J Neurophysiol
87: 1426 –1439, 2002; 10.1152/jn.00627.2001.
Mitochondrial Ca2⫹ Buffering Regulates Synaptic Transmission
Between Retinal Amacrine Cells
KATHRYN MEDLER AND EVANNA L. GLEASON
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
Received 30 July 2001; accepted in final form 9 November 2001
Address for reprint requests: E. L. Gleason, Dept. of Biological Sciences,
202 Life Sciences Building, Louisiana State University, Baton Rouge, LA
70803 (E-mail: [email protected]).
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.
INTRODUCTION
1426
0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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In the vertebrate retina, amacrine cells make synapses onto
bipolar cells, ganglion cells, and other amacrine cells, and the
output of these synapses can be critical for shaping the visual
signal. With a culture system containing identified GABAergic
amacrine cells, we have begun to elucidate the physiological
properties of the synapses formed between these cells. Unlike
many other CNS neurons, but like retinal bipolar cells and
photoreceptors, amacrine cells use L-type Ca2⫹ channels at
their synapses to control neurotransmitter release (Gleason et
al. 1994). One of the consequences of using L-type Ca2⫹
channels at the synapse is that both the Ca2⫹ channels and the
exocytotic machinery are sensitive to cytosolic Ca2⫹. It has
been established that L-type Ca2⫹ channels are inactivated by
the Ca2⫹-calmodulin complex (Peterson et al. 1999). Thus we
would predict that the effects of Ca2⫹ buffering in the presynaptic terminal represents a balance between limiting exocytosis
and promoting the activity of L-type Ca2⫹ channels.
At least five Ca2⫹-buffering mechanisms are thought to
coexist in most cells: the plasma membrane Ca2⫹ ATPase and
Na-Ca exchanger, endoplasmic reticulum Ca2⫹ATPases, cytosolic Ca2⫹-buffering proteins, and mitochondria. In amacrine
cells, it has been estimated that ⬃60% of a Ca2⫹ load admitted
through voltage-gated Ca2⫹ channels is removed by the plasma
membrane Na-Ca exchanger (Gleason et al. 1995). This fraction was unaffected by inhibition of Ca2⫹ATPases, indicating
that the balance of the Ca2⫹ must be taken up by temporary
Ca2⫹ removal systems within the cell, possibly mitochondria,
prior to export. We have also demonstrated that in the absence
of Na-Ca exchanger activity, synaptic transmission between
amacrine cells is dramatically prolonged (Gleason et al. 1994).
However, the role of the other Ca2⫹-buffering mechanisms in
synaptic transmission has not previously been explored.
Although it has long been understood that significant Ca2⫹
flux occurs across the mitochondrial membranes, more recent
studies have demonstrated that this flux can have a significant
impact on the amplitude and time course of Ca2⫹ transients in
neurons (Friel and Tsien 1994; Thayer and Miller 1990; Werth
and Thayer 1994). Although the outer mitochondrial membrane is freely permeable to Ca2⫹ ions, the inner mitochondrial
membrane contains machinery that controls the flux of Ca2⫹
into and out of the matrix (for review, see Babcock and Hille
1998; Duchen 1999; Simpson and Russell 1998). The Ca2⫹
uniporter transports Ca2⫹down its electrochemical gradient
into the mitochondrial matrix and is dependent on the mitochondrial membrane potential generated by the proton gradient
across the inner mitochondrial membrane. The Na-Ca exchanger on the inner mitochondrial membrane usually moves
Ca2⫹ back into the cytoplasm and is the primary efflux mechanism thought to operate during normal cellular function. Another pathway for Ca2⫹ efflux is the permeability transition
pore. This conductance is activated at relatively high mito-
Medler, Kathryn and Evanna L. Gleason. Mitochondrial Ca2⫹
buffering regulates synaptic transmission between retinal amacrine
cells. J Neurophysiol 87: 1426 –1439, 2002; 10.1152/jn.00627.2001.
The diverse functions of retinal amacrine cells are reliant on the
physiological properties of their synapses. Here we examine the role
of mitochondria as Ca2⫹ buffering organelles in synaptic transmission
between GABAergic amacrine cells. We used the protonophore ptrifluoromethoxy-phenylhydrazone (FCCP) to dissipate the membrane
potential across the inner mitochondrial membrane that normally
sustains the activity of the mitochondrial Ca2⫹ uniporter. Measurements of cytosolic Ca2⫹ levels reveal that prolonged depolarizationinduced Ca2⫹ elevations measured at the cell body are altered by
inhibition of mitochondrial Ca2⫹ uptake. Furthermore, an analysis of
the ratio of Ca2⫹ efflux on the plasma membrane Na-Ca exchanger to
influx through Ca2⫹ channels during voltage steps indicates that
mitochondria can also buffer Ca2⫹ loads induced by relatively brief
stimuli. Importantly, we also demonstrate that mitochondrial Ca2⫹
uptake operates at rest to help maintain low cytosolic Ca2⫹ levels.
This aspect of mitochondrial Ca2⫹ buffering suggests that in amacrine
cells, the normal function of Ca2⫹-dependent mechanisms would be
contingent upon ongoing mitochondrial Ca2⫹ uptake. To test the role
of mitochondrial Ca2⫹ buffering at amacrine cell synapses, we record
from amacrine cells receiving GABAergic synaptic input. The Ca2⫹
elevations produced by inhibition of mitochondrial Ca2⫹ uptake are
localized and sufficient in magnitude to stimulate exocytosis, indicating that mitochondria help to maintain low levels of exocytosis at rest.
However, we found that inhibition of mitochondrial Ca2⫹ uptake
during evoked synaptic transmission results in a reduction in the
charge transferred at the synapse. Recordings from isolated amacrine
cells reveal that this is most likely due to the increase in the inactivation of presynaptic Ca2⫹ channels observed in the absence of
mitochondrial Ca2⫹ buffering. These results demonstrate that mitochondrial Ca2⫹ buffering plays a critical role in the function of
amacrine cell synapses.
ROLE OF MITOCHONDRIA IN AMACRINE CELLS
chondrial matrix Ca2⫹ concentration, and its opening may be
key in Ca2⫹-mediated cell death.
Measurements of relative Ca2⫹ concentration and electrophysiological techniques were employed to reveal the role of
mitochondrial Ca2⫹ buffering in retinal amacrine cells and
their synapses. The protonophore p-trifluoromethoxy-phenylhydrazone (FCCP) was used to dissipate the proton gradient
across the inner mitochondrial membrane, disabling uniporter
activity and preventing Ca2⫹ uptake by the mitochondria.
Because the role of mitochondria in buffering cytosolic Ca2⫹
transients has not previously been examined in amacrine cells,
we first use cytosolic Ca2⫹ measurements to determine how
Ca2⫹ transients in amacrine cells are shaped by mitochondrial
Ca2⫹ buffering. We then examine the impact of mitochondrial
Ca2⫹ buffering on synaptic transmission using perforatedpatch voltage-clamp recordings.
Cell culture
Chick retinal cells were dissociated and cultured as previously
described (Gleason et al. 1993). Cells were maintained in culture at
37°C under 5% CO2 atmosphere until they were used for experiments,
7–14 days after plating. For both Ca2⫹ measurement and electrophysiology experiments, amacrine cells were identified based on their
morphology. Cells with relatively large cell bodies (10 –15 ␮M)
bearing two to five primary processes have been previously identified
as amacrine cells using both immunocytochemical and physiological
criteria (Gleason et al. 1993; Huba and Hofmann 1990, 1991; Huba et
al. 1992).
