1II. Physiologisches Institut, Universität Göttingen, D-37073 Göttingen; and 2Institut für Physiologie, Humboldt-Universität Berlin, Universitätsklinikum Charité, D-10117 Berlin, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 () 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
, 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
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
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
. 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
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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
) 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
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
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 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
(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
in whole cell
recorded hippocampal neurons in situ. For comparison of the effects of
plasma membrane depolarization with
responses due to direct
modulation of energy metabolism, we have analyzed the effects on
[Ca2+]i and
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
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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M
. 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 G
, and series resistance was between 10 and 25 M
. 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 ( measurements). Emission was measured at 510 nm
([Ca2+]i measurements) or
530 nm (
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
. 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
).
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 M
), 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 G
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 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
(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.
|
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
to become maximally hyperpolarized. The
protonophore p-trifluoromethoxy-phenylhydrazone (FCCP), by directly
dissipating the mitochondrial transmembrane H+
gradient, depolarizes
. The right-hand inset axis is
scaled to assumed values of
200 and
60 mV for
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
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
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
(compare Neher 1989
; Trapp et al.
1996a
).
signals due to plasmalemmal depolarization
The origin of the RFS increase due to plasmalemmal
depolarization that indicates depolarization of 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
recovered to baseline
with a mono-exponential time course (
= 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
(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).
|
|
Role of Ca2+ influx in voltage-dependent
depolarization
The preceding results suggest that the 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
depolarization (n = 5; Fig.
4). Furthermore the
Ca2+-free superfusate led to a reversible
decrease in the RFS by
25%, representing
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
(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
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).
|
|
Eosin-induced potentiation of voltage-dependent
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
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).
|
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
response in ~60% of observations
(n = 17; Fig. 7,
A and B). In individual cells, no
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
depolarization was due to a methodological artifact resulting in
incapability of Rh-123 to respond to
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
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).
|
|
|
Despite the moderate effects of short application of CN
on basic membrane properties, the drug produced a prominent increase in
RFS indicating depolarization of
. In the experiment of Fig. 7A, the cell was depolarized repeatedly for 15 s
and then exposed to CN
. This revealed that the
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
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
depolarization
The experiments described so far showed that reproducible
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the first part of RESULTS, which contains a summary
of the principal of 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
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 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
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
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
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 and the
[Ca2+]i transient on
plasmalemmal depolarization. The potentiation of the peak
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
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
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
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
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
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
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
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
depolarization
In contrast to a maximal 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
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
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
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
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
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
(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
in different compartments of mammalian neurons in situ. In the
present study, spatial resolution was hampered by the fact that
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
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|