbance from background illumination. The diaphragm limited the region from which light was collected from the cell soma and proximal dendrites to a circular spot of 20 µm diam.

Fluorescence ratios were converted into [Ca2+]i by using Eq. 1: [Ca2+]i = K(R - Rmin)/(Rmax - R) (Grynkiewicz et al. 1985), in which R is the fluorescence ratio (360 nm/390 nm) and K is the effective dissociation constant of fura-2. In vivo calibration to determine Rmin, Rmax, and K was performed according to the method described by Neher (1989). Briefly, measurements were performed with three different pipette solutions (pH 7.1) that contained (in mM) 130 KCl, 1 MgCl2, 10 BAPTA, 10 HEPES, and 1 Na2-ATP (low calcium; Rmin); 130 KCl, 1 MgCl2, 3 CaCl2, 4 BAPTA, 10 HEPES, and 1 Na2-ATP [intermediate Ca2+; 300 nM, according to a KD of 107 nM for BAPTA (Tsien 1980)]; or 130 KCl, 1 MgCl2, 10 CaCl2, 10 HEPES, and 1 Na2-ATP (high Ca2+; Rmax). To each solution, 100 µM fura-2 was added. The resulting intracellular fluorescence ratios were calculated according to Eq. 1. K was calculated as K =300 nM (Rmax - R)/(R - Rmin).

Data analysis

Fluorescence and electrophysiological signals were sampled at 3 Hz by the PowerPC (Apple) via the ITC-16 interface of the EPC-9 amplifier using the X-Chart extension of the Pulse/Pulsefit software (HEKA). Analysis of the data was done with IGOR software (Wavemetrics, Lake Oswego, OR). Images were sampled at a rate of 1-10 Hz on an IBM-compatible computer using T.I.L.L. vision software. Further image processing was done using Adobe Photoshop software (Adobe Systems, Mountain View, CA) and CANVAS (Deneba software, Miami, FL). Values are means ± SE.


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ABSTRACT
INTRODUCTION
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Dialysis of CA1 pyramidal neurons with Rh-123

The permeant dye Rh-123 accumulates in polarized intracellular compartments such as mitochondria (Chen 1988; Duchen 1992, 1999; Johnson et al. 1980; Scaduto and Grotyohann 1999). At appropriate concentrations, the accumulated dye will self-quench, thereby reducing its quantum yield. Mitochondrial depolarization induces release of Rh-123 into the cytoplasm where, because of its dilution, it will produce a larger fluorescence signal. So far the dye has been used almost exclusively in cells that were loaded with the dye on bath application. In the present study, we have elaborated whether Rh-123 can be applied via the patch electrode to neurons within brain slices to continuously measure relative changes of mitochondrial membrane potential (Delta Psi ).

In previous reports on acutely dissociated or cultured neurons, 10 µg/ml was typically used for bulk-loading with Rh-123 (Nowicky and Duchen 1998; Schuchmann et al. 1998; see also Bindokas et al. 1998; Chen 1988). With this concentration of Rh-123 in the patch electrode, a stable rhodamine fluorescence signal (RFS), which indicated a rather constant concentration of the dye in the cytoplasm, was observed within 166 ± 72 s after establishing the whole cell configuration. In contrast, stabilization of the fluorescence signal was delayed three- to fourfold while dialyzing the cells with 5 µg/ml of the dye. In particular, when series resistance was relatively high (>15 MOmega ), the RFS continued to drift throughout the whole cell recording period with 5 µg/ml Rh-123 in the electrode. Because of the even greater time delay to establish a stable intracellular fluorescence signal, it was not appropriate to dialyze the cells with Rh-123 at a concentration of <5 µg/ml. Accordingly, because of the more rapid equilibration time and the better signal-to-noise ratio, a concentration of 10 µg/ml of the dye was used routinely for this study. Under these conditions, Im and gm (-21 ± 12 pA; 2.9 ± 1.9 nS; n = 39) as well as the RFS were stable for time periods of <= 2 h, indicating lack of cytotoxic effects of the drug as well as lack of photodamage (see also Chen 1988; Johnson et al. 1980). Similar stable whole cell recordings were obtained on dialysis of the cells with either fura-2 alone or on simultaneous administration of both dyes. In some slices, leakage of Rh-123 from the patch electrode before establishing a GOmega seal produced dye labeling of tissue in the vicinity of the recorded neuron. Such unspecific labeling was avoided by prefilling of the electrodes with 2 µl of intracellular solution that did not contain the dye.

Calibration of Rh-123 fluorescence signal

A depolarizing change of Delta Psi is indicated by an increase in RFS in response to dequenching of the dye after release from mitochondria. In isolated mitochondria, the magnitude of the Rh-123 signal had been shown to vary linearly with Delta Psi (Duchen 1992; Emaus et al. 1986). Additional factors, including total cell volume and mitochondrial volume fraction, influence the intensity of the Rh-123 fluorescence monitored in intact cells. Even among neurons of the same population, these factors may in principal vary considerably. Most of this variability is removed when rhodamine fluorescence intensity, monitored as the output voltage of the photomultiplier tube (Fig. 1), is normalized to the baseline observed in the resting cell that is examined after equilibration of dye from the patch pipette but before stimulus.