Ca2⫹ measurements
Cells were loaded with 2 ␮M fluo-3 acetoxymethylester (AM,
Molecular Probes, Eugene, OR) for 1 h at room temperature and in the
dark. At the end of the loading period, cells were washed with normal
external solution (see following text) and maintained in the dark until
they were used, routinely within 20 min. Culture dishes were mounted
on the stage of an upright Nikon Optiphot II microscope, and the cells
were visualized using a Noran laser confocal imaging system with a
40⫻ water-immersion objective. Fluo-3 fluorescence was visualized
using the 488-nm laser line with a 515-nm long-pass filter. Image and
data acquisition were controlled by the InterVision (Noran) software
and hardware. Images consisting of an average of eight frames were
collected every 5 s. Shorter shuttering intervals often resulted in
damage to the cells as determined by inspection with transmitted
illumination at the end of an experiment. Because our shuttering rate
was relatively slow (0.2 Hz), faster sampling (0.33 Hz) was done on
a subset of cells to test whether we were accurately measuring the
peak fluorescence increases. When the shutter was opened and fluorescence was measured every 3 s, 10-s applications of 100 mM K⫹
generated responses with 5–10 data points at the peak of the response,
indicating that peak fluorescence amplitudes could be accurately
measured with a 0.2-Hz sampling rate. Mean pixel intensity was
sampled over a rectangular region (⬃25 ␮M2) of the cell body at 28
Hz. Peak pixel intensity values collected during shutter openings were
subsequently extracted from the raw data using the peak detection
analysis portion of the Origin software package (Microcal). Background fluorescence was sampled from a cell-free region of the field
and remained constant over the duration of the experiments (10 –20
min). For analysis of the spatial and temporal aspects of FCCPdependent Ca2⫹ elevations (Fig. 7), fluorescence intensity values
were obtained with a sampling rate of 28 Hz and without shuttering.
For display purposes, most of the fluorescence intensity values were
normalized to baseline values and reported as F/Fo.
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All solutions were delivered through gravity-driven bath perfusion
(3– 4 ml/min). Ten seconds of the delay between agonist application
and the cellular response was estimated to be due to the dead space in
the perfusion line. Composition of the normal external solution was
(in mM) 5.3 KCl, 135.0 NaCl, 3.0 CaCl2, 0.41 MgCl2, 5.6 glucose,
and 3.0 HEPES, pH 7.4. Fifty and 100 mM K external solutions were
made by replacement of NaCl. External solutions were adjusted to a
final pH of 7.4 with NaOH. FCCP was dissolved in DMSO to form a
10 mM stock and then added to external solution for a final concentration of 1 ␮M.
Electrophysiological recording
Tissue culture dishes were mounted on the stage of an Olympus
IX70 inverted microscope. A reference Ag/AgCl pellet served to
ground the dish. Patch electrodes were pulled from thick-walled
borosilicate glass (1.5 mm OD, 0.86 mm ID; Sutter Instruments,
Novato, CA) on a Flaming-Brown micropipette puller (Sutter Instruments). Electrode resistance values ranged from 3 to 5 M⍀. Most
voltage-clamp recordings were made using the perforated-patch technique (Horn and Marty 1988). The pipette solution contained 200
␮g/ml amphotericin B. Internal solutions consisted of the following
(in mM): solution A (high Cl⫺): 12.0 Cs acetate, 133.0 CsCl, 1.0
NaCl, 2.0 MgCl2, 0.1 CaCl2, 1.1 EGTA, and 10.0 HEPES; solution B
(low Cl⫺): 135.0 Cs acetate, 10.0 CsCl, 1.0 NaCl, 2.0 MgCl2, 0.1
CaCl2, 1.1 EGTA, and 10.0 HEPES. Voltage-clamp experiments
performed in the ruptured-patch configuration used the following
internal solution: 10.0 Cs acetate, 100.0 CsCl, 1.0 NaCl, 2.0 MgCl2,
0.1 CaCl2, 1.1 EGTA, 10.0 HEPES, 3.0 K ATP, 1.0 Na ATP, and 20.0
phosphocreatine and 50.0 IU/ml creatine phosphokinase. Currentclamp experiments were done using K⫹-containing internal solution:
150.0 K acetate, 10.0 KCl, 2.0 MgCl2, 0.1 CaCl2, 10.0 HEPES, 1.1
EGTA, and 1.0 Na ATP. All internal solutions were adjusted to pH
7.4 with CsOH.
For perforated-patch experiments, gigaohm seals were obtained by
briefly hyperpolarizing the patch (⫺140 mV) prior to gaining electrical access to the cell. Within 5 min of obtaining a seal, the resistance
had usually fallen below 50 M⍀ and stabilized. Series resistance was
monitored throughout all experiments. and if the series resistance was
unstable during the recording, data from that cell were eliminated
from the data set. The measured liquid junction potentials between the
normal external and internals A and B were ⫺2 and ⫺7 mV, respectively. No corrections for errors due to series resistance or junction
potentials were applied to these data.
External solutions were delivered by gravity-flow perfusion (2–3
ml/min) from one of two types of inlets. For some electrophysiology
experiments (Figs. 3 and 5), a 1-mm diameter inlet tube was positioned ⬃5 mm from the cell. Solutions were manually switched
upstream from the inlet. We estimated from dye experiments that the
dead space in the perfusion accounted for ⬃7–9 s of the delay
between test solution application and the cellular response. For all
other experiments (Figs. 2 and 7–9), a double-barreled inlet was
positioned ⬃3 mm from the cell. One of the barrels contained normal
external and the second was manually switchable between test solutions. The inlet was positioned such that turning off the normal
solution would allow the plume of test solution to quickly (⬍1 s)
cover the cell. Based on control experiments with dye-containing
FCCP solution, we determined that the delay between FCCP arrival
and elevation of cytosolic Ca2⫹ (estimated by increased exchanger
activity or stimulation of exocytosis) was ⬍5 s. External solutions
consisted of the following (in mM): normal external: 116.7 NaCl, 5.3
KCl, 20.0 TEA Cl, 3.0 CaCl2, 0.41 MgCl2, 5.6 glucose, and 3.0
HEPES. Ba external: normal external with 3 mM BaCl2 replacing 3
mM CaCl2. Li external: normal external with LiCl2 replacing NaCl2.
Ca2⫹-free external: normal external with no added Ca2⫹. Bis-(oaminophenoxy)-N,N,N⬘,N⬘-tetraacetic acid (BAPTA) loading of cells
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METHODS
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K. MEDLER AND E. L. GLEASON
was achieved by incubating cultures in 10 ␮M BAPTA AM (Biomol,
Plymouth Meeting, PA) for 30 min at 37°C.
Unless synaptic currents were being recorded, 10 ␮M bicuculline
methiodide (Research Biochemicals, Natick, MA) was added to the
external solutions to block receptor-mediated GABAA currents arising
from autapses (Frerking et al. 1995; Gleason et al. 1993). In all
voltage-clamp experiments, tetrodotoxin (150 nM, Alomone Labs,
Jerusalem, Israel) was included in external solutions to block currents
through voltage-gated Na⫹ channels and TEA was included to block
voltage-gated K⫹ channels. Statistical analyses were done using the
paired t-test and are reported as means ⫾ SE. Unless otherwise
specified, all reagents were obtained from Sigma (St. Louis, MO). All
experiments were carried out at room temperature (24 –27°C).
Specificity of FCCP
Mitochondria buffer Ca2⫹ loads in amacrine cells
To determine whether mitochondrial Ca2⫹ uptake normally
plays a role in buffering Ca2⫹ loads in amacrine cells, cultured
amacrine cells were loaded with fluo-3, and fluorescence was
monitored at the cell body. Cytosolic Ca2⫹ elevations were
elicited by depolarizing the cells with high K⫹ external solution, and mean fluorescence intensity was sampled from cell
bodies. Most experiments were performed using 50 mM K⫹
external but the effects of 100 mM K⫹ external were examined
on a subset of amacrine cells. Under normal conditions, depolarization produced two kinetic classes of Ca2⫹ responses in
amacrine cells (Fig. 1, A–C). In response to 50 mM K⫹, all
cells exhibited an initial steep rise in cytosolic Ca2⫹ (n ⫽ 43).
