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1 Department of Neurophysiology, Charité, Humboldt University, D-10117 Berlin, Germany; 2 Department of Neurochemistry, Hungarian Academy of Sciences, Budapest 1025, Hungary
Submitted 15 January 2003; accepted in final form 17 April 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-methyl-5-hydroxytryptamine, and carbachol, transiently increase
mitochondrial Ca2+ concentration
([Ca2+]m) as recorded by changes in Rhod-2
fluorescence, stimulate mitochondrial oxidative metabolism as revealed by
elevations in NAD(P)H fluorescence, and induce K+ outward currents
as monitored by rapid increases in extracellular K+ concentration
([K+]o). Carbachol (1–1,000 µM) elevated
NAD(P)H fluorescence by 
14%
F/F0 and
increased [K+]o by 4.3 mM in a dose-dependent
manner. Carbachol-induced responses persisted in
Ca2+-free solution and blockade of ionotropic
glutamatergic and nicotinic receptors. Under similar conditions caffeine,
known to cause Ca2+-induced Ca2+
release (CICR), also evoked elevations in
[Ca2+]m, NAD(P)H fluorescence and
[K+]o that, in contrast to carbachol-induced responses,
displayed oscillations. After depletion of intracellular
Ca2+ stores by carbachol in
Ca2+-free solution, re-application of 1.6 mM
Ca2+-containing solution triggered marked elevations in
[Ca2+]m, NAD(P)H fluorescence and
[K+]o. These data indicate that metabotropic
transmission effectively regulates mitochondrial oxidative metabolism via
diverse receptor types in hippocampal cells and that inonitol
1,4,5-trisphosphate-induced Ca2+ release (IICR) or CICR
or capacitative Ca2+ entry might suffice in stimulating
oxidative metabolism by elevating [Ca2+]m.
Thus activation of metabotropic receptors might significantly contribute to
generation of ATP within neurons and glial cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-aminobutyric acid (GABA) and glutamate. One of the
intracellular signaling cascades activated by metabotropic receptors is
generation of second-messenger inositol 1,4,5-trisphosphate
(InsP3), which acts on InsP3 receptors to release
Ca2+ from endoplasmatic reticulum (ER;
InsP3-induced Ca2+ release, IICR). In concert
with ryanodine receptors, which are also localized at the ER and potentially
mediate Ca2+-induced Ca2+ release
(CICR), complex spatiotemporal Ca2+ signals can be
generated within excitable and nonexcitable cells
(Clapham 1995
;
Meldolesi 2001;
Verkhratsky et al. 1998).
It is assumed that 60% of ATP consumption within the CNS is used for ion
transport across plasma membranes to maintain or restore neuronal excitability
and another 10–20% for the process of neurotransmission
(Ames 2000). Most of ATP is
generated within mitochondria by oxidative metabolism that primarily uses
pyruvate to generate NADH and FADH2 in the Krebs' cycle
(Ames 2000;
Duchen 1999). These reduced
cofactors are essential to establish a potential across the inner
mitochondrial membrane that drives F1F0-ATP synthase to
phosphorylate ADP, thereby generating ATP
(Ames 2000;
Mitchell 1966). The current
concept concerning the regulation of oxidative metabolism primarily emerges
from studies of isolated mitochondria and cultures of nonexcitable cells
(Hansford and Zorov 1998;
McCormack et al. 1990;
Pralong et al. 1994;
Robb-Gaspers et al. 1998;
Voronina et al. 2002). This
concept involves mitochondrial Ca2+, which regulates the
activity of different dehydrogenases within the Krebs' cycle in the nanomolar
to micromolar range (Denton et al.
1978; McCormack
1985; Rutter
1990), as well as the ratios of substrates like ADP/ATP,
NAD+/NADH or CoA/acetyl CoA
(Hansford 1980;
Reed and Yeaman 1987).
The capability of diverse metabotropic transmitters in generating complex
spatiotemporal Ca2+ signals within nerve and glial cells
has been well documented (Berridge
1998; Blaustein and Golovina
2001; Verkhratsky et al.
1998). Moreover, via intracellular
Ca2+-signaling metabotropic transmitters trigger several
cellular processes and influence membrane excitability, dendritic integration,
and synaptic plasticity (Nakamura et al.
1999; Tsubokawa and Ross
1997; Vanderklish and Edelman
2002). Surprisingly, less is known about the role of metabotropic
transmitter signaling in regulation of energy homeostasis, namely generation
of ATP within cells of the CNS. However, it is essential for our understanding
of neurophysiology as well as of pathophysiology of various neurological
diseases (Kovács et al.
2001; Mattson
2000; Schapira
1999; Schuchmann et al.
1998) whether metabotropic receptor-evoked increases in
cytoplasmic Ca2+ concentration
([Ca2+]c) are sufficient to elevate
mitochondrial Ca2+ concentration
([Ca2+]m) and to have, thereby, functional
consequences on mitochondrial oxidative metabolism.