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Fig. 1. Calibration of Rh-123 fluorescence signal (RFS) in voltage-clamped CA1 pyramidal neurons of hippocampal slices. A: after stabilization of the RFS ~12 min subsequent to establishing the whole cell recording, plasmalemmal depolarization for 15 s from -60 to 0 mV led to an increase in rhodamine fluorescence intensity that is proportional to the output voltage of the photomultiplier tube (numbers represent mV). Increased rhodamine fluorescence intensity corresponds to a relative depolarization of mitochondrial membrane potential (Delta Psi ). RFS routinely is calibrated (or rather normalized) to percent of control, where 100% corresponds to the signal prior to stimulation. As further possibility for normalization, maximal mitochondrial hyperpolarization (to about -200 mV) can be evoked by the antibiotics oligomycin (10 µg/ml), whereas maximal depolarization (to around -60 mV) is induced by 1 µM of the mitochondrial uncoupler p-trifluoromethoxy-phenylhydrazone (FCCP). [Values are taken from the literature (for references, see Emaus et al. 1986; Scaduto and Grotyohann 1999).] B: mean values for RFS intensity baseline, for the peak response to plasmalemmal depolarization, and for oligomycin with or without FCCP (n = 5). C: statistical representation of the evoked changes of RFS in B normalized as percent of control.

The inset axis in Fig. 1A (left) illustrates application of this procedure to produce the RFS (%), the normalized rhodamine fluorescence signal in percent of control, that we routinely analyzed. Stimulus by depolarization of the plasma membrane from -60 to 0 mV for 15 s evoked a >1-nA outward current and a transient increase in the rhodamine intensity by <= 50% indicating a relative depolarization (see also Fig. 1, B and C). Figure 1A, right, illustrates a more elaborate procedure, not used routinely, for calibration (or rather normalization) of Rh-123 fluorescence intensity to the maximal responses observed for fully energized and fully depolarized mitochondria. Oligomycin, by inhibiting the mitochondrial ATP synthase, allows Delta Psi to become maximally hyperpolarized. The protonophore p-trifluoromethoxy-phenylhydrazone (FCCP), by directly dissipating the mitochondrial transmembrane H+ gradient, depolarizes Delta Psi . The right-hand inset axis is scaled to assumed values of -200 and -60 mV for Delta Psi of fully hyperpolarized and -depolarized mitochondria (Scaduto and Grotyohann 1999). On average oligomycin (10 µg/ml) led to a decrease of RFS by 52 ± 7%, whereas subsequent addition of FCCP (1 µM) produced an RFS increase of 295 ± 17% (n = 7; Fig. 1, A and C). The latter type of normalization gives a rough estimate for the activity of the ATP synthase and therefore of basic metabolic activity. As evident by the small standard error bars in Fig. 1, B and C, baseline levels as well as experimentally evoked changes of Delta Psi were rather uniform. These results show on the one hand that the variability in steady-state levels of dye distribution is very low, suggesting that resting Delta Psi is rather similar between individual cells. On the other hand, it is evident that dialysis with "fresh" dye via the patch electrode does not buffer or impede dynamic changes of Delta Psi (compare Neher 1989; Trapp et al. 1996a).

Delta Psi signals due to plasmalemmal depolarization

The origin of the RFS increase due to plasmalemmal depolarization that indicates depolarization of Delta Psi was analyzed further. Depolarization to 0 mV for 15 s led to a slowly inactivating outward current with a magnitude that varied in individual cells between 0.3 and 3 nA (n = 48). With a delay of 3.3 ± 0.8 s (n = 11) after onset of the outward current, a mean RFS increase of 36 ± 14% was observed (n = 11; Fig. 2, A and B). After termination of the plasmalemmal depolarization, the RFS increase and thus Delta Psi recovered to baseline with a mono-exponential time course (tau  = 19.2 ± 8.9 s; n = 11). In four cells analyzed, hyperpolarization from -60 to -120 mV for 15 s evoked a sustained inward current by between -300 and -900 pA without an effect on Delta Psi (Fig. 2B). The voltage threshold for the depolarization-evoked RFS differed in individual cells between -45 and -40 mV and a pulse duration of >1 s was necessary to evoke an RFS signal in response to depolarization to 0 mV. The rise of RFS saturated at depolarizations to between 0 and +30 mV, and the mean RFS increase for depolarization to +30 mV was 38 ± 6.9% (n = 4). A maximum RFS increase was seen in response to a 10-s depolarization (Fig. 3A). For longer plasmalemmal depolarizations, <= 30 s in length, the RFS transient stabilized at a maximal value for the duration of the stimulus (Fig. 3A). The depolarization-evoked RFS responses were accompanied by changes of [Ca2+]i baseline (101 ± 5 nM; n = 9). On 15-s depolarization to -40, -20, 0, and +20 mV, [Ca2+]i rose by 23 ± 9, 120 ± 28, 281 ± 168.1, and 358 ± 12 nM, respectively, in these cells after a delay of <1 s (Fig. 2C). Little or no recovery occurred until the stimulus terminated, even when the pulses had a duration of 30 s (Fig. 3B). Hyperpolarization of three cells to -120 mV did not affect [Ca2+]i baseline (Fig. 2D).