After reaching a peak, calcium concentration began to decline
quickly; however, some cells (7%) had a second, slower plateau phase of recovery (Fig. 1, A and C). The plateau response
was more prevalent in cells depolarized by 100 mM K⫹
external (39%; n ⫽ 23).
To quantify the role of mitochondria in buffering amacrine
cell calcium loads, the peaks and durations of the Ca2⫹ elevations in response to depolarization were measured and compared in cells before and after the Ca2⫹ uniporter was blocked
by the addition of 1 ␮M FCCP. Only data from cells stimulated
with 50 mM K⫹ for 10 s were included in these analyses. The
duration of the response was measured at half height to exclude
the plateau phase data in the measurements. When mitochondrial Ca2⫹ uptake was impaired, we observed a significantly
longer duration of Ca2⫹ elevations in response to FCCP (Fig.
1, A and B; normal, 47.1 ⫾ 5.0 s; FCCP, 72.3 ⫾ 6.6 s; n ⫽ 42;
P ⬍ 0.0006). In amacrine cells that normally produced a
plateau phase, inhibition of mitochondrial Ca2⫹ uptake always
2⫹
FIG. 1. Mitochondria buffer Ca
elevations induced by depolarization. Cells were
loaded with fluo-3 Ca2⫹ indicator and images
collected every 5 s. A–D: vertical arrows denote
initiation of high K⫹ application and gray boxes
denote duration of p-trifluoromethoxy-phenylhydrazone (FCCP) application. A: a 10-s application of 100 mM K⫹ generates a cytosolic
Ca2⫹ elevation. After the initial rapid clearance
of calcium, a plateau phase of Ca2⫹ concentration is observed. In the presence of 1 ␮M
FCCP, the initial clearance of calcium is slowed
and the plateau phase is lost. B: in a different
cell from A, application of 50 mM K⫹ for 10 s
produces a Ca2⫹ transient with no plateau
phase. In the presence of FCCP, recovery to
baseline Ca2⫹ levels is slowed. Note that in this
cell, an FCCP-dependent Ca2⫹ increase is observed (arrow) prior to depolarization. C: addition of FCCP immediately after a 30-s application of 100 mM K⫹ produces a plateau phase in
a cell that did not exhibit a pronounced plateau
phase in response to depolarization alone. D: a
35-s application of FCCP produces a transient
increase in cytosolic Ca2⫹ in the absence of any
previous depolarization. A subsequent 10 s application of 50 mM K⫹ also produces a Ca2⫹
elevation.
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We chose to use FCCP for these experiments because it is fast
acting and rapidly reversible and its effects are well characterized.
Importantly, both carbonyl cyanide m-chlorophenylhydrazone
(CCCP) and FCCP have been demonstrated to inhibit mitochondrial
Ca2⫹ elevations in intact cells (Boitier et al. 1999; David et al. 1998;
Herrington et al. 1996; Ricken et al. 1998). Although there is evidence
that prolonged exposure (⬎10 min) of these compounds can have
secondary effects in invertebrate neurons (Jensen and Rehder 1991),
in numerous other preparations, brief exposures to low concentrations
(1–5 ␮M) have effects on cytosolic Ca2⫹ consistent with perturbation
of mitochondrial function exclusively (Babcock et al. 1996; David and
Barrett 2000; David et al. 1998; Friel and Tsien 1994; Herrington et
al. 1996; Scotti et al. 1999; Werth and Thayer 1994; White and
Reynolds 1995, 1997). Furthermore, we have tested the effect of
pretreatment of cells with antimycin A1(1 ␮M), an electron transport
chain inhibitor, and oligomycin (1 ␮M), an ATPsynthase inhibitor.
We found that in all cells tested (n ⫽ 9), this pretreatment occluded
the FCCP-dependent Ca2⫹ elevations that normally occur in 78% of
these cells (see RESULTS) indicating that the effects of FCCP on
cytosolic Ca2⫹ are due to its action on mitochondria exclusively
(Colegrove et al. 2000; David and Barrett 2000).
RESULTS
ROLE OF MITOCHONDRIA IN AMACRINE CELLS
Relatively small Ca2⫹ loads can be buffered by
mitochondria
With these measurements of cytosolic Ca2⫹, we have established that mitochondrial Ca2⫹ buffering plays a role in clearing cytosolic Ca2⫹ elevations in response to prolonged (ⱖ10 s)
depolarizations. In amacrine cells, we have the opportunity to
measure Ca2⫹ influx through voltage-gated Ca2⫹ channels and
to compare that to subsequent efflux on the plasma membrane
Na-Ca exchanger (Gleason et al. 1995). Thus it is possible to
determine whether mitochondria normally remove a fraction of
a Ca2⫹ load imposed by relatively brief activation of voltagegated Ca2⫹ channels by asking whether the fraction of the load
removed by the exchanger is larger when mitochondrial Ca2⫹
uptake is inhibited. For these experiments, cells were voltage
clamped in the perforated-patch configuration and depolarized
from ⫺70 to 0 mV for 700 ms. To estimate the fraction of the
load removed by the exchanger, the inward current was integrated during the voltage step and during the subsequent exchange current (for an example, see Fig. 8B). The ratio of the
charge moved on the exchanger to the charge entering during
the voltage step was calculated (n ⫽ 7). As previously reported
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(Gleason et al. 1995), under control conditions, the fraction of
a load removed by the exchanger varied considerably from cell
to cell, ranging in value from 25 to almost 100%. For three of
seven cells analyzed, blocking mitochondrial Ca2⫹ uptake
shifted the normal balance and increased the fraction of the
load removed by the Na-Ca exchanger (by 15, 42, and 70%,
respectively). It is probably significant that the three cells
whose ratios were shifted were also the three cells with the
lowest control ratios, indicating that the plasma membrane
Na-Ca exchanger normally played less of a role in these cells.
These data suggest that in a subset of amacrine cells, mitochondrial Ca2⫹ buffering normally contributes to Ca2⫹ handling on this time frame.
Depolarization-induced Ca2⫹ current inactivation limits the
amplitude of high-K⫹-dependent Ca2⫹ elevations
In our initial examination of the effects of inhibiting mitochondrial Ca2⫹ uptake, we found that depolarization in the
presence of FCCP did not significantly increase the amplitude
of Ca2⫹ elevations. This was unexpected because we would
predict that normally, mitochondrial Ca2⫹ uptake would be
removing some fraction of the Ca2⫹ entering while Ca2⫹
concentration is reaching its peak amplitude and that blocking
this uptake would increase the amplitude of the Ca2⫹ elevation.
However, a confounding factor for these cells is that the first
high-K⫹ depolarization almost always produces a larger response than a second high-K⫹ depolarization in these cells,
even in the absence of FCCP application (50 mM K⫹, 24 ⫾
0.76% reduction, n ⫽ 10; 100 mM K⫹, 50 ⫾ 0.05% reduction,
n ⫽ 10). This is may be due to the expression of primarily
L-type voltage-gated Ca2⫹ channels in these cells that are
susceptible to Ca2⫹-dependent inactivation. To examine this
possibility, we attempted to mimic ten second applications of
50 and 100 mM K⫹ with a “test depolarization” in an electrophysiology experiment so that we could observe the effects of
this sort of depolarization on the Ca2⫹ current amplitude.
Using ruptured patch whole cell recordings in the currentclamp configuration, we recorded the voltage changes engendered by either 50 or 100 mM K⫹ external solution. The
averaged voltage responses to high K⫹ were used to establish
the time course of the voltage excursions. Amplitude information from these recordings was disregarded because it is unlikely that we could mimic the conditions of an intact cell in
this recording configuration. Instead we used peak amplitudes
of ⫺30 and ⫺10 mV to approximate the predicted values
derived from calculations of EK in 50 and 100 mM K⫹ externals. The time course and predictions of voltage amplitude
were used to construct the waveform of the test depolarization
shown in Fig. 2C. To determine whether amacrine cell Ca2⫹
currents were being inactivated by pulses of high external K⫹,
Ca2⫹ currents were recorded in the perforated-patch configuration before and after the test depolarization. Ca2⫹ currents
were elicited by a voltage step from ⫺70 to ⫺10 mV for 250
ms (Fig. 2A). These voltage steps were applied once every 60 s.
