II. Physiologisches Institut, Universität Göttingen, D-37073 Göttingen, Germany
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ABSTRACT |
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Kulik, Anna,
Stefan Trapp, and
Klaus Ballanyi.
Ischemia But Not Anoxia Evokes Vesicular and
Ca2+-Independent Glutamate Release In the Dorsal Vagal
Complex In Vitro.
J. Neurophysiol. 83: 2905-2915, 2000.
Whole cell recordings of fura-2 dialyzed
vagal neurons of brain stem slices were used to monitor interstitial
glutamate accumulation within the dorsal vagal complex. Anoxia produced
a sustained outward current (60 pA) and a moderate
[Ca2+]i rise (40 nM). These responses were
neither mimicked by [1S,3R]-1-aminocyclo-pentane-1,3-dicarboxylic acid nor affected by Ca2+-free solution,
6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide
in glucose-free saline (in vitro ischemia) as well as ouabain or
iodoacetate elicited an initial anoxia-like
[Ca2+]i increase that turned after several
minutes into a prominent Ca2+ transient (0.9 µM) and
inward current (1.8 nA). APV plus CNQX (plus methoxyverapamil)
inhibited this inward current as well as accompanying spontaneous
synaptic activity, and reduced the secondary
[Ca2+]i rise to values similar to those
during anoxia. Each of the latter drugs delayed onset of both ischemic
current and prominent [Ca2+]i rise by several
minutes and attenuated their magnitudes by up to 40%.
Ca2+-free solution induced a twofold delay of the ischemic
inward current and suppressed the prominent Ca2+ increase
but not the initial moderate [Ca2+]i rise.
Cyclopiazonic acid or arachidonic acid in Ca2+-free saline
delayed further the ischemic current, whereas neither inhibitors of
glutamate uptake (dihydrokainate,
D,L-threo-
-hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the
Cl
channel blocker 5-nitro-2-(3-phenylpropyl-amino)
benzoic acid had any effect. In summary, the response to metabolic
arrest is due to activation of ionotropic glutamate receptors causing
Ca2+ entry via
N-methyl-D-aspartate receptors and
voltage-activated Ca2+ channels. An early
Ca2+-dependent exocytotic phase of ischemic glutamate
release is followed by nonvesicular release, not mediated by reversed
glutamate uptake or Cl
channels. The results also show
that glycolysis prevents glutamate release during anoxia.
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INTRODUCTION |
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Excitotoxicity related to extracellular
accumulation of glutamate is believed to be a major cause for the low
tolerance of most mammalian neurons to anoxia (Benveniste et al.
1984; Choi 1988
). The anoxia-induced
vulnerability, in particular of cortical neurons, appears to be related
to the profound rise of the free intracellular concentration of
Ca2+
([Ca2+]i) that is
associated with increased interstitial levels of glutamate. Such
neuronal [Ca2+]i
elevation results primarily from influx via
Ca2+-permeable glutamate receptors and
voltage-activated Ca2+ channels (Kristian
and Siesjö 1996
; Silver and Erecinska
1990
). Also impairment of Ca2+ extrusion
and sequestration due to suppression of ion pump activity and
mitochondrial function contributes to the excessive anoxic rise of
[Ca2+]i (Kristian
and Siesjö 1996
; Schinder et al. 1996
).
The neuronal Ca2+ overload in combination with
cellular changes such as ATP consumption by depolarized mitochondria
activates a cascade of events that leads to cell death (Budd and
Nicholls 1996
; Schanne et al. 1979
). Besides the
complexity of cellular events that mediate glutamate-induced neuronal
death, it is not clear which processes are responsible for interstitial
accumulation of glutamate during anoxia. There is disagreement on
whether Ca2+-dependent exocytosis or reduced
uptake is the major constituent of the initial phase of the
ischemia-related glutamate rise (Drejer et al. 1985
;
Ikeda et al. 1989
; Katayama et al. 1991
;
Szatkowski and Attwell 1994
). It is also under
discussion whether the late phase of the anoxic glutamate release is
associated with reversed action of the transporters, unspecific
membrane damage, or other processes (Bickler and Hansen
1994
; Madl and Burgesser 1993
; Roettger and Lipton 1996
; Sanchez-Prieto and Gonzales
1988
).