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Fig. 2. Voltage-dependent Delta Psi depolarizations and cytosolic Ca2 rises. A and B: outward current (Im) in response to plasmalemmal depolarization from -60 mV to potentials more positive than about -45 mV was accompanied by a sustained depolarization of mitochondrial potential (Delta Psi ) as reflected by the percentage increase of RFS (A), whereas the inward current due to hyperpolarisation to -120 mV did not change Delta Psi (B). C: plasmalemmal depolarization evoked a graded increase of intracellular-free Ca2+ ([Ca2+]i), whereas hyperpolarization had no effect (D).



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Fig. 3. Delta Psi depolarization and cytosolic [Ca2+]i rise during sustained depolarization. A: increasing the duration of depolarizing voltage pulses to a maximal of 30 s resulted in a stable mitochondrial (Delta Psi ) depolarization as represented by the RFS that was maximal for pulses with a duration of >10 s. B: persistent Delta Psi depolarizations were reflected by a sustained rise of intracellular Ca2+ ([Ca2+]i) by ~400 nM.

Role of Ca2+ influx in voltage-dependent Delta Psi depolarization

The preceding results suggest that the Delta Psi depolarization is related causally to the depolarization-induced [Ca2+]i transient that results from Ca2+ entry through voltage-gated Ca2+ channels (for references, see Trapp et al. 1996b). To support this assumption, the slices were superfused with Ca2+-free saline that also contained 5 mM Mg2+ and 1 mM of the Ca2+ chelator EGTA. This solution not only blocked the stimulus-induced [Ca2+]i rises (n = 4) but also abolished the concomitant Delta Psi depolarization (n = 5; Fig. 4). Furthermore the Ca2+-free superfusate led to a reversible decrease in the RFS by <= 25%, representing Delta Psi hyperpolarization (Fig. 4A) and a decrease in [Ca2+]i baseline by 42.1 ± 1.5 nM (Fig. 4B). Besides these effects, the Ca2+-free solution attenuated the slowly inactivating plateau value of the depolarization-induced outward current by ~20% (Fig. 4). Very similar effects on Delta Psi (n = 5), [Ca2+]i (n = 4), and Im were observed on bath application of a mixture of 200 µM of the blockers of voltage-activated Ca2+ channels Ni2+ and Cd2+ (not shown) (compare Trapp et al. 1996b). For an estimation of the temporal correlation of the depolarization-evoked Delta Psi and [Ca2+]i transient, the cells were dialyzed with both fura-2 and Rh-123 in one series of experiments. Also under these nonratiometric conditions, a decrease in the fura-2 fluorescence (390-nm excitation) corresponds to a rise of [Ca2+]i (see METHODS) (Nowicky and Duchen 1998). The evoked [Ca2+]i increase was found to precede the depolarization-evoked RFS rise by 1.6 ± 0.2 s (n = 4). After termination of the depolarizing stimulus, RFS had recovered by between 45 and 60% at the time when [Ca2+]i had almost returned to baseline (Fig. 5).



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Fig. 4. Ca2+ influx is pivotal for voltage-dependent Delta Psi depolarization and cytosolic [Ca2+]i rises. A: plasmalemmal depolarization from a holding potential of -60 to 0 mV evoked the typical Delta Psi depolarization, expressed as the RFS. Subsequent administration of a Ca2+-free solution that also contained 5 mM Mg2+ and 1 mM EGTA resulted in hyperpolarization of Delta Psi baseline and abolished the voltage-evoked Delta Psi depolarization. B: Ca2+-free solution also decreased [Ca2+]i baseline and blocked the depolarization-evoked Ca2+ rise.



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Fig. 5. Simultaneous measurement of voltage-dependent Delta Psi depolarization and [Ca2+]i rise. Cell was dialysed via the recording patch electrode with both Rh-123 for measurement of Delta Psi and with fura-2 to monitor [Ca2+]i. Depolarization from a holding potential of -60 to 0 mV produced the typical Delta Psi depolarization measured in arbitrary units as the fluorescence at 530 nm (excitation at 485 nm). Inset: illustration at a higher time resolution showing that the mitochondrial depolarization was preceded by >1 s by a rise of intracellular Ca2+ that is displayed as the inverted fura-2 fluorescence measured at 530 nm (excitation at 390 nm). Note that the change in fura-2 fluorescence (which was almost maximal before any change of the Rh-123 signal) did not interfere with the Delta Psi measurement.