Thirty seconds after the second voltage step, the test depolarization (mimicking high K⫹ application) depicted in Fig. 2C
was applied. Following this, the Ca2⫹ current was retested
(once every 60 s) for ⱖ7 min to encompass the time span
normally separating high K⫹ pulses in the fluo-3 experiments.
As shown in Fig. 2B, the mean peak Ca2⫹ current amplitudes
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removed that component of the response (Fig. 1A). This is
consistent with the plateau phase being generated by release of
Ca2⫹ from mitochondria subsequent to uptake via the
uniporter. In cells without a plateau phase, the response duration increased, and no other consistent effect on the waveform
of the response was observed (Fig. 1B). In contrast to observations in other neurons (Friel and Tsien 1994; Thayer and
Miller 1990; Werth and Thayer 1994), peak response amplitudes were not significantly different in the presence of FCCP
[control, 183.6 ⫾ 7.8% (of baseline values); FCCP, 189.6 ⫾
8.0%, P ⫽ 0.14; n ⫽ 41]. The underlying cause for this
difference will be explored in a later section.
If mitochondria normally take up Ca2⫹ during depolarization, then application of FCCP after the depolarization should
impair uniporter activity and shift the balance between uptake
and release, resulting in a cytosolic calcium elevation. When
FCCP was added after the depolarization, during the recovery
phase, either generation or enhancement of a plateau phase was
produced in all cells examined (Fig. 1C; n ⫽ 57). These results
indicate that in response to depolarization, mitochondria in
amacrine cells can sequester a significant amount of Ca2⫹ and
that with some Ca2⫹ loads, subsequent release of mitochondrial calcium can have a substantial and long-lasting effect on
cytosolic Ca2⫹ levels in these cells.
In some cells, an increase in Ca2⫹ levels was detectable in
the presence of FCCP before addition of the K⫹ stimulus (Fig.
1B; arrow), indicating that inhibition of the mitochondrial
uniporter alone results in cytosolic Ca2⫹ elevations. To test
this, previously unstimulated cells were exposed to FCCP. By
itself, FCCP generated cytosolic Ca2⫹ increases in 78% of
cells examined (Fig. 1D; n ⫽ 51). On average, the peak
amplitudes of FCCP-dependent Ca2⫹ elevations were 43.0%
(⫾9.2; n ⫽ 23) of the peak amplitudes of Ca2⫹ elevations
produced in the same cell by depolarization with 50 mM K⫹.
These results imply that under resting conditions, mitochondria
store a measurable amount of Ca2⫹ and/or that a constant leak
of Ca2⫹ into the cytosol is normally offset by mitochondrial
uniporter activity.
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K. MEDLER AND E. L. GLEASON
were initially reduced by ⬃50 and 70% by depolarizations to
⫺30 and ⫺10 mV, respectively. The degree of inhibition
declined with time, but even 7 min after the test depolarization,
the effects on the Ca2⫹ current amplitudes were substantial
(⫺30 mV protocol, ⬃14% inhibited; ⫺10 mV protocol, ⬃25%
inhibited). These results indicate that Ca2⫹ channel inactivation has the potential to mask an increase in amplitude of
depolarization-dependent Ca2⫹ elevations in the presence of
FCCP. Although the magnitude of Ca2⫹ influx is not the only
factor determining free cytosolic Ca2⫹ concentration, we used
our data on the time course of Ca2⫹ current inactivation to
generate a correction factor to offset the effect of inactivation
on the amplitude of the Ca2⫹ elevation. With this manipulation, we estimated what probably represents the upper limit of
the role of mitochondrial Ca2⫹ uptake in determining the peak
Ca2⫹ elevation engendered by depolarization. Using the value
for sustained inhibition (average of last 3 data points for Ca2⫹
amplitude after 50 mM K⫹), we scaled the data collected with
50 mM K⫹ stimulation in the presence of FCCP. Re-analysis
with scaled amplitude values gave FCCP values significantly
larger than control (normal, 183.6 ⫾ 7.8%; FCCP, 216.16 ⫾
9.2%, P ⬍ 0.0001, n ⫽ 41) suggesting that mitochondrial Ca2⫹
J Neurophysiol • VOL
buffering normally does limit the amplitude of depolarizationinduced Ca2⫹ elevations.
Inhibition of the mitochondrial uniporter stimulates Na-Ca
exchange in amacrine cells
Having established that mitochondrial Ca2⫹ buffering can
shape the time course of Ca2⫹ elevations in amacrine cells,
electrophysiological methods were used to examine whether
Ca2⫹-dependent processes were also under the influence of
mitochondrial function. Initial recordings from single, isolated
amacrine cells revealed that application of 1 ␮M FCCP usually
caused a reversible change in the holding current when cells
were held at ⫺70 mV (Fig. 3A). This current was not elicited
by DMSO alone (n ⫽ 4). Two possible sources of the inward
current were considered. One possibility was that FCCP either
directly or indirectly activated a plasma membrane conductance. The other possibility was that the electrogenic plasma
membrane Na-Ca exchanger (Gleason et al. 1995) was being
activated by the FCCP-dependent increases in cytosolic Ca2⫹
that were seen for most cells in the Ca2⫹ measurement experiments.
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FIG. 2. Ten-second depolarizations have
long-term inhibitory effects on calcium current amplitudes. A: data from a single amacrine cell recorded in the perforated-patch
voltage-clamp configuration. The cell was
held at ⫺70 mV and stepped to ⫺10 mV for
250 ms once every minute. Between a and b,
the test depolarization depicted in C was applied to the cell to mimic application of high
K⫹. a– c: obtained at the time points indicated in B. B: the time course for the changes
in peak current amplitude is shown. ●, averaged data from 5 cells that were depolarized
to ⫺30 mV with the protocol in C. ■, averaged data from 4 cells that were depolarized
to ⫺10 mV with a protocol similar to the one
shown in C. C: the voltage protocol applied
and the inward current elicited in the same
cell as shown in A.
ROLE OF MITOCHONDRIA IN AMACRINE CELLS
1431
To determine whether the plasma membrane Na-Ca exchanger generated this FCCP-dependent current, the effect of
replacing external Na⫹ with Li⫹, a manipulation well established to inhibit exchanger function, was examined. Replacement of external Na⫹ with Li⫹ reversibly abolished this inward
current (Fig. 3B) in all cells examined (n ⫽ 15). On return to
normal (Na⫹-containing, FCCP-free) external solution, we observed a transient increase in inward current before it returned
to baseline values. The appearance of a transient current is
consistent with the recovery of normal cytosolic Ca2⫹ levels
once the plasma membrane Na-Ca exchanger has been reenabled. These results strongly suggest that the FCCP-dependent inward current was due to the electrogenic activity of the
plasma membrane Na-Ca exchanger.
If the FCCP-dependent current is due to the activity of the
Na-Ca exchanger working to transport Ca2⫹ originating from
the inside of the cell, then this current should persist in the
absence of external Ca2⫹. In 0 mM Ca2⫹ external solution and
with no previous Ca2⫹ load imposed, the FCCP-dependent
inward current was still observed (Fig. 3C). When the FCCPdependent current amplitude was measured in both normal and
J Neurophysiol • VOL
in Ca2⫹-free external in the same cell, there was only a 24 ⫾
10.5% (n ⫽ 5) reduction in current amplitude in the absence of
external Ca2⫹. The persistence of this current in the absence of
extracellular Ca2⫹ indicates that external Ca2⫹ is not the primary source of Ca2⫹contributing to the production of the
current. Additionally, blockade of voltage-gated Ca2⫹ channels
specifically with 200 ␮M Cd2⫹ was ineffective in reducing
the amplitude of the FCCP-dependent current (not shown;
FCCP, 28.3 ⫾ 5.0 pA; FCCP/Cd2⫹, 27.8 ⫾ 5.6 pA; n ⫽ 4, P ⫽
0.7).