We therefore investigated the effects of different
Ca2+-mobilizing metabotropic receptor ligands and, in
detail, cellular Ca2+ release and
Ca2+ entry pathways by combining microfluorimetric and
electrophysiological techniques. Rhod-2-based fluorimetry was used to monitor
changes in [Ca2+]m
(Babcock et al. 1997;
Billups and Forsythe 2002;
Rutter et al. 1996). We
recently verified this method in organotypic hippocampal slice cultures by
demonstrating that mitochondrial uncoupler, carbonyl cyanide
m-chlorophenyl hydrazone (CCCP), strongly reduced stimulus-induced
elevations in Rhod-2 fluorescence (Kann et
al. 2003). NAD(P)H fluorescence signals provide an intrinsic
parameter of mitochondrial metabolic function within living cells
(Hajnóczky et al. 1995;
Schuchmann et al. 1998). When
excited by UV light, NAD(P)H fluorescence originates from fluorescing reduced
forms of nicotinamide adenine dinucleotides, NADH, and NADPH, whereas the
oxidized forms are nonfluorescent (Aubin
1979). Simultaneously to Rhod-2 or NAD(P)H fluorescence
recordings, we monitored changes in extracellular K+ concentration
([K+]o) using ion-sensitive microelectrodes
(Cordingley and Somjen 1978;
Heinemann and Lux 1975) to get
insight into K+ fluxes occurring at the plasma membranes on
application of the ligands.
Our data provide evidence that ligands of metabotropic glutamatergic, serotonergic, and muscarinic receptors and more in detail IICR, CICR, as well as capacitative Ca2+ entry, increase [Ca2+]m effectively, stimulate mitochondrial oxidative metabolism, and induce K+ outward currents.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Organotypic hippocampal slice cultures were prepared as described
previously (Stoppini et al.
1991). In brief, hippocampal slices (400 µm) were cut from 7-
to 9-day-old Wistar rats under sterile conditions in gassed (95%
O2-5% CO2), ice-cold minimal essential medium (MEM,
Gibco, Invitrogen, Karlsruhe, Germany). Slices were maintained on a
biomembrane surface (0.4 µm, Millicell-CM, Millipore, Eschborn, Germany)
between culture medium [50% MEM, 25% Hank's balanced salt solution (Sigma,
Taufkirchen, Germany), 25% horse serum (Gibco), and 2 mM
L-glutamine at pH 7.3] and humidified atmosphere (5%
CO2, 36.5°C) in an incubator (Unitherm 150, UniEquip,
Martinsried, Germany). Culture medium was completely replaced twice a week.
Slice cultures were used for experiments after 7–10 days in vitro. All
animals were housed, cared and killed in accordance with the recommendations
of the European Commission and the Berlin Animal Ethics Committee.
Solutions and recordings
Slice cultures on excised membranes were superfused in the recording
chamber with gassed (20% O2-5% CO2) artificial
cerebrospinal fluid (ACSF) at 34 ± 1°C that contained (in mM) 129
NaCl, 3 KCl, 1.25 NaH2PO4, 1.8 MgSO4, 1.6
CaCl2, 21 NaHCO3, and 10 glucose (Sigma); pH 7.35.
Ca2+-free ACSF was similar but with 1.6 mM
MgCl2 in place of CaCl2 and 0.5 mM EGTA. Stock solutions
of (ADA), 2-amino-5-phosphonopentanoic acid (D-AP5),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
-methyl-5-hydroxytryptamine maleate (-Me-5-HT),
(S)--methyl-4-carboxyphenylglycine (MCPG), mianserin
(Biotrend/Tocris, Köln, Germany), atropine, mecamylamine, carbachol, and
caffeine (Sigma) were freshly dissolved in ACSF and applied via the
superfusion system (rate at 4.5 ml/min). To avoid the induction of artifacts
in NAD(P)H fluorescence signals by applying hyperosmolar, 20 mM caffeine
containing ACSF, slice cultures were allowed to adapt to hyperosmolar
(Ca2+-free) ACSF (addition of 20 mM saccharose, Sigma)
before and after caffeine application. The recording chamber was mounted on an
epifluorescence microscope (Axioskop, Zeiss, Jena, Germany) equipped with a
x20 water-immersion objective (0.5 numerical aperture). For recordings,
a double-barrelled K+-sensitive/field potential recording
microelectrode was placed in stratum pyramidale of area CA3, and a monopolar
stimulation electrode (glass pipette filled with ACSF, tip diameter:
5–10 µm, resistance: <10 M
) was positioned >350 µm
away in st. radiatum. Slice cultures were accepted for experiments when evoked
postsynaptic field potentials (single pulse of 0.1 ms) displayed amplitudes of
>0.5 mV.
Ion-sensitive microelectrodes
DC-coupled recordings of field potentials and changes in
[K+]o were performed with double-barrelled
K+-sensitive and reference microelectrodes manufactured and
calibrated as described previously
(Heinemann and Arens 1992). In
brief, electrodes were pulled from double-barrelled theta glass (Science
Products, Hofheim, Germany). The reference barrel was filled with 154 mM NaCl
solution, the ion-sensitive barrel with potassium ionophore I cocktail A
(60031, Fluka Chemie, Buchs, Switzerland) and 100 mM KCl. Ion-sensitive
microelectrodes with a sensitivity of 59 ± 2 mV to a 10-fold increase
in [K+] were used for experiments. The amplifier was equipped with
negative capacitance feedback control, which permitted recordings of changes
in [K+]o with time constants of 50–200 ms. Signals
from the electrode were digitised at 10 Hz using a standard PC and FeliX
software (Photon Technology Instruments, Wedel, Germany).