Eosin-induced potentiation of voltage-dependent Delta Psi depolarization

Previous studies established that a plasmalemmal Ca2+/H+ pump (Carafoli 1991) has a major contribution to the recovery from a cytosolic Ca2+ load due to voltage-gated Ca2+ channels (Benham et al. 1992; Werth et al. 1996). Accordingly, it recently was demonstrated that block of this Ca2+ extrusion mechanism with eosin (Choi and Eisner 1999; Gatto et al. 1995) produces a considerable delay of recovery of [Ca2+]i rises evoked by membrane depolarization such as that used in the present study (Trapp et al. 1996b). In accordance with the latter study, we have added 100 µM eosin-B to the Rh-123-containing intracellular solution to block the Ca2+/H+ pump. In six neurons tested, depolarization of the plasma membrane to 0 mV for 15 s resulted in a RFS increase that was up to fourfold larger than that of control measurements. Furthermore, as exemplified in Fig. 6A, repetitive administration of depolarizing pulses resulted in both, a consecutive increase in the peak of the Delta Psi signal and delayed recovery to baseline level. Addition of the drug to the fura-2-containing patch electrode revealed that recovery from depolarization-evoked [Ca2+]i rises also was delayed considerably (Fig. 6, B and C). In the eosin-dialyzed cells, recovery of Ca2+ from the plasmalemmal depolarization reached 90% after 83.8 ± 10.3 s (n = 5) versus 8.6 ± 0.8 s in four control cells. In four of six CA1 neurons, this impairment of [Ca2+]i recovery led to a consecutive rise in [Ca2+]i baseline by between 30 and 120 nM. However, this effect was not as pronounced as the stepwise elevation of RFS evoked by eosin-B (compare Fig. 6, A and B).



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Fig. 6. Inhibitory effects of intracellular eosin on recovery from voltage-dependent Delta Psi depolarization and [Ca2+]i rise. A: continuous recording started ~5 min after establishing the whole cell configuration with a patch electrode that contained 100 µM of the Ca2+/H+ pump blocker eosin-B in addition to Rh-123. Plasmalemmal depolarization from -60 to 0 mV led to a progressive increase in the RFS, indicating consecutive diminution of mitochondrial potential. B: in a different neuron that was dialysed with eosin-B and fura-2, recovery from plasmalemmal depolarization as in A was delayed considerably with regard to control measurements without intracellular eosin as exemplified for 1 cell (gray traces) in C.

Comparison of voltage-dependent and metabolism-related RFS changes

In contrast to the potentiating effect of eosin-B on RFS increases in response to consecutive application of depolarizing pulses, the voltage-dependent RFS rise tended to decrease in magnitude under control conditions. This became evident when the cells were depolarized repeatedly from -60 to 0 mV at a stimulus interval of 1-6 min. The mitochondrial depolarization during the second stimulus was potentiated by <= 60% (Figs. 2 and 4), whereas further periods of depolarization attenuated the Delta Psi response in ~60% of observations (n = 17; Fig. 7, A and B). In individual cells, no Delta Psi depolarization could be elicited by the fourth or fifth depolarization although the membrane current response was not altered (not shown). This attenuating effect was not due to washout of Ca2+ currents and subsequent decrease in the magnitude of depolarization-induced [Ca2+]i transients as these were stable or even increased over time periods of >1 h in 13 cells tested (Fig. 7, C and D). To investigate, whether the decrease in the magnitude of the depolarization-induced Delta Psi depolarization was due to a methodological artifact resulting in incapability of Rh-123 to respond to Delta Psi changes, the effects of bath application of CN- (1 mM) were compared with those of depolarization of the plasma membrane. CN- is established to cause a major Delta Psi depolarization by blocking aerobic metabolism (Duchen and Biscoe 1992; McCormack et al. 1990; Miller 1991). In 13 of 35 CA1 pyramidal cells, application of CN- for 30 s evoked an outward current of 50 ± 11 pA and a concomitant gm increase by between 20 and 90% (Figs. 7 and 8). In the remaining 22 neurons, application of CN- for 30 s either did not change (60% of cases) resting Im or gm (Fig. 9) or evoked an inward current (<50 pA; Fig. 10) and gm rise (<30% of cells).



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Fig. 7. Effects of consecutive periods of plasma membrane depolarization and metabolic inhibition on Delta Psi and [Ca2+]i. A: cell was subjected to regular application of depolarizing pulses (from -60 to 0 mV) and bath application of CN- (1 mM). Delta Psi depolarization (evident as an increase in RFS) as evoked by voltage pulses was attenuated consecutively in this cell. In contrast, the depolarizing Delta Psi response to block of aerobic metabolism by CN- progressively increased. B: statistical analysis revealed attenuation of the voltage-dependent () Delta Psi depolarization (after a slight potentiation during the 2nd stimulation) whereas the mitochondrial response to CN- () increased in particular during the 2nd administration (n = 5). C: in this pyramidal neuron, consecutive depolarization led to potentiation of the rise of intracellular Ca2+ ([Ca2+]i), whereas the moderate [Ca2+]i increase due to CN- slightly decreased in magnitude. D: statistical analysis shows that the rises of [Ca2+]i during depolarization and CN- were rather stable (n = 5).



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Fig. 8. Effects of oligomycin and FCCP on CN--induced Delta Psi depolarization and [Ca2+]i increase. A: preincubation with oligomycin (10 µg/ml) led to an increase in the peak response with albeit faster recovery of Delta Psi in response to block of aerobic metabolism with CN- (1 mM). Subsequent exposure to FCCP (1 µM) led to an almost identical mitochondrial depolarization. B: in a different cell [Ca2+]i remained almost unchanged by CN-, oligomycin, or FCCP. Note that CN- evoked a moderate outward current (Im), whereas FCCP induced an inward shift of Im.