These data lead to two predictions that could be tested with
Ca2⫹ measurements. First, because the inward current can
remain above baseline for minutes in the continued presence of
FCCP, we would expect that prolonged (⬎2 min) applications
of FCCP would produce sustained rather than transient Ca2⫹
elevations in amacrine cells. This was tested by applying FCCP
to cells for 2.5 min and measuring cytosolic Ca2⫹. Although
the amplitude of the Ca2⫹ elevation declined in many cells
over the 2.5-min period, Ca2⫹ did remain well above basal
levels for the duration of the application in all cells examined
(n ⫽ 19; Fig. 3D). The persistence of the FCCP-induced Ca2⫹
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2⫹
FIG. 3. Inhibition of mitochondrial Ca
uptake activates the plasma membrane
Na-Ca exchanger and reveals Ca2⫹ efflux
from internal sources. A: a whole cell, perforated-patch recording from a representative isolated amacrine cell. Application of 1
␮M FCCP induces an inward current that is
sustained over the duration of FCCP application. B: in a different cell from A, the
FCCP-dependent inward current is elicited
by 1 ␮M FCCP. This current is abolished by
subsequent replacement of external Na⫹
with Li⫹. The dependence of the inward
current on external Na⫹ is consistent with
the electrogenic activity of the plasma membrane Na-Ca exchanger. On removal of
FCCP and replacement of Li⫹ with Na⫹, the
inward current rebounds before returning to
baseline. C: data from another isolated amacrine cell for which external Ca2⫹ was removed 30 s prior to application of FCCP.
The presence of the FCCP-dependent Na-Ca
exchange current is not dependent on the
influx of Ca2⫹ across the plasma membrane.
D: in the absence of any previous stimulus,
application of 1 ␮M FCCP stimulates an
elevation in cytosolic Ca2⫹. Prolonged application of FCCP (2.5 min) generates a
Ca2⫹ elevation that is sustained well above
baseline over the duration of exposure to
FCCP. E: data are shown for a different
amacrine cell that is first stimulated with
FCCP (1.5 min) in normal external solution.
After the initial Ca2⫹ transient, the external
solution was changed to Ca2⫹-free external.
Subsequent application of FCCP (1.5 min)
also produced a Ca2⫹ elevation, indicating
an intracellular Ca2⫹ source. The 2nd response was generally smaller that the first
regardless of whether it was elicited in normal external or 0 mM Ca2⫹ external. F: data
from a different cell in an experiment similar
to that shown in B but where the first response was elicited in 0 Ca2⫹.
1432
K. MEDLER AND E. L. GLEASON
Ca2⫹ elevations are not due to ATP depletion
Because FCCP dissipates the proton gradient across the
inner mitochondrial membrane, ATP production via oxidative
phosphorylation is also inhibited and, under these conditions,
the ATPsynthase will consume ATP. It has been demonstrated
in several neuronal cell types that the glycolytic pathway can
maintain the ATP/ADP ratio for tens of minutes (Kauppinen
and Nicholls 1986; Peng 1998; Werth and Thayer 1994; White
and Reynolds 1995). Nonetheless, it was necessary to establish
that the FCCP-dependent Ca2⫹ elevations we were detecting
were not due to loss of ATP and inhibition of the Ca2⫹
ATPases. Oligomycin was used to directly inhibit the activity
of the mitochondrial ATPsynthase. FCCP and oligomycin were
co-applied to see whether, in the absence of ATP consumption,
an FCCP-dependent rise in cytosolic Ca2⫹ would be observed
(Colegrove et al. 2000). All cells producing Ca2⫹ elevations in
response to FCCP produced similar Ca2⫹ elevations in response to FCCP in the presence of oligomycin (n ⫽ 21; Fig.
4A). To address whether inhibition of the ATPsynthase alone
would generate Ca2⫹ elevations, oligomycin was applied to
cells for 3 min (Fig. 4B). In these experiments, oligomycindependent Ca2⫹ elevations were never observed (n ⫽ 21),
indicating that on the time frame of our experiments, ATP
depletion alone was not sufficient to generate an elevation in
cytosolic Ca2⫹. As an additional control, we recorded from
amacrine cells in the ruptured-patch configuration with an
internal solution containing 4 mM ATP and an ATP regeneration system. Under these conditions, all cells examined still
exhibited the FCCP-dependent Na-Ca exchange current (n ⫽
6, not shown) indicating that ATP depletion is not the source of
this current.
Inhibition of mitochondrial Ca2⫹ buffering stimulates
exocytosis
We have demonstrated that disruption of mitochondrial
Ca2⫹ uptake produces Ca2⫹ elevations in amacrine cells; howJ Neurophysiol • VOL
2⫹
FIG. 4. FCCP-dependent Ca
elevations are not due to ATP depletion. A:
a previously unstimulated amacrine cell was exposed to FCCP in the presence
of 1 ␮M oligomycin to determine if the rise in Ca2⫹ was due to ATP
consumption by the ATP synthase. Even when the synthase was inhibited by
oligomycin, FCCP still evoked an elevation in cytosolic Ca2⫹ in all cells tested
(n ⫽ 21). Further stimulation with FCCP alone indicates that cells can produce
a similar response to FCCP in the presence or absence of oligomycin. B: to
determine if inhibition of ATP production alone produces an elevation in
cytosolic Ca2⫹, amacrine cells were exposed to oligomycin only for 3 min.
Over that time period, no elevation of calcium was seen, although subsequent
cytosolic Ca2⫹ elevations were elicited in response to FCCP in all cells tested
(n ⫽ 21). These data indicate that the FCCP-dependent Ca2⫹ elevations are not
due to inhibition of ATP-dependent processes.
ever, because the imaging data were collected from cell bodies
only and because the FCCP-dependent Na-Ca exchange current was recorded from the whole cell, these experiments do
not address any site-specific mitochondrial function in amacrine cells. Specifically, we wanted to know if mitochondria
were important at synaptic release sites, so we asked whether
FCCP-induced Ca2⫹ elevations could stimulate exocytosis. We
recorded from amacrine cells in contact with three to five other
unclamped amacrine cells (Fig. 5). Previous studies have demonstrated that after ⬃8 days in culture, amacrine cells can
make functional Ca2⫹-dependent GABAergic synapses with
themselves (autapses) and with other amacrine cells that employ postsynaptic GABAA receptors (Frerking et al. 1995;
Gleason et al. 1993). When cells were held at 0 mV, synaptic
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elevation indicates that the mitochondria themselves are probably not the sole source of the Ca2⫹.
A second prediction from our electrophysiology experiments
would be that FCCP-dependent Ca2⫹ elevations could be generated in zero external Ca2⫹. For these experiments, FCCP was
applied in the presence and absence of external Ca2⫹ (Fig. 3,
E and F). Seven of nine cells examined produced Ca2⫹ elevations in the absence of external Ca2⫹. The lack of response in
two of the cells is probably due to the loss of Ca2⫹ from
internal stores that can occur fairly quickly when these cells are
bathed in Ca2⫹-free external (not shown). The smaller amplitude of the FCCP response in zero Ca2⫹ shown in Fig. 3E does
not necessarily reflect a large contribution from extracellular
Ca2⫹ because when the zero Ca2⫹ responses were measured
first, they were often (5 of 8 cells, Fig. 3F) larger than the
responses elicited in normal external Ca2⫹. These results support a scenario where in the absence of uniporter activity,
cytosolic Ca2⫹ concentration rises and stimulates an efflux of
Ca2⫹ via the plasma membrane Na-Ca exchanger. Furthermore, the persistence of the cytosolic Ca2⫹ elevation in the
absence of external Ca2⫹ indicates that the source of Ca2⫹ is
largely internal.