Microfluorimetric measurements of [Ca2+]m and NAD(P)H
Slice cultures were stained with cell-permeable Rhod-2 AM (5 µM,
Molecular Probes, Leiden, The Netherlands) in ACSF for 60 min at 36.5°C;
this facilitates accumulation of this positively charged
Ca2+ indicator into mitochondria
(Minta et al. 1989). To allow
for hydrolysis of the dye-esters and to promote mitochondrial
compartmentalization of Rhod-2 AM, slice cultures were then maintained in ACSF
for 60–90 min at 36.5°C. Excitation wavelengths (NAD(P)H, 360 nm;
Rhod-2, 530 nm) were set by a monochromator system (Photon Technology
Instruments). Emission light from st. pyramidale and st. radiatum of area CA3
was detected by a photomultiplier (SMT, Seefeld, Germany) at >590 nm
(Rhod-2) or 460 nm (NAD(P)H) (Aubin
1979). Because the emission spectra of NADH and NADPH overlap,
NAD(P)H indicates that the recorded fluorescence might have originated from
either one or both. Photomuliplier data were recorded on computer disk at 10
Hz simultaneously with signals of the K+-sensitive electrode.
Fluorescence signals of Rhod-2 and NAD(P)H are presented as changes in
%F/F0
(F/F0 * 100) where F0
is the averaged fluorescence of a 30-s period before bath application of an
agonist. Illustrated traces of Rhod-2 fluorescence were corrected for
bleaching and washout of the dye.
Calculations and statistics
To translate the recorded potential values (mV) in
[K+]o, a modified Nernst equation was used
 | (1) |
Experimental data from slice cultures (n) were obtained from at least three different preparations. All results of a particular experiment were pooled and are given as means ± SE. Groups were compared by ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Three different metabotropic receptor agonists, namely, the glutamatergic
ligand, ADA (Kapur et al.
2001; Manahan-Vaughan et al.
1996), the serotonergic ligand, -Me-5-HT
(Baxter et al. 1995;
Rainnie 1999), and the
muscarinic ligand, carbachol (Irving and
Collingridge 1998; Müller
and Connor 1991), were tested for their capacity to evoke changes
in [Ca2+]m, NAD(P)H fluorescence, and
[K+]o in area CA3 of organotypic hippocampal slice
cultures. In these experiments, each ligand was applied via the bath solution
for 60 s, while Rhod-2 or NAD(P)H fluorescence signals from st. pyramidale and
st. radiatum were recorded simultaneously to electrophysiological monitoring
of [K+]o from st. pyramidale.
All three ligands (ADA, 1 mM; -Me-5-HT, 40 µM; carbachol, 10
µM) evoked reversible monotonic elevations in
[Ca2+]m [each: 3 of 3 slice cultures tested
(n = 3/3)] and in NAD(P)H fluorescence (n = 4/4, each)
(Figs. 1,
2,
3). Elevations in both
fluorescence signals were tightly associated with transient increases in
[K+]o, ranging from 
0.1 mM (-Me-5-HT, 40
µM) to 1.8 mM (ADA, 1 mM) from the baseline of 3 mM. Interestingly, on any
application of metabotropic receptor ligands,
[Ca2+]m and NAD(P)H fluorescence were still
elevated at times when [K+]o had decreased to
preapplication levels (Figs. 1,
2,
3). The specificity of the
effects of metabotropic receptor ligands on changes in NAD(P)H fluorescence
and [K+]o was tested by measurements in presence of the
respective antagonists. Elevations in both signals induced by ADA (1 mM),
-Me-5-HT (40 µM), or carbachol (100 µM) were completely blocked
in the presence of MCPG (500 µM, not shown), mianserin (10 µM, not
shown), or atropine (1 µM, Fig.
4C), respectively (n = 3; each). The ADA-evoked
elevations in [Ca2+]m, NAD(P)H fluorescence
and in [K+]o persisted during blockade of ionotropic
glutamate receptors by CNQX (60 µM) and D-AP5 (60 µM) (not
shown).
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To characterize the relationship between metabotropic ligand-induced
elevations in NAD(P)H fluorescence and increases in
[K+]o more in detail, we focused on carbachol. Carbachol
was applied at different concentrations over the range of 1–1,000 µM
in 14 slice cultures. Maximal responses in NAD(P)H fluorescence and
[K+]o were evoked by 1,000 µM carbachol whereas a
concentration of 1 µM carbachol was effective only in one of three slice
cultures. The dose-response curve had an EC50 value of 34.9 µM
(Fig. 4A). When
elevations in NAD(P)H fluorescence evoked by application of carbachol at
different concentrations were plotted as a function of increases in
[K+]o, linear regression analysis revealed a positive
correlation between both parameters (r = 0.62; P < 0.001;
n = 39; Fig.
4B). Because we recently observed strong positive
correlations between stimulus-induced increases in [K+]o
as a measure of neuronal activation, NAD(P)H signals, and rises in
[Ca2+]m
(Kann et al. 2003), the
positive correlation between carbachol-induced elevations in
[K+]o and NAD(P)H signals might also provide indirect
evidence for a fine-tuned coupling of elevations in
[Ca2+]c/[Ca2+]m
and stimulation of mitochondrial oxidative metabolism.
The data from these experiments indicate that different InsP3-coupled, metabotropic receptor ligands increase [Ca2+]m effectively to stimulate mitochondrial oxidative metabolism, and induce K+ outward currents.
IICR and CICR elevate [Ca2+]m, NAD(P)H fluorescence and [K+]o
Because metabotropic receptor-evoked elevations in [Ca2+]c might be due to activation of Ca2+ entry and Ca2+ release pathways, we investigated next whether release of Ca2+ from ER was sufficient to mimic the effects of metabotropic receptor ligands on [Ca2+]m, NAD(P)H fluorescence and [K+]o.
To isolate IICR from capacitative Ca2+ entry, a
mechanism that is considered to refill depleted Ca2+
stores (Nilius and Droogmans
2001; Parekh and Penner
1997), carbachol was applied in Ca2+-free
ACSF. Before application of carbachol, slice cultures were superfused with
Ca2+-free ACSF for 
6 min. This superfusion period
sufficed to completely block evoked postsynaptic field potentials and
stimulus-induced elevations in
[Ca2+]c/[Ca2+]m
in hippocampal slice cultures (Kann et al.