Here we have investigated the origin and functional consequence of
glutamate release during anoxia/ischemia in a brain stem slice
preparation that contains the dorsal vagal complex (Loewy and
Spyer 1990). This preparation was chosen as the response of dorsal vagal neurons (DVN) to different types of metabolic disturbances is well described. In contrast to the glutamate-related vulnerability of most neuronal structures of the mature brain, DVN are extremely tolerant to anoxia (Cowan and Martin 1992
; Trapp
and Ballanyi 1995
). Block of aerobic metabolism by either
anoxia or cyanide (CN
) hyperpolarizes DVN due
to a sustained outward current through ATP-sensitive
K+ channels (Karschin et al. 1998
;
Trapp and Ballanyi 1995
). Sulfonylurea-induced block of
the anoxic outward current unmasks a moderate inward current that
remains stable during anoxia periods of at least 20 min
(Ballanyi and Kulik 1998
; Trapp and Ballanyi
1995
). Thus, in the DVN, oxygen depletion alone does not evoke
a progressive inward current that underlies the "terminal"
depolarization of anoxia-vulnerable neurons (Bures and Buresova
1957
; Haddad and Jiang 1993a
,b
; Hansen et
al. 1982
). However, block of glycolysis with iodoacetate
(Ballanyi and Kulik 1998
) and anoxic exposure after
preincubation in glucose-free solution that mimics ischemia (Ballanyi et al. 1996
) lead to a progressive
depolarization of DVN that is accompanied by profound perturbation of
ion homeostasis. In the present study, we have used whole cell
recordings to indirectly monitor changes of extracellular glutamate
(Warr et al. 1999
) during anoxia or ischemia by
activation of postsynaptic ionotropic glutamate receptors of DVN
(Travagli et al. 1991
; Willis et al. 1996
). In addition, the cells were dialyzed via the patch
electrode with fura-2 to assess pathways of putative glutamate
receptor-associated [Ca2+]i increases during
anoxia and ischemia.
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METHODS |
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Preparation and solutions
The experiments were performed on brain stem slices obtained
from 14- to 20-day-old Wistar rats of either sex. Rats were
anesthetized with ether and decapitated. The brain stem was isolated
and kept for 5 min in ice-cold artificial cerebrospinal fluid (standard solution; composition see below; Ca2+
concentration reduced to 0.5 mM). Afterward, six to eight transverse slices (200 µm) were cut from the brain stem around the obex level in
the 0.5 mM Ca2+ solution. Before transfer to the
recording chamber, the slices were stored at 30°C in standard
solution. The recording chamber (volume 3 ml) was superfused at 30°C
with oxygenated standard solution (flow rate 5 ml/min) of the following
composition (in mM): 118 NaCl, 3 KCl, 1 MgCl2,
1.5 CaCl2, 25 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose. pH was adjusted to 7.4 by gassing with 95%
O2 and 5% CO2. Tissue
anoxia was evoked by hypoxic superfusate gassed with 95%
N2 instead of O2
(Ballanyi et al. 1996; Trapp and Ballanyi 1995
). Chemical anoxia was induced by addition of
CN
(1 mM) to the standard solution
(Ballanyi and Kulik 1998
; Karschin et al.
1998
). In vitro ischemia was elicited by either anoxic exposure
of the brain stem slices after preincubation in glucose-free solution
for at least 30 min or by application of CN
subsequent to 30 min preincubation in glucose-free superfusate (Ballanyi et al. 1996
). Ca2+-free
saline contained 1 mM EGTA and 5 mM Mg2+.