ROLE OF MITOCHONDRIA IN AMACRINE CELLS
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2⫹
FIG. 5. Inhibition of mitochondrial Ca
buffering stimulates exocytosis. A: a recording from a single amacrine cell in contact with
other, unclamped amacrine cells. The cell was recorded with internal solution B and held at 0 mV. Prior to application of FCCP, quantal
events are detectable and most likely arise from autaptic input stimulated by influx through voltage-gated Ca2⫹ channels. In the presence
of FCCP, no inward current is generated which is consistent with the voltage-dependence of the Na-Ca exchanger. After FCCP
application, the frequency of quantal events increases dramatically. Inset: a segment of the current (denoted by dotted lines) displayed in
A. B: recording from another amacrine cell contacting other unclamped amacrine cells. This cell was held at ⫺70 mV with solution B
in the pipette. At ⫺70, quantal events were rare but in the presence of FCCP, the inward exchange current develops and quantal events
appear. Inset: a segment of data from this recording displayed on a longer time scale. These results indicate that in the absence of
mitochondrial Ca2⫹ buffering, cytosolic Ca2⫹ levels increase in the presynaptic cells and exocytosis is either enhanced (A) or initiated
(B), depending on the holding potential of the cell. Note that most of the delay for the increase in exchanger current is due to the dead
space in the perfusion system (see METHODS). C and D: for this analysis, a data set from an amacrine cell connected to other, unclamped
amacrine cells was selected for analysis on the following criteria. First, quantal events in normal solution had to be detectable but not so
frequent that they were indistinguishable from one another. Second, a substantial Na-Ca exchange current had to be present (ⱖ10 pA)
to avoid counting events during solution changes. These data were recorded at ⫺40 mV with solution A internally. C: peaks of quantal
events were manually identified and counted from 20 discontinuous traces (5 s in duration) in normal external and from 15 traces in FCCP.
*, a statistical difference (P ⫽ 0.0007, unpaired t-test) between normal and FCCP quantal frequency. D: peak quantal current amplitudes
were measured from these same recordings. Peaks were measured only if the onset of the quantal current began at baseline to avoid
considering multiple, nearly coincident quantal events as one. These data indicate that although the frequency of quantal events is
increased in FCCP, their amplitude distribution is unchanged.
1434
K. MEDLER AND E. L. GLEASON
J Neurophysiol • VOL
2⫹
FIG. 6. Cytolosic Ca
elevations elicited by FCCP are local. Measurements of fluorescence intensity over time are shown for 2 fluo-3-loaded
amacrine cells. Data were collected with a sampling rate of 28 Hz. A: a
representative example of recordings from an amacrine cell body and one of its
processes (*). FCCP was applied just prior to the beginning of the record and
remained on for 1 min. Inset: the fluorescence data from the process have been
scaled to the average of the values during the first 4 s of the recording at the
cell body. 䡠 䡠 䡠 , the baseline. The increases in fluorescence begin at the same
time in both the cell body and the process. B: fluorescence measurements from
a different amacrine cell body and process (*). FCCP application is the same
as in A. In this cell, the FCCP produced an elevation in the cell body but not
at the site recorded from in the process. The data in A and B have been
smoothed with 10 point adjacent averaging for display purposes.
and do not propagate. Given these results, we favor the hypothesis that mitochondria normally maintain low Ca2⫹ at
presynaptic terminals and that disruption of this mechanism
leads to exocytosis.
Inhibition of mitochondrial Ca2⫹ uptake alters evoked
synaptic transmission
The role of mitochondrial Ca2⫹ buffering during evoked
synaptic transmission was examined by recording from isolated pairs of amacrine cells. For these experiments, both cells
of a pair were voltage clamped and held at ⫺70 mV. The
presynaptic cell was depolarized to ⫺10 mV by a 100-ms
voltage step. In the presence of FCCP, the postsynaptic currents were reversibly reduced in amplitude (Fig. 7A). Because
the currents are so noisy, a useful measure of synaptic transmission is to integrate the postsynaptic current to measure the
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currents (presumably mostly autaptic) were usually observed
(Fig. 5A). In these experiments, the Cl⫺ equilibrium potential
is estimated to be about ⫺57 mV (internal solution B), and
accordingly, the synaptic currents were outward at 0 mV.
Exposure to FCCP generated a dramatic increase in synaptic
activity. At 0 mV, no FCCP-dependent inward currents were
seen; consistent with the voltage dependence of the Na-Ca
exchanger previously described for amacrine cells (Gleason et
al. 1995). When cells were held at ⫺70 mV, quantal events
were rare until FCCP was applied (Fig. 5B). For these experiments, a high Cl⫺ internal solution (solution A) was used that
gave a calculated Cl⫺ equilibrium potential of ⫺2 mV and thus
inward synaptic currents at ⫺70 mV. As expected from previous experiments, application of FCCP at this holding potential was accompanied by the inward Na-Ca exchange current.
The increase in synaptic activity followed the development of
the Na-Ca exchange current as would be expected if this
current is tracking cytosolic Ca2⫹ levels near release sites.
Another consequence of disrupting mitochondrial function is
that acidification of the cytosol can occur, which has been
shown to stimulate Ca2⫹-independent exocytosis (Drapeau and
Nachshen 1988). To address this possibility, experiments were
also conducted on cells loaded with 10 ␮M BAPTA-AM to
minimize Ca2⫹ elevations. With BAPTA-loaded cells, FCCP
failed to elicit exocytosis, indicating that the release stimulated
by FCCP was Ca2⫹ dependent (n ⫽ 4, not shown).
To determine if the effects of FCCP were entirely presynaptic, we analyzed the effects of FCCP on GABA-gated current amplitude. GABA-gated (100 ␮M) whole cell currents
were not significantly affected by FCCP (normal, ⫺75.4 ⫾ 6.7
pA; FCCP, ⫺70.2 ⫾ 5.8 pA, P ⫽ 0.18, n ⫽ 5). To quantify the
effects of FCCP on postsynaptic GABAA receptors specifically, we analyzed quantal events recorded from an amacrine
cell connected to other unclamped amacrine cells. The cell that
provided the data in Fig. 5 (C and D) was selected for analysis
because quantal events were frequent enough in normal external to generate a reasonable sample size but not so frequent as
to preclude accurate measurements of individual quanta. First
we confirmed that the frequency of the quantal events increased significantly in the presence of FCCP (Fig. 5C; P ⫽
0.0007). We then analyzed the amplitude distribution of the
quantal events and found that the distribution was unaffected
by the presence of FCCP (Fig. 5D). These results indicated that
the effects of FCCP were presynaptic.
These results indicate either that mitochondria normally
keep Ca2⫹ concentration low at release sites or that in the
absence of mitochondrial Ca2⫹ buffering, Ca2⫹ diffuses from
elsewhere, possibly the cell body, out to active sites on the
processes. To discriminate between these two possibilities, we
collected fluorescence intensity data from cell bodies and multiple sites in the processes of six amacrine cells. In all cases,
perfusion of FCCP generated Ca2⫹ elevations in cell bodies. In
some cells (3 of 6, Fig. 6A), Ca2⫹ elevations were also seen at
all of the sites recorded in the processes. The other three cells
had both responding and nonresponding sites (Fig. 6B). Comparisons of the time courses of Ca2⫹ elevations in cell bodies
and processes indicate that Ca2⫹ elevations in processes do not
follow those measured in at the cell body but instead are
initiated coincidently (Fig. 6A, inset). This, as well as the
absence of Ca2⫹ elevations in some sites in processes, provides
evidence that FCCP-dependent Ca2⫹ elevations occur locally
ROLE OF MITOCHONDRIA IN AMACRINE CELLS
1435
charge transferred at the synapse. The postsynaptic currents at
these synapses normally exhibit considerable variability in
peak amplitude and duration (Gleason et al. 1993). Because of
this intrinsic variability, we attempted to apply FCCP multiple
times to each cell pair to confirm our observations. In one pair
of amacrine cells, we were able to apply FCCP four times and
on each occasion, the charge transfer was reduced (Fig. 7B).