2003) as well as depolarization-induced intracellular
Ca2+ increases in pyramidal cells of acute hippocampal
slices (Schuchmann et al.
2000). Because Ca2+ release from
intracellular stores might trigger release of neurotransmitters
(Cochilla and Alford 1998;
Emptage et al. 2001),
Ca2+-free ACSF additionally contained antagonists, CNQX
and D-AP5 (60 µM, each) to block any putative activation of
ionotropic glutamate receptors. Under these conditions, application of
carbachol (500 µM, 60 s) induced a biphasic NAD(P)H fluorescence signal in
which an initial drop became more apparent as compared with lower carbachol
concentrations (Fig.
5A, see also Fig.
3). The initial drop was followed by a long-lasting, monotonic
elevation of the signal (7.9 ±
0.7%F/F0) for 15 min (n =
3/3). Initially, these changes in NAD(P)H fluorescence were associated with
increases in [K+]o by 1.6 ± 0.1 mM from basal
levels (n = 3/3; Fig.
5A). These carbachol-induced biphasic changes in NAD(P)H
fluorescence and the increases in [K+]o were also evoked
by prolonged carbachol application (500 µM) for 180 s, and they were
resistant to additional application of the nonspecific nicotinic acetylcholine
receptor antagonist, mecamylamine (20 µM), still indicating the muscarinic
nature of the responses at higher concentrations of carbachol (not shown).
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Caffeine, in millimolar concentrations, is known to cause CICR by
activation of ryanodine receptors located on the membrane of ER. Thus caffeine
provides an alternative tool in initiating Ca2+ release
from intracellular stores (McPherson et
al. 1991; Shmigol et al.
1996). To avoid artificial changes in NAD(P)H fluorescence due to
ATP consumption associated with cellular responses to changes of osmolarity
under 20 mM caffeine, slice cultures were superfused with hyperosmolar
Ca2+-free ACSF (substitution of 20 mM saccharose instead
of caffeine) before and after application of the ligand. Caffeine (20 mM, 180
s) evoked long-lasting elevations in NAD(P)H fluorescence (11.7 ±
0.9%F/F0), and transient increases in
[K+]o by 1.9 ± 0.1 mM (n = 3/3). In
contrast to application of carbachol, caffeine-induced elevations in NAD(P)H
fluorescence displayed marked oscillations with slow and fast decreasing as
well as increasing components in the range of seconds
(Fig. 5B). Carbachol
and caffeine also induced elevations in
[Ca2+]m (not shown, but see also
Fig. 6).
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These data indicate that Ca2+ mobilization from intracellular Ca2+ stores due to either CICR or IICR is sufficient to induce oscillatory as well as nonoscillatory elevations in [Ca2+]m, NAD(P)H fluorescence and [K+]o.
Ca2+ entry after store depletion increases [Ca2+]m, NAD(P)H fluorescence and [K+]o
Ca2+ entry after depletion of intracellular
Ca2+ stores provides a further mechanism that elevates
[Ca2+]c and that is thought to replenish the
Ca2+ stores (Emptage
et al. 2001; Parekh and Penner
1997). Thus we next focused on the effects of
Ca2+ entry after Ca2+ store
depletion on [Ca2+]m, NAD(P)H fluorescence
and [K+]o. Store depletion was achieved by application
of carbachol in Ca2+-free ACSF. In some slice cultures,
application of the ligand was performed twice, while first Rhod-2 fluorescence
and [K+]o and subsequently NAD(P)H fluorescence and
[K+]o were recorded to monitor fluorescence parameters
under identical conditions. Recordings of stimulus-induced postsynaptic field
potentials (single pulse of 0.1 ms) before and after such experiments
documented that the viability of the slice cultures was only slightly affected
by the experimental procedure (Fig.
6B).
Interestingly, application of Ca2+-free ACSF did not
only decrease [Ca2+]c (e.g.,
Schuchmann et al. 2000) but
also [Ca2+]m
(Fig. 6A,
top). Application of carbachol in Ca2+-free
ACSF induced elevations in [Ca2+]m (2.4
± 0.2%F/F0; n = 4/4),
NAD(P)H fluorescence (11.2 ±
1.3%F/F0; n = 5/5), and
[K+]o (1.9 ± 0.3 mM; n = 7/7) that
displayed different time courses of recovery
(Fig. 6A).
Re-application of Ca2+-containing ACSF after 5 min
triggered rapid elevations in [Ca2+]m (6.5
± 0.9%F/F0; n = 4/4) that,
in comparison to the onset of the experiments, exceeded their basal levels
(Fig. 6A,
top). These rapid elevations in
[Ca2+]m were associated with additional,
monotonic elevations in NAD(P)H fluorescence (3.9 ±
0.7%F/F0; n = 5/5) that arose
from carbachol-induced elevated levels
(Fig. 6A,
middle). Elevations in both signals were accompanied by rises in
[K+]o, whose amplitudes were only 37% (0.7 ± 0.2
mM; n = 7/7) of those evoked during application of carbachol in
Ca2+-free ACSF (1.9 ± 0.3 mM; P <
0.05; Fig. 6A,
bottom). Notably, the increase in [K+]o was
transient and displayed a faster recovery than elevations in
[Ca2+]m and NAD(P)H fluorescence. The effects
of carbachol application in Ca2+-free solution as well
as of reapplication of Ca2+-containing ACSF on
[Ca2+]m, NAD(P)H fluorescence, and
[K+]o were also present during combined blockade of
ionotropic glutamate receptors by CNQX/D-AP5 (60 µM, each) and
nicotinic acetylcholine receptors by mecamylamine (20 µM) (not shown).