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Fig. 9. Digital imaging of voltage-dependent and CN--evoked mitochondrial (Delta Psi ) depolarization. A: continuous recording of RFS showed the typical mitochondrial depolarization in response to plasmalemmal depolarization from -60 to 0 mV. Imaging series displayed in the top right (numbers correspond to those in the continuous RFS trace) illustrate that the Rh-123 fluorescence (yellow and red pseudo color as indicative of successively larger Delta Psi depolarization) is concentrated locally in soma regions that are not occupied by the nucleus (pink symbol). B: in the same cell, 1 mM CN- produced a considerably larger increase in Rh-123 fluorescence that was observed primarily in somatic regions that were not next to the plasma membrane (see imaging series, bottom right).

Fig. 10. Digital imaging of voltage-dependent and CN--evoked intracellular Ca2+ ([Ca2+]i) rises. A: continuous recording of [Ca2+]i showed the typical Ca2+ transient in response to depolarization of the plasma membrane from -60 to 0 mV. Imaging series, top right, (numbers correspond to those in the continuous [Ca2+]i trace) illustrates that the peak fura-2 fluorescence change (yellow and red pseudo color as an indicative of a succesively larger rise in [Ca2+]i) is distributed uniformely over the soma. Note that block of aerobic metabolism by CN- (in B) does not evoke a major rise of [Ca2+]i, not even next to the membrane.

Despite the moderate effects of short application of CN- on basic membrane properties, the drug produced a prominent increase in RFS indicating depolarization of Delta Psi . In the experiment of Fig. 7A, the cell was depolarized repeatedly for 15 s and then exposed to CN-. This revealed that the Delta Psi response to depolarization progressively decreased in magnitude. In contrast, the response to CN-, which was initially more than twofold larger than the response to depolarization, was more and more potentiated (Fig. 7A). As exemplified with the neuron of Fig. 7C, both these experimental procedures had opposite effects on [Ca2+]i. CN- elevated [Ca2+]i by <50 nM, whereas depolarization to 0 mV caused a steadily increasing rise of intracellular Ca2+. The mean CN--induced RFS increase for the third and fourth application stabilized at values of between 200 and 270% of control (Fig. 7B), and the concomitant [Ca2+]i rise ranged between 10 and 45 nM (Fig. 7D). To exclude that the RFS responses to CN- are due to a nonspecific action of the agent, the effects of block of aerobic metabolism by rotenone, an inhibitor of complex-I NADH dehydrogenase, were tested. Bath application of 1 µM rotenone (1 min) produced a RFS increase that was very similar with that evoked by CN- (178 ± 87%, n = 15; not shown).

Relation between metabolism-induced RFS changes and [Ca2+]i

The preceding experiments showed that the prominent RFS increase in response to block of aerobic metabolism with CN- occurs without a major change of [Ca2+]i. For further characterization of the relation of Delta Psi and intracellular Ca2+ during metabolic manipulation, the effects of oligomycin and FCCP were analyzed. The cell illustrated in Fig. 8A responded to repetitive application of CN- with an almost identical increase in RFS. Subsequent exposure of oligomycin resulted in the typical decrease of RFS (compare Fig. 1). In the presence of oligomycin, the recovery of the RFS rise due to a further application of CN- was faster than under control although the peak response was potentiated. Addition of FCCP to the oligomycin-containing superfusate after recovery from CN- induced a RFS increase to the same level as seen during CN- in the presence of oligomycin. On average, the CN--evoked RFS rise in this series of four experiments was 202 ± 20%, whereas the potentiated response in the presence of oligomycin was 285 ± 25%. Oligomycin produced a hyperpolarization by 46 ± 10%, whereas FCCP evoked a RFS increase by 300 ± 20% in the presence of oligomycin. In five different neurons, CN- elevated [Ca2+]i by <30 nM. Exposure of these cells to oligomycin neither had an effect on [Ca2+]i baseline nor were the very moderate CN--evoked intracellular Ca2+ rises potentiated. Also FCCP only produced a [Ca2+]i rise of <30 nM in the presence of oligomycin (Fig. 8B). Oligomycin did not affect Im or gm, whereas FCCP induced an inward current in two of the five CA1 neurons.