The effect of FCCP was examined on six cell pairs and in each
case, we observed a reduction in the postsynaptic currents. On
average, inhibition of the mitochondrial uniporter significantly
reduced the charge transfer at the synapse (Fig. 7C; P ⫽
0.0001). These results indicate that although loss of mitochondrial Ca2⫹ buffering can increase cytosolic Ca2⫹ and provoke
exocytosis in an unstimulated cell, inhibition of this same
process has the opposite effect during evoked synaptic transmission.
If inhibition of Ca2⫹ uptake into mitochondria was the sole
outcome of FCCP application, then we might expect that in the
presence of FCCP, presynaptic Ca2⫹ levels would be enhanced
and that the charge transfer during synaptic transmission would
be augmented rather than diminished during synaptic transmission. One implication of this inhibitory effect of FCCP is that
FCCP is either directly or indirectly inhibiting some other
aspect of synaptic function.
Inhibition of mitochondrial Ca2⫹ uptake inhibits
voltage-gated Ca2⫹ currents
These amacrine cells primarily express L-type voltage-gated
Ca2⫹ channels that have been previously demonstrated to
control evoked synaptic transmission between these cells
(Gleason et al. 1993). Because it is well established that L-type
Ca2⫹ channels undergo Ca2⫹-dependent inactivation (Imredy
and Yue 1992, 1994; Peterson et al. 1999; Zong et al. 1994),
we considered the possibility that the Ca2⫹ elevations provoked by inhibition of mitochondrial Ca2⫹ uptake might be
sufficient in magnitude to contribute to Ca2⫹-dependent inactivation of the channels. Unfortunately, recordings of presynaptic Ca2⫹ currents from pairs of amacrine cells during synaptic transmission are often contaminated by autaptic currents,
J Neurophysiol • VOL
thus compromising accurate measurements of current amplitude. However, in those amacrine cell pairs that had low levels
of autaptic currents, FCCP-dependent decreases in presynaptic
Ca2⫹ current amplitude were discernable. To confirm this,
Ca2⫹ currents were recorded from single, isolated amacrine
cells in the presence of 10 ␮M bicuculline to block GABAA
receptor activation at autapses during depolarization. For initial
experiments, cells were stepped from ⫺70 to 0 mV for 500 ms
to activate voltage-gated Ca2⫹ channels. In all cases, application of FCCP produced inhibition of the voltage-dependent
Ca2⫹ current (38 ⫾ 8%, n ⫽ 8).
Inhibition of mitochondrial Ca2⫹ uptake also inhibits
Ba2⫹ currents
It is well known that Ba2⫹ carries a larger current through
L-type Ca2⫹ channels than Ca2⫹ does but is a poor substitute
for Ca2⫹ on the plasma membrane Na-Ca exchanger. As expected, when Ba2⫹ was present externally, the voltage-dependent inward current was larger than in external Ca2⫹. However,
the FCCP-dependent plasma membrane Na-Ca exchange current was not significantly enhanced (Ca2⫹, 45.3 ⫾ 8.9 pA;
Ba2⫹, 41.0 ⫾ 6.5 pA; n ⫽ 4; P ⫽ 0.36; Fig. 8). This observation agrees with our experiments in Cd2⫹ and provides
additional evidence that the FCCP-dependent inward current is
not due to the flux of Ca2⫹ through L-type Ca2⫹ channels. In
the presence of FCCP, the voltage-dependent inward Ba2⫹
current was also inhibited (Fig. 8, A and C), consistent with the
hypothesis that cytosolic Ca2⫹ elevations are largely derived
from internal stores and can be sufficient to inhibit these
channels.
Under normal conditions, when Ca2⫹ enters during a voltage
step, the charge transferred during the subsequent plasma
membrane Na-Ca exchange tail current is directly related to the
charge transferred during the preceding voltage-dependent
Ca2⫹ current. Accordingly, the Na-Ca exchange tail current in
FCCP is smaller (after subtracting the FCCP-dependent component) than the Na-Ca exchange tail current in normal external (Fig. 8B). In external Ba2⫹, the Na-Ca exchange tail current
is nearly abolished because Ba2⫹ is not easily transported on
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2⫹
FIG. 7. Inhibition of mitochondrial Ca
buffering affects the
magnitude of evoked synaptic transmission. A: postsynaptic currents recorded from an isolated pair of voltage-clamped amacrine
cells. The presynaptic cell is held at ⫺70 mV and stepped to ⫺10
mV for 100 ms. The postsynaptic cell is held at ⫺70 mV with
internal solution A in the pipette. The FCCP-sensitive exchange
current has been subtracted so that the evoked currents can be
compared. In the presence of FCCP, the postsynaptic currents are
reduced. B and C: because the postsynaptic currents are noisy and
can be quite variable, the effect of FCCP was quantified by
measuring the total charge of the postsynaptic current measured
from the beginning of the presynaptic voltage step to the end of the
record (⬃1.2 s) and normalizing this to the 1st record in normal
external. In B, the normalized charge transfer is plotted over time
for a cell pair that was recorded for ⬃15 min and had 4 exposures
to FCCP (1). Although the amount of charge transferred at the
synapse was quite variable, FCCP was consistently inhibitory. C:
normalized data for 6 cell pairs where postsynaptic currents recorded just prior to FCCP application are compared with postsynaptic currents recorded during FCCP application and for 4 cell
pairs after washout of the FCCP. The normalization precludes a
statistical comparison between normal and FCCP data but a comparison between FCCP and wash data shows them to be significantly different (P ⫽ 0.0002, unpaired t-test).
1436
K. MEDLER AND E. L. GLEASON
the exchanger. However, in the presence of FCCP, although
the inward Ba2⫹ current is reduced, the Na-Ca exchange tail
current is actually enhanced (Fig. 8D). This observation is
consistent with FCCP-dependent Ca2⫹ accumulation (probably
leakage from stores) occurring during the 500-ms voltage step
to 0 mV when the Na-Ca exchanger is not working to transport
Ca2⫹ out of the cell.
Ca2⫹ influx can influence the magnitude and time course of
the FCCP-dependent inhibition of Ca2⫹ currents
To test the hypothesis that inhibition of mitochondrial Ca2⫹
uptake enhances Ca2⫹-dependent inactivation of Ca2⫹ current,
we looked at the effects of FCCP over time with two different
Ca2⫹-loading conditions. Cells were depolarized to 0 mV for
either 100 or 700 ms. The onsets of the voltage step protocols
were separated by 1 min. Regardless of the duration of the
voltage step, the Ca2⫹ current was always suppressed in the
presence of FCCP (Fig. 9; n ⫽ 15), and this effect persisted for
minutes after FCCP was washed out of the bath. The duration
of the effect indicated that the inhibition of the current is not a
direct effect of FCCP on the channels because we know from
our other experiments that FCCP washes out of the cells within
seconds. When cells were depolarized for 100 ms, the effect of
FCCP took longer to develop, was less pronounced, and had a
shorter duration than when cells were depolarized for 700 ms
(Fig. 9C). Thus the recent history of Ca2⫹ influx (previous
control voltage steps) determined the magnitude and the time
course of the FCCP effect, providing further evidence that
cytosolic Ca2⫹ was mediating the inhibition. Control experiJ Neurophysiol • VOL
ments done on the same time frame, either without any treatment (n ⫽ 3) or with pulses of 0.01% DMSO alone (n ⫽ 4), did
not produce decreases in Ca2⫹ current amplitude (not shown).
These results do not rule out the possibility that mitochondria
normally play a role in shaping the time course of the presynaptic Ca2⫹ elevation during synaptic transmission. Instead,
they point to an additional role of mitochondria in helping to
offset the effects of Ca2⫹-dependent inactivation on the Ca2⫹
channels.