These data indicate that even capacitative Ca2+ entry after depletion of intracellular Ca2+ stores by IICR triggers not only elevations in [Ca2+]m but also in NAD(P)H fluorescence and [K+]o.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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;
Irving and Collingridge 1998;
Jaffe and Brown 1994) but also
in their mitochondria, which results in stimulation of mitochondrial oxidative
metabolism. Moreover, different pathways that contribute to metabotropic
receptor-mediated Ca2+ signaling, namely IICR, CICR, and
capacitative Ca2+ entry, suffice in elevating
[Ca2+]m and in stimulating a metabolic
response. Thus activation of metabotropic receptors does not only affect
modulation of membrane excitability, dendritic integration, and synaptic
plasticity (Nakamura et al.
1999; Tsubokawa and Ross
1997; Vanderklish and Edelman
2002) but also does effectively regulate oxidative metabolism
providing the generation of ATP within the cells. Activation of metabotropic receptors in hippocampal neurons
In extensive and profound studies, it has been described that activation of
metabotropic glutamatergic (Jaffe and
Brown 1994; Kapur et al.
2001; Rae et al.
2000; Shirasaki et al.
1994) and muscarinic receptors
(Beier and Barish 2000;
Egorov et al. 1999;
Irving and Collingridge 1998;
Power and Sah 2002) generates
cytoplasmic Ca2+ signals in hippocampal neurons. Here,
we demonstrate that metabotropic glutamatergic and muscarinic receptor
ligands, ADA and carbachol, also induce elevations in
[Ca2+]m that are tightly associated with
elevations in NAD(P)H fluorescence and transient increases in
[K+]o (Figs.
1 and
3). Using the selective agonist
-Me-5-HT (Fig. 2), we
extend these observations to a third metabotropic receptor family, namely
serotonergic 5-HT2 receptors that, so far, have been described to
evoke intracellular Ca2+ signals in ovary cells
(Porter et al. 1999),
astrocytes (Sanden et al.
2000), and a neuronal-like cell line
(Jerman et al. 2001).
In our pharmacological approach, concentrations of the agonists applied via
the bath solution were used in the micromolar to millimolar range, and it has
been reported that, e.g., the concentration of glutamate peaked at 1.1 mM at
cultured hippocampal synapses (Clements et
al. 1992). However, further studies in neuronal tissue employing
metabotropic receptor antagonists and stimulation in the physiological range
might strengthen our observations with respect to functional meaning.
Rhod-2 fluorescence and [Ca2+]m
We used the fluorescence indicator Rhod-2 AM, the positive charge of which
facilitates its accumulation within mitochondria and that has been used in a
variety of preparations to monitor mitochondrial Ca2+
signaling (Billups and Forsythe
2002; Hajnóczky et al.
1995; Hoth et al.
1997; Rutter et al.
1996). It should be noted that the positive charge does not
necessarily imply the exclusive presence of Rhod-2 within mitochondria after
the loading procedure (Bindokas et al.
1998; Kaftan et al.
2000). However, we recently verified in organotypic hippocampal
slice cultures that, in comparison to Fluo-3, Rhod-2 primarily reflects
changes in [Ca2+]m by demonstrating that
Rhod-2 fluorescence signals evoked by repetitive stimulation displayed
different kinetics and that the mitochondrial uncoupler, CCCP strongly reduced
stimulus-induced elevations in Rhod-2 fluorescence
(Kann et al. 2003; see also
Kovács et al.
2001).
The three metabotropic receptor ligands, ADA, carbachol, and
-Me-5-HT induced monotonic elevations in Rhod-2 fluorescence signals
that declined over minutes reflecting transient elevations in
[Ca2+]m (Figs.
1,
2,
3). These data demonstrate that
InsP3-coupled activation of metabotropic receptors in a neuronal
preparation is sufficient to induce Ca2+ uptake by
mitochondria, which extends observations in nonneuronal cells
(Hajnóczky et al. 1995;
Robb-Gaspers et al. 1998;
Voronina et al. 2002).
Similarly, IICR and CICR, evoked under Ca2+-free
conditions and blockade of ionotropic receptors, as well as capacitative
Ca2+ entry after store depletion (Figs.
5 and
6), that have been reported to
increase [Ca2+]c in many cell types including
neurons (Clapham 1995;
Parekh and Penner 1997;
Pozzan et al. 1994;
Verkhratsky and Shmigol 1996),
caused elevations in Rhod-2 fluorescence, indicating substantial mitochondrial
Ca2+ uptake (see also
Collins et al. 2001;
Hoth et al. 1997). Thus
activation of metabotropic, InsP3-coupled receptors in hippocampal
cells might recruit different Ca2+ sources in generating
spatiotemporal Ca2+ signals that will still affect
[Ca2+]m and, thereby, will have functional
implications on shaping cytoplasmic Ca2+ dynamics as
well as on stimulating oxidative metabolism.
NAD(P)H fluorescence and mitochondrial oxidative metabolism
The metabotropic receptor ligands also induced monotonic elevations in
NAD(P)H fluorescence signals that were tightly coupled to the elevations in
[Ca2+]m and [K+]o
(Figs. 1,
2,
3 and
6). These data indicate the
efficacy of metabotropic receptor-induced elevations in
[Ca2+]m to stimulate mitochondrial
dehydrogenases, a process that results in an enhanced generation of NADH and
NADPH (Hansford and Zorov
1998; McCormack et al.