Imaging of voltage-dependent and CN--induced Delta Psi depolarization

The experiments described so far showed that reproducible Delta Psi changes can be recorded for extended time periods with a photomultiplier-based optical system in voltage-clamped CA1 neurons in situ. In a final approach, imaging was used in 12 (Rh-123 measurements) and 11 ([Ca2+]i measurements) neurons to determine the extent to which spatial resolution of the intracellular fluorescent signals could be resolved. In the example of Fig. 9, the Rh-123 fluorescence under resting conditions was more prominent in somatic regions close to the nucleus. On plasmalemmal depolarization, the Rh-123 fluorescence increased primarily in this area and invaded neither the region of the nucleus nor sites close to the plasma membrane. On exposure of the cell to CN-, the Rh-123 fluorescence increase, which was considerably larger than that on depolarization of the plasma membrane, again appeared rather localized in somatic regions that were not occupied by the nucleus. CN- did not produce a major signal in the vicinity of the plasma membrane (Fig. 9B). In contrast, the depolarization-evoked rise of [Ca2+]i distributed rapidly and uniformly over the entire soma (Fig. 10A), whereas the CN--induced minor rise of [Ca2+]i was diffusely distributed over the somatic region (Fig. 10B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the first part of RESULTS, which contains a summary of the principal of Delta Psi measurements with Rh-123, it was demonstrated that mammalian neurons of brain slices can be dialyzed via the whole cell recording patch electrode with Rh-123 for long-term recording of mitochondrial membrane potential and therefore metabolic activity. It also was shown that different ways of normalization of the Rh-123 fluorescence signal are possible. Furthermore it was found that FCCP induces a prominent increase in RFS indicating mitochondrial depolarization whereas oligomycin evokes a noticeable hyperpolarization similar to previous studies on isolated cells (Duchen 1992, 1999; Duchen and Bisoe 1992). This indicates a wide dynamic range of activity-related mitochondrial membrane potential changes in neurons in situ. Accordingly we demonstrated that depolarization of the plasma membrane elicits a reversible Delta Psi depolarization that is likely to be due to stimulation of Ca2+ uptake into mitochondria. In contrast, a prominent CN--induced mitochondrial depolarization developed in the absence of a major cytosolic Ca2+ increase as a consequence of block of the electron transport chain. On the basis of methodological considerations, future applications of the technique for simultaneous measurement of cell metabolism and excitability are considered.

Ca2+-dependent Delta Psi depolarization

Previous studies using extracted mitochondria (Gunter et al. 1994; McCormack et al. 1990) or isolated cells (Duchen 1992, 1999; Duchen and Biscoe 1992; Loew et al. 1994; Nowicky and Duchen 1998) have established that a rise of cytosolic [Ca2+]i depolarizes mitochondria. As shown in detail in the latter studies, this mitochondrial depolarization is secondary to influx of Ca2+ into these organelles via an electrogenic Ca2+ uniporter conductive pathway (see also Babcock et al. 1997; David et al. 1998). At present, it is thought that the increase of intramitochondrial Ca2+ is not only important for buffering of cytosolic Ca2+ loads (Babcock and Hille 1998; Herrington et al. 1996; Werth et al. 1996) but also serves to stimulate aerobic energy production (Gunter et al. 1994; McCormack et al. 1990). This view gains support from the finding of an initial oxidation of NAD(P)H and FADH, which turns into a secondary increase in the reduced state of both enzymes (Duchen 1992, 1999; Schuchmann et al. 1998). In the present study, a robust rise of [Ca2+]i by 200-500 nM was evoked in the CA1 pyramidal cells by depolarization of the plasma membrane. The magnitudes of the depolarization-induced [Ca2+]i rise and Delta Psi depolarization were parallel with the voltage dependence of voltage-gated Ca2+ channels in CA1 cells (for references, see Trapp et al. 1996b; see also Duchen 1992). That the Delta Psi depolarization was indeed caused by Ca2+ entry through voltage-gated Ca2+ channels and was not due to a direct effect of the plasmalemmal depolarization is indicated by the blocking effect of Ni2+/Cd2+ and Ca2+-free superfusate on both the [Ca2+]i and Delta Psi response (Duchen 1992).

The fall of Rh-123 fluorescence on omission of extracellular Ca2+ could be due to a decrease in the demand for mitochondrial ATP production (see preceding text). As an alternative, mitochondria might mediate buffering of cytosolic Ca2+ even under resting conditions. Involvement of mitochondria in Ca2+ regulation in the hippocampal neurons is suggested by the finding that inhibition of the plasmalemmal Ca2+/H+ pump by eosin-B prolonged recovery of both the Delta Psi and the [Ca2+]i transient on plasmalemmal depolarization. The potentiation of the peak Delta Psi response on repetitive periods of plasmalemmal depolarization in the presence of eosin-B can be explained by the observation that recovery of RFS from a single stimulus was much slower than that of the [Ca2+]i transient. These results support previous assumptions that a plasmalemmal Ca2+/H+ pump plays a major role in cellular Ca2+ homeostasis (Benham et al. 1992; Choi and Eisner 1999; Trapp et al. 1996b; Werth et al. 1996). They also suggest that impairment of Ca2+ homeostasis results in a pronounced Ca2+ load of mitochondria in the CA1 pyramidal neurons. Future monitoring of mitochondrial Ca2+ with dyes such as rhod-2 will elucidate the role of mitochondria in Ca2+ homeostasis in these central neurons as previously demonstrated for other excitable cells (Babcock and Hille 1998; Babcock et al. 1997; David et al. 1998; Pivovarova et al. 1999; Werth and Thayer 1994).