Drugs were purchased from Sigma (Munich, Germany), Biomol (Cologne,
Germany) or Tocris Cookson (Bristol, UK). The following drugs were kept
as stock solution in standard saline: glutamate (100 mM), the blocker
of N-methyl-D-aspartate (NMDA) glutamate receptors 2-amino-5-phosphonovalerate (APV, 10 mM),
N-(4-hydroxyphenyl-propanolyl)-spermine (NHPPS, 1 mM),
L-transpyrrolidone-2,4-dicarboxylate (PDC, 10 mM), dihydrokainate (DHK, 10 mM), and
D,L-threo--hydroxyaspartate (THA, 10 mM). The blocker of
-amino-3-hydroxy-5-methyl-4-isoxalone/kainate (AMPA/KA)
glutamate receptors 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 mM),
methoxyverapamil (D-600, 100 mM), tolbutamide (100 mM), gliquidone (10 mM), cyclopiazonic acid (CPA, 10 mM), and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB, 100 mM) were dissolved in DMSO. [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD, 1 mM) was dissolved in 0.1 M NaOH and arachidonic acid (100 mM) in ethanol. The other drugs were added to the superfusate.
Patch clamp
Patch pipettes were produced from borosilicate glass capillaries
(GC 150TF, Clark Electromedical Instruments, Pangbourne, UK) using a
horizontal electrode puller (Zeitz, Munich, Germany). Standard patch
pipette solution contained (in mM) 140 potassium gluconate, 1 MgCl2, 10 HEPES, 1 Na2ATP,
pH 7.3-7.4. The resistance of the patch electrodes ranged from 4 to 6 M 100 µM fura-2 (Molecular Probes, Eugene, OR) was added
to the pipette solution before the experiment. Whole cell recordings
were performed on superficial DVN under visual control using an EPC-9
patch-clamp amplifier (HEKA, Lambrecht, Germany). Membrane conductance
(gm) was measured by application of
hyperpolarizing current (10-100 pA) or voltage (
20 mV) pulses with a
duration of 500 ms. The holding potential (Vh) in voltage clamp was
50 mV
[corresponding to mean resting membrane potential
(Vm)] (Ballanyi et al.
1996
; Trapp and Ballanyi 1995
) unless stated otherwise.
Ca2+ measurements
[Ca2+]i was
measured fluorometrically using a fura-2-based single-detector system.
Cells were loaded with fura-2 by dialysis via the patch electrode. An
upright microscope (Standard 16, Zeiss, Oberkochen, Germany) was
equipped with epifluorescence optics, a monochromator (Polychrome II,
T.I.L.L. photonics, Planegg, Germany) to allow for alternating
fluorescent excitation at 360 and 390 nm, and a photomultiplier system
(Luigs and Neumann, Ratingen, Germany). Emission was measured at 510 nm
through a pinhole diaphragm, which limited the region from which light
was collected to a circular spot of 20 µm diam. Fluorescence ratios
were converted into
[Ca2+]i by using
Eq. 1, [Ca2+]i = K(R Rmin)/(Rmax
R) (Grynkiewicz et al. 1985
), in which R is the fluorescence ratio (360 nm/390 nm) and K
is the effective dissociation constant of fura-2. In vivo calibrations
to determine Rmin,
Rmax, and K were done
according to the method described by Neher (1989)
.
Measurements were performed with three pipette solutions (in mM):
1) 130 KCl, 1 MgCl2, 10 BAPTA, 10 HEPES, and 1 Na2ATP (low calcium;
Rmin); 2) 130 KCl, 1 MgCl2, 3 CaCl2, 4 BAPTA, 10 HEPES, and 1 Na2ATP [intermediate calcium; 300 nM, according to a KD of 107 nM for
BAPTA (Tsien 1980
)]; and 3) 130 KCl, 1 MgCl2, 10 CaCl2, 10 HEPES,
and 1 Na2ATP (high calcium;
Rmax); to each solution 100