1990). Thus our observations confirm the current concept on
Ca2+ regulation in mitochondrial oxidative metabolism
that, on the cellular level, is based on studies with InsP3-coupled
hormones in nonneuronal cells
(Hajnóczky et al. 1995;
Robb-Gaspers et al. 1998;
Voronina et al. 2002). For
neuronal cells, the Ca2+ dependence of stimulus-induced
NAD(P)H fluorescence signals has been exclusively established by applying
electrical depolarizing stimuli to acutely isolated sensory neurons and
hippocampal slice cultures (Duchen
1992; Kann et al.
2003). However, the capacity of metabotropic receptor-mediated
Ca2+ signaling in affecting
[Ca2+]m and stimulating a metabolic response
has not been tested. Thus our data extend the concept of
Ca2+ regulation in mitochondrial oxidative metabolism
for neuronal preparations to InsP3-coupled, metabotropic
transmitter signaling.
Interestingly, application of carbachol and caffeine under
Ca2+-free conditions resulted in differing shapes of
elevations in NAD(P)H fluorescence signals
(Fig. 5). Higher concentrations
of carbachol induced biphasic NAD(P)H signals that were composed of an initial
decline and a prolonged monotonic elevation similar to those obtained by
depolarisation of brain slices (Lipton
1973), organotypic slice cultures
(Kann et al. 2003;
Kovács et al. 2001), or
dissociated sensory neurons (Duchen
1992). In contrast, application of caffeine induced elevations in
NAD(P)H signals displaying slow and fast decreasing as well as increasing
components. Due to technical limitation, that is, the lack of simultaneous
temporal recordings of NAD(P)H and Rhod-2 fluorescence as well as the lack of
simultaneous spatial recordings of fluorescence signals and
[K+]o (see following text), we were not able to fully
correlate decreasing and increasing components of caffeine-induced changes in
NAD(P)H fluorescence with those in Rhod-2 and [K+]o.
However, it is likely that the oscillatory nature of caffeine-induced changes
in NAD(P)H signals reflects an overlay of several biphasic signals that were
evoked, e.g., by enhancement of synchronized Ca2+
release and/or Ca2+ waves due to
activation/sensitization of ryanodine receptors by caffeine.
Surprisingly, not only Ca2+ release from ER but also
Ca2+ entry after depletion of intracellular
Ca2+ stores (Fig.
6) evoked elevations in Rhod-2 and additional elevations in
NAD(P)H fluorescence. This suggests that capacitative
Ca2+ entry results in mitochondrial
Ca2+ uptake that is functionally integrated into a
metabolic response (Rohács et al.
1997, adrenal glomerulosa cells). This observation might extend
the knowledge on functional implications of capacitative
Ca2+ entry (Nilius
and Droogmans 2001; Parekh and
Penner 1997).
By inducing metabotropic receptor-mediated Ca2+
signaling, we recorded long-lasting elevations in NAD(P)H fluorescence that,
by applying diverse stimuli, have been also observed in neuronal and
nonneuronal cells in vitro (Duchen
1992; Hajnóczky et al.
1995; Kovács et al.
2001; Robb-Gaspers et al.
1998; Rohács et al.
1997; Voronina et al.
2002). However, epileptiform neuronal activity or cortical
spreading depression has been reported to predominantly correlate with
decreases in NAD(P)H fluorescence in vivo that were sparsely followed by
elevations (Jöbsis et al.
1971; Mayevsky and Chance
1975). These deviant findings in the in vitro and in vivo
situation might relate to cellular oxygen supply, changes in the vascular
compartment with alterations of cerebral blood flow and effects of anesthetics
on mitochondria (Anderson et al.
2002; Hertsens et al.
1984).
[K+]o and ionic changes at the plasma membranes
In general, K+ release from neurons results from membrane
depolarizations that increase the driving force for K+ currents
through different types of K+ channels, like voltage-dependent
K+ channels and Ca2+-activated K+
channels. Thus during neuronal activity [K+]o does
increase by <3 mM from basal levels under physiological conditions
(Amzica and Steriade 2000;
Heinemann and Lux 1975,
1977;
Lothman and Somjen 1975) or
might exceed tens of millimolar under pathophysiological conditions
(Lux et al. 1986;
Nicholson et al. 1978). The
fast recovery of increases in [K+]o reflects clearance
of K+ from the extracellular space that is determined by active as
well as passive uptake mechanisms of neurons and glial cells
(D'Ambrosio et al. 2002;
Newman 1995;
Somjen 1995).
In the present study, metabotropic receptor ligands evoked transient increases in [K+]o displaying a faster recovery than elevations in NAD(P)H fluorescence (Figs. 1, 2, 3). Moreover, application of carbachol or caffeine, even in the presence of Ca2+-free solution and ionotropic receptor antagonists, as well as re-application of Ca2+-containing solution after store depletion by carbachol, resulted in transient increases in [K+]o of <2 mM from basal levels (Figs. 5 and 6). These changes in [K+]o were monitored by K+-sensitive microelectrodes that measure accumulation of K+ in a restricted extracellular space, irrespective of whether K+ is released from dendrites, somata, axons, or presynaptic terminals.
Different types of K+ channels and mechanisms might have
contributed to these increases in [K+]o: 1)
Ca2+-activated K+ channels
(Knaus et al. 1996;
Poolos and Johnston 1999) that
were activated by IICR and CICR (Sah and
Faber 2002; Vergara et al.