That influx of Ca2+ initiates the mitochondrial depolarization and not vice versa, that mitochondrial depolarization promotes the cytosolic Ca2+ signal, is indicated by the results from the simultaneous fura-2 and Rh-123 measurements. These recordings showed that the [Ca2+]i rise precedes the Delta Psi response on average by 1.6 s. As suspected by Nowicky and Duchen (1998) on the basis of similar simultaneous measurements in fura-2 and Rh-123 bulk-loaded dissociated hippocampal cells, the time lag might be smaller because the Rh-123 signal reflects unquenched dye that left the mitochondrial compartment on depolarization of the organelles (see also Chen 1988). As the fura-2 signal during the simultaneous recordings was not ratiometrically measured in this series of experiments of the present study, the Ca2+ transient could not be quantified. Ratiometric [Ca2+]i measurements are, in principal, possible during simultaneous monitoring of Delta Psi and [Ca2+]i provided that the optical set up allows for excitation with three alternating wavelengths (Nowicky and Duchen 1998). The fura-2 signal did not appear to interfere with the RFS, but the extent to which a major change in Rh-123 fluorescence might influence the fura-2 signal (Duchen 1992) needs to be analyzed in future studies.

It was found with long-term recording that the average magnitude of the Delta Psi depolarization decreased in ~50% of cells and even was abolished in individual neurons on consecutive administration of depolarizing voltage steps. Nevertheless, the persistence and even potentiation of the Delta Psi response in response to CN- (see following text) excludes that wash-out or bleaching of Rh-123 is responsible for this effect. It is probable, but deserves further experimental analysis, that the attenuation of the depolarization-evoked Delta Psi response represents saturation of mitochondrial Ca2+ stores, which inhibits further Ca2+ entry (Duchen 1992; Pivovarova et al. 1999; Werth and Thayer 1994).

Depolarizing voltage steps with a duration of >1 s were necessary to reveal a mitochondrial depolarization. We tested here the suitability of the method of dialysis of neurons with Rh-123 with a simple experimental procedure that is not hampered by signals originating from synaptic and metabolic interactions within the slice. We chose depolarization of the plasma membrane as a test protocol because the effects of [Ca2+]i rises due to voltage-activated Ca2+ channels on mitochondrial energetics have been elucidated in detail in isolated neurons and glia (Duchen 1992, 1999; Duchen and Biscoe 1992; Nowicky and Duchen 1998). More prominent Delta Psi depolarizations are likely to be revealed in future studies on synaptic activation for several reasons. On tetanic stimulation, that is necessary to evoke synaptic plasticity (Andersen et al. 1977; Bliss and Collingridge 1993; Edwards 1995; Tang and Zucker 1997) not only intracellular Ca2+ is elevated but also intracellular Na+ increases by several tens of millimolar (Ballanyi et al. 1984; Yu and Salter 1998). As indicated by a concomitant prominent fall of tissue oxygen, that correlates with the kinetics of Na+/K+ pump activation (for references, see Brockhaus et al. 1993), the activity-related Na+ rise also should stimulate aerobic metabolism and thus dissipate mitochondrial potential.

Furthermore the [Ca2+]i rise associated with synaptic activity of hippocampal neurons typically exceeds that evoked by exclusive activation of voltage-gated Ca2+ channels due to activation of both ionotropic and metabotropic glutamate receptors (Alford et al. 1993; Magee et al. 1995; Murphy and Miller 1988; Regehr and Tank 1994; Regehr et al. 1989). Accordingly, it was shown that activation of ionotropic glutamate receptors evokes a profound Delta Psi depolarization in ester-loaded neurons (Bindokas et al. 1998; Budd and Nicholls 1996; Hoyt et al. 1998; Schinder et al. 1996; Schuchmann et al. 1998; White and Reynolds 1996). The recovery from glutamate receptor-dependent mitochondrial depolarization was sometimes considerably slower than that of the concomitant [Ca2+]i rise (Schuchmann et al. 1998) and even could be incomplete, in particular if the N-methyl-D-aspartate type of glutamate receptors was involved (Khodorov et al. 1996; Schinder et al. 1996; White and Reynolds 1996). This led to the assumption by the latter authors that Ca2+-dependent mitochondrial disfunction is a primary event in glutamate-induced neurotoxicity due to anoxic/ischemic insults (Budd and Nicholls 1996; Choi 1988; Kristian and Siesjö 1996; Schanne et al. 1979; Stout et al. 1998).