DISCUSSION
Mitochondria and the control of resting cytosolic Ca2⫹
Our results indicate that regulation of Ca2⫹-dependent processes in amacrine cells, including exocytosis and voltagegated Ca2⫹ channel function, depends on the Ca2⫹ uptake
capabilities of mitochondria. Measurements of cytosolic Ca2⫹
concentration demonstrated that in the absence of a previously
imposed Ca2⫹ load, FCCP stimulated a Ca2⫹ elevation in 78%
of amacrine cells examined. This observation suggests that
mitochondria store some Ca2⫹ at rest and that uniporter function is required to maintain this store. Because FCCP-dependent Ca2⫹ elevations are not usually transient in the continued
presence of FCCP, it is unlikely that the mitochondria themselves are the sole source of Ca2⫹. One possibility is that in
amacrine cells, mitochondrial Ca2⫹ uptake normally functions
to offset a leak of Ca2⫹ into the cytosol. If mitochondria do
normally offset a Ca2⫹ leak, then the relative insensitivity of
this leak to removal of extracellular Ca2⫹ implies that its
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2⫹
FIG. 8. Inward currents through voltage-gated Ca
channels are inhibited in FCCP. A–D: gray lines denote the current recorded
in the presence of FCCP. These recordings were made in the perforated-patch recording configuration. A: in normal external
containing 3 mM Ca2⫹, a 500-ms depolarization from ⫺70 to 0 mV produced an inward current followed by a prolonged tail
current that was due to the activity of the plasma membrane Na-Ca exchanger. In the presence of FCCP, the inward exchange
current is present and the Ca2⫹ current is reduced in amplitude. Offsets in baseline current here and in C are due to the
FCCP-dependent exchange current. B: the Na-Ca exchange tail current from A is shown. Here, the FCCP-dependent exchange
current has been subtracted from the tail recorded in FCCP so that the portion of the exchange current due to the preceding
depolarization could be isolated. The dotted line represents the level of the leak current in normal external. C: currents from the
same cell were recorded in external solution with 3 mM Ba2⫹ replacing Ca2⫹. As expected, the Ba2⫹ currents are larger than the
Ca2⫹ currents but in the presence of FCCP, the inward Na-Ca exchange current appears and the voltage-dependent Ba2⫹ current
is reduced in amplitude. D: the Na-Ca exchange tail currents are depicted as in B but were greatly reduced in external Ba2⫹. The
small tail currents that persist are presumably due to the accumulation of Ca2⫹ that occurs in the cytosol during the voltage step
when the exchanger is inactive. This effect is larger when mitochondrial Ca2⫹ uptake is inhibited with FCCP.
ROLE OF MITOCHONDRIA IN AMACRINE CELLS
1437
Mitochondria as activity sensors
2⫹
FIG. 9. FCCP-dependent Ca
current inhibition is enhanced by increased
Ca2⫹ influx. For these experiments Ca2⫹ currents were elicited at 1-min
intervals by depolarization from ⫺70 to 0 mV for either 100 (A) or 700 (B) ms.
Five identical voltage steps were delivered, separated by 5 s. Only data from
the 1st of these voltage steps are shown in this figure. A and B: Ca2⫹ currents
recorded from 2 different isolated amacrine cells. Recordings were made with
solution B internal. For both recordings, the FCCP-dependent exchange current has been subtracted from the currents recorded at ⫺70 mV but not during
the voltage steps to 0 mV when the exchanger is known not to function. C: the
effects of FCCP on peak Ca2⫹ current amplitude are shown over the time
course of a 9-min (100-ms steps, n ⫽ 6) or 13-min (700-ms steps, n ⫽ 9)
experiment. Approximately 1.5 min after removal of FCCP, the current amplitude began to recover; however, with this duration of FCCP application,
complete recovery was usually not achieved.
source is intracellular, possibly the endoplasmic reticulum
(ER). Previous studies on Ca2⫹ transport into the ER and
sarcoplasmic reticulum (SR) have demonstrated that elimination of trans-ER or -SR proton gradients with protonophores
do not inhibit the uptake of Ca2⫹ into these organelles (Bode
et al. 1994; Levy et al. 1990). These observations argue against
the possibility that there is a direct effect of FCCP on Ca2⫹
J Neurophysiol • VOL
Because of the glutamatergic input that amacrine cells receive from bipolar cells, amacrine cells in the intact retina are
potentially subjected to substantial and fairly long-lasting Ca2⫹
elevations. Two mechanisms have now been identified that
make a significant contribution to recovery from Ca2⫹ elevations in amacrine cells: the plasma membrane Na-Ca exchanger (Gleason et al. 1994) and mitochondria. One benefit of
using mitochondria as a Ca2⫹-buffering organelle would be
that a Ca2⫹ elevation in mitochondria increases the activity of
several mitochondrial dehydrogenases leading to an increase in
NADH levels (Rizzuto et al. 1994; Rutter et al. 1998). As
originally proposed by Denton and McCormack (1980), transmission of a Ca2⫹ signal to the mitochondria could allow
metabolic activity to be matched to the current energy demands
of the cell. Activation of glutamate receptors and depolarization produce an influx of Na⫹ as well as Ca2⫹. Transmission of
the Ca2⫹ signal to mitochondria could ensure that sufficient
ATP is available to power the Na-K ATPase, thus keeping Na⫹
levels in the cell low enough to maintain the Ca2⫹ export
activity of the Na-Ca exchanger.
Mitochondria at the amacrine cell synapse
Our results also demonstrate that mitochondria are important
regulators of cytosolic Ca2⫹ at the amacrine cell synapse. The
ability of FCCP to induce Ca2⫹-dependent exocytosis from
amacrine cells implies that normal mitochondrial function
serves to limit the rate of exocytosis in the absence of presyn-
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transport into the ER. Instead, we propose that FCCP causes a
release of mitochondrial Ca2⫹, and then, in the absence of
mitochondrial buffering, the leak of Ca2⫹ from the ER elevates
resting cytosolic Ca2⫹. In support of this hypothesis, preliminary experiments indicate that pretreatment of the cells with
thapsigargin reduces the duration of FCCP-dependent Ca2⫹
elevations (unpublished observations).
In other preparations, mitochondrial Ca2⫹ uptake does not
usually become significant until cytosolic Ca2⫹ levels reach
⬃500 nM (Friel and Tsien 1994; Herrington et al. 1996; Werth
and Thayer 1994). A recent study in sympathetic neurons,
however, demonstrated mitochondrial Ca2⫹ uptake at significantly lower (200 –300 nM) cytosolic Ca2⫹ concentrations
(Colegrove et al. 2000). Previous quantitative Ca2⫹ imaging of
these amacrine cells demonstrated resting cytosolic Ca2⫹ levels between 50 and 100 nM (Borges et al. 1996), but it may be
that mitochondria are sensing higher local Ca2⫹ levels. Consistent with this interpretation, both anatomical (Rizzuto et al.
1998; Simpson et al. 1997) and physiological interactions
between mitochondria and IP3 receptor sites on the endoplasmic reticulum have been demonstrated, and the physiological
studies have suggested that mitochondria can buffer the local
Ca2⫹ efflux from activated IP3 receptors in nonneuronal cells
(Csordás et al. 1999; Hajnóczky et al. 1999). As suggested by
Duchen (1999), it might be that this local buffering is required
to maintain Ca2⫹ levels in the permissive range for IP3 receptor
activation. The organization of mitochondria and ER in amacrine cells is unknown, but imaging of the subcellular architecture should be possible with a combination of antibodies and
organelle-specific dyes.
1438
K. MEDLER AND E. L. GLEASON
J Neurophysiol • VOL
al. 1996), whereas in profiles of presynaptic amacrine cell
processes, mitochondria are generally situated closer to the
release sites and can occupy more than up to one-half of the
cross sectional area of the process (Dowling and Boycott
1966). Thus the mechanisms that regulate the distribution of
mitochondria may play an important role in determining the
functional properties of synaptic transmission between amacrine cells.
We thank M. Wilson and B. Hoffpauir for critical reading of the manuscript
and T. Deitz for valuable discussions.
This work was supported by National Eye Institute Grant EY-12204 to E. L.
Gleason.
Present address of K. Medler: Dept. of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523.
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