1998), 2) voltage-dependent K+ channels
(Klee et al. 1995;
Mitterdorfer and Bean 2002),
and 3) modulation of two-pore domain K+ channels
determining resting (background) conductances
(Goldstein et al. 2001;
Talley et al. 2001),
carbachol-mediated suppression of M-currents
(Marrion 1997), activation of
Ca2+-release activated channels
(Hoth and Penner 1992;
Nilius and Droogmans 2001;
Parekh and Penner 1997),
and/or activation of Ca2+-activated nonselective cation
channels (Partridge and Valenzuela
2000; Petersen
2002) that all favor neuronal depolarization. However, the precise
nature of the ion channels and mechanisms involved in the increases in
[K+]o that we report on has to be left to further
studies.
Because the amplitude of transient increases in [K+]o provides an indirect measure of the degree of depolarisation and/or elevations in [Ca2+]c, the significant positive correlation between increases in [K+]o and elevations in NAD(P)H fluorescence (Fig. 4) might indicate a tight coupling between metabotropic receptor-mediated neuronal activity and mitochondrial metabolic function. This coupling seems to be fine-tuned and not characterized by an all-or-nothing response.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests: O. Kann, Abteilung Neurophysiologie, Johannes-Müller-Institut für Physiologie, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Tucholskystrasse 2, D-10117 Berlin, Germany (E-mail: oliver.kann{at}charite.de).
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
Amzica F and
Steriade M. Neuronal and glial membrane potentials during sleep and
paroxysmal oscillations in the neocortex. J Neurosci
20: 6648–6665,
2000.
Anderson CM,
Norquist BA, Vesce S, Nicholls DG, Soine WH, Duan S, and Swanson RA.
Barbiturates induce mitochondrial depolarization and potentiate excitotoxic
neuronal death. J Neurosci 22:
9203–9209, 2002.
Aubin JE. Autofluorescence of viable cultured mammalian cells. J Histochem Cytochem 27: 36–43, 1979.[Abstract]
Babcock DF,
Herrington J, Goodwin PC, Park YB, and Hille B. Mitochondrial
participation in the intracellular Ca2+ network.
J Cell Biol 136:
833–844, 1997.
Baxter G, Kennett G, Blaney F, and Blackburn T. 5-HT2 receptor subtypes: a family re-united? Trends Pharmacol Sci 16: 105–110, 1995.[Medline]
Beier SM and
Barish ME. Cholinergic stimulation enhances cytosolic calcium ion
accumulation in mouse hippocampal CA1 pyramidal neurons during short action
potential trains. J Physiol
526: 129–142,
2000.
Berridge MJ. Neuronal calcium signaling. Neuron 21: 13–26, 1998.[ISI][Medline]
Billups B and
Forsythe ID. Presynaptic mitochondrial calcium sequestration influences
transmission at mammalian central synapses. J Neurosci
22: 5840–5847,
2002.
Bindokas VP,
Lee CC, Colmers WF, and Miller RJ. Changes in mitochondrial function
resulting from synaptic activity in the rat hippocampal slice. J
Neurosci 18:
4570–4587, 1998.
Blaustein MP and Golovina VA. Structural complexity and functional diversity of endoplasmic reticulum Ca2+ stores. Trends Neurosci 24: 602–628, 2001.[ISI][Medline]
Carmant L, Woodhall G, Ouardouz M, Robitaille R, and Lacaille JC. Interneuron-specific Ca2+ responses linked to metabotropic and ionotropic glutamate receptors in rat hippocampal slices. Eur J Neurosci 9: 1625–1635, 1997.[ISI][Medline]
Clapham DE. Calcium signaling. Cell 80: 259–268, 1995.[ISI][Medline]
Clements JD,
Lester RA, Tong G, Jahr CE, and Westbrook GL. The time course of glutamate
in the synaptic cleft. Science
258: 1498–1501,
1992.
Cochilla AJ and Alford S. Metabotropic glutamate receptor-mediated control of neurotransmitter release. Neuron 20: 1007–1016, 1998.[ISI][Medline]
Collins TJ,
Lipp P, Berridge MJ, and Bootman MD. Mitochondrial
Ca2+ uptake depends on the spatial and temporal profile
of cytosolic Ca2+ signals. J Biol
Chem 276:
26411–26420, 2001.
Cordingley GE and Somjen GG. The clearing of excess potassium from extracellular space in spinal cord and cerebral cortex. Brain Res 151: 291–306, 1978.[ISI][Medline]
D'Ambrosio R,
Gordon DS, and Winn HR. Differential role of KIR channel and
Na+/K+-pump in the regulation of extracellular
K+ in rat hippocampus. J Neurophysiol
87: 87–102,
2002.
Denton RM, Richards DA, and Chin JG. Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues. Biochem J 176: 899–906, 1978.[ISI][Medline]
Duchen MR.
Contributions of mitochondria to animal physiology: from homeostatic sensor to
calcium signaling and cell death. J Physiol
516: 1–17,
1999.
Duchen MR. Ca2+-dependent changes in the mitochondrial energetics in single dissociated mouse sensory neurons. Biochem J 283: 41–50, 1992.[Medline]
Egorov AV,
Gloveli T, and Müller W. Muscarinic control of dendritic excitability
and Ca2+ signaling in CA1 pyramidal neurons in rat
hippocampal slice. J Neurophysiol
82: 1909–1915,
1999.