CN--induced Delta Psi depolarization

In contrast to a maximal Delta Psi response of 50% RFS increase on depolarization of the plasma membrane, block of aerobic metabolism produced a mitochondrial depolarization that could amount to 300% RFS of control. One explanation of these differences might be that only those mitochondria close to the cell membrane contribute to the changes of RFS due to plasmalemmal depolarization. [Ca2+]i rises in more central microdomains of the cytosol might be too small to induce mitochondrial depolarization (for references, see Duchen 1999; Pivovarova et al. 1999). Nevertheless, the CN- effects substantiate the preceding assumption that the depolarization-induced Rh-123 signals are not at saturating levels of the dynamic range of cellular Delta Psi changes. This suggests that the expected changes in mitochondrial potential during intense neuronal activity could in principal be well resolved. Because of the strong temperature dependence of Delta Psi depolarizations associated with metabolic blockade (Duchen and Biscoe 1992) even larger responses to CN- are probably inducible at 36-37°C instead of 30°C used in the present study. The reduced in vitro temperature also might explain the moderate effects of CN- on membrane current and [Ca2+]i (for references, see Morris et al. 1991). Also the short application time of 30 s might contribute to the small effects on current and intracellular Ca2+. Accordingly, exposure of isolated CA1 neurons to CN- for 2 min was found to promote a [Ca2+]i rise of 100 nM, leading to a pronounced hyperpolarization by opening of Ca2+-activated K+ channels (Nowicky and Duchen 1998). It is also possible that the moderate CN--induced [Ca2+]i rise is partly due to dialysis of the cells (compare Bickler and Hansen 1998; Yamagushi et al. 1998). However, no difference in the CN--related [Ca2+]i increase was found between whole cell recorded and intact dorsal vagal medullary neurons (Ballanyi and Kulik 1998). In the latter study, it was suggested that the small initial [Ca2+]i rise in the vagal neurons is mediated by Ca2+ release from mitochondria as also was suggested for Purkinje cells of cerebellar slices (Ballanyi et al. 1999). Although the mechanism of the initial moderate rise of intracellular Ca2+ in the CA1 neurons during CN- remains to be elucidated, our measurements clearly show that changes in mitochondrial function, and therefore of metabolism, can occur in the absence of a major cytosolic Ca2+ signal.

The CN--induced Delta Psi signal appears to represent mitochondrial depolarization and not an artificial response to the drug as block of aerobic metabolism by rotenone produced a very similar effect. The prominent Delta Psi responses on blockade of aerobic metabolism occurred despite dialysis of the neurons with ATP via the patch electrode. This supports previous assumptions that the mitochondrial depolarization is not primarily related to a decrease in the ATP/ADP.Pi ratio (Duchen 1992; Duchen and Biscoe 1992). In agreement with conclusions from the latter studies, the present results suggest that the Delta Psi depolarization induced by the metabolic inhibitors is caused by block of electron transport through the respiratory chain. Nevertheless, dialysis of the cells with ATP-containing patch pipette solution might partly counteract the depolarizing effect of CN- on mitochondrial potential by providing the fuel for reverse-mode operation of the ATP synthase, which then would hyperpolarize Delta Psi (Babcock et al. 1997). However, this mechanism does not appear to have a major contribution as block of the ATP synthase with oligomycin only led to a modest potentiation of the CN--induced mitochondrial depolarization. Interestingly, the oligomycin-potentiated CN--evoked depolarization was almost identical with the expected maximal depolarization induced by FCCP (Duchen 1992).

Future applications

High-resolution imaging techniques using fluorophores that are only sensitive to very high Ca2+ levels demonstrated that [Ca2+]i rises can amount to several µM in dendrites during tetanic stimulation that is necessary to induce long-term changes of synaptic plasticity (Regehr and Tank 1992). Because these activity-related Ca2+ transients last for up to several seconds, it can be assumed on the basis of the present results that they induce depolarization of mitochondria. It is established that mitochondria are present not only in the soma but also along the entire dendritic tree of CA1 neurons (Nafstad and Blackstad 1966; Siklos and Kuhnt 1994). Thus it should be possible to monitor localized metabolic activity by means of dynamic changes of Delta Psi in different compartments of mammalian neurons in situ. In the present study, spatial resolution was hampered by the fact that Delta Psi imaging was not done with confocal or two-photon optical techniques. Accordingly, resolution of mitochondrial signals or of [Ca2+]i rises in small dendrites or even spines was not possible. Nevertheless it was revealed that Rh-123 fluorescence under resting conditions was distributed nonuniformly in spots and that the region of the nucleus did not show fluorescence. This is consistent with findings in cultured hippocampal neurons in which mitochondria were found to be clustered in particular in the perinuclear somatic region (Bindokas et al. 1998; Schinder et al. 1996) as also shown for other cell types (Chen 1988; Duchen 1999; White and Reynolds 1996). Ester loading of hippocampal slices with Delta Psi -sensitive dyes recently was demonstrated as a powerful tool to study the relation of metabolism and synchronized electrical activity. In extension of this work, we have presented here a method for selective labeling of individual neurons within functionally intact neuronal networks. This allows for simultaneous recording of membrane excitability, energy metabolism and [Ca2+]i. The method also should be applicable to presynaptic boutons (David et al. 1998; Tang and Zucker 1997) as well as to single glial cells in brain slices (Kulik et al. 1999). Therefore it should be possible to elaborate in future studies how stimulated neuronal and/or glial metabolism (Tsacopoulos and Magistretti 1996) contributes to synaptic plasticity (Edwards 1995; Tang and Zucker 1997) or pathological processes such as epilepsy (Duchen 1992; Lee et al. 1984; Schuchmann et al. 1999) or ischemia-related neurotoxicity (Budd and Nicholls 1996; Choi 1988; Kristian and Siesjö 1996; Schanne et al. 1978).


    ACKNOWLEDGMENTS

We thank A.-A. Grützner for expert technical assistance.

This study was supported by the Deutsche Forschungsgemeinschaft and the Hermann und Lilly Schilling-Stiftung.


    FOOTNOTES

Address for reprint requests: K. Ballanyi, II. Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany. E-mail: kb{at}neuro-physiol.med.uni-goettingen.de

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.

Received 3 June 1999; accepted in final form 22 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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