Emptage NJ, Reid CA, and Fine A. Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release. Neuron 29: 197–208, 2001.[ISI][Medline]
Goldstein SA, Bockenhauer D, O'Kelly I, and Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2: 175–184, 2001.[ISI][Medline]
Hajnóczky G, Robb-Gaspers LD, Seitz MB, and Thomas AP. Decoding of cytosolic calcium oscillations in the mitochondria. Cell 82: 415–424, 1995.[ISI][Medline]
Hansford RG. Control of mitochondrial substrate oxidation. Curr Top Bioenerg 10: 217–277, 1980.
Hansford RG and Zorov D. Role of mitochondrial calcium transport in the control of substrate oxidation. Mol Cell Biochem 184: 359–369, 1998.[ISI][Medline]
Heinemann U and Arens J. Production and calibration of ion-sensitive microelectrodes. In: Practical Electrophysiological Methods, edited by Kettenmann H and Grantyn R. New York: Wiley-Liss, 1992, p. 206–212.
Heinemann U and Lux HD. Undershoots following stimulus-induced rises of extracellular potassium concentration in cerebral cortex of cat. Brain Res 93: 63–76, 1975.[ISI][Medline]
Heinemann U and Lux HD. Ceiling of stimulus-induced rises in extracellular potassium concentration in the cerebral cortex of cat. Brain Res 120: 231–249, 1977.[ISI][Medline]
Hertsens R, Jacob W, and Van Bogaert A. Effect of hypnorm, chloralosane and pentobarbital on the ultrastructure of the inner membrane of rat heart mitochondria. Biochim Biophys Acta 769: 411–418, 1984.[Medline]
Hoth M, Fanger
CM, and Lewis RS. Mitochondrial regulation of store-operated calcium
signaling in T lymphocytes. J Cell Biol
137: 633–648,
1997.
Hoth M and Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355: 353–356, 1992.[Medline]
Irving AJ and
Collingridge GL. A characterization of muscarinic receptor-mediated
intracellular Ca2+ mobilization in cultured rat
hippocampal neurons. J Physiol
511: 747–759,
1998.
Jaffe DB and
Brown TH. Metabotropic glutamate receptor activation induces calcium waves
within hippocampal dendrites. J Neurophysiol
72: 471–774,
1994.
Jerman JC, Brough SJ, Gager T, Wood M, Coldwell MC, Smart D, and Middlemiss DN. Pharmacological characterisation of human 5-HT2 receptor subtypes. Eur J Pharmacol 414: 23–30, 2001.[ISI][Medline]
Jöbsis FF,
O'Connor M, Vitale A, and Vreman H. Intracellular redox changes in
functioning cerebral cortex. I. Metabolic effects of epileptiform activity.
J Neurophysiol 34:
735–749, 1971.
Kaftan EJ, Xu
T, Abercrombie RF, and Hille B. Mitochondria shape hormonally induced
cytoplasmic calcium oscillations and modulate exocytosis. J Biol
Chem 275:
25465–25470, 2000.
Kann O, Schuchmann S, Buchheim K, and Heinemann U. Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat. Neuroscience 119: 87–100, 2003.[ISI][Medline]
Kapur A, Yeckel M, and Johnston D. Hippocampal mossy fiber activity evokes Ca2+ release in CA3 pyramidal neurons via a metabotropic glutamate receptor pathway. Neuroscience 107: 59–69, 2001.[ISI][Medline]
Klee R, Ficker
E, and Heinemann U. Comparison of voltage-dependent potassium currents in
rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3.
J Neurophysiol 74:
1982–1995, 1995.
Knaus HG,
Schwarzer C, Koch RO, Eberhart A, Kaczorowski GJ, Glossmann H, Wunder F, Pongs
O, Garcia ML, and Sperk G. Distribution of high-conductance
Ca2+-activated K+ channels in rat brain:
targeting to axons and nerve terminals. J Neurosci
16: 955–963,
1996.
Kovács R, Schuchmann S, Gabriel S, Kardos J, and Heinemann U. Ca2+ signaling and changes of mitochondrial function during low-Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures. Eur J Neurosci 13: 1311–1319, 2001.[ISI][Medline]
Lipton P. Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices. Biochem J 136: 999–1009, 1973.[ISI][Medline]
Lothman EW and
Somjen GG. Extracellular potassium activity, intracellular and
extracellular potential responses in the spinal cord. J
Physiol 252:
115–136, 1975.
Lux HD, Heinemann U, and Dietzel I. Ionic changes and alterations in the size of the extracellular space during epileptic activity. Adv Neurol 44: 619–639, 1986.[Medline]
Manahan-Vaughan D, Reiser M, Pin JP, Wilsch V, Bockaert J, Reymann KG, and Riedel G. Physiological and pharmacological profile of transazetidine-2, 4-dicarboxylic acid: metabotropic glutamate receptor agonism and effects on long-term potentiation. Neuroscience 72: 999–1008, 1996.[ISI][Medline]
Marrion NV. Control of M-current. Annu Rev Physiol 59: 483–504, 1997.[ISI][Medline]
Mattson MP. Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1: 120–129, 2000.[ISI][Medline]
Mayevsky A and Chance B. Metabolic responses of the awake cerebral cortex to anoxia hypoxia spreading depression and epileptiform activity. Brain Res 98: 149–165, 1975.[ISI][Medline]
), while Rhod-2 or NAD(P)H fluorescence signals from st.
pyramidale and st. radiatum were recorded simultaneously to
electrophysiological monitoring of [K+]o from st.
pyramidale. Changes in fluorescence signals were expressed as
%
).
) in
[Ca2+]m (Rhod-2 fluorescence), NAD(P)H
fluorescence, and [K+]o in area CA3 of an organotypic
hippocampal slice culture. Changes in fluorescence signals were expressed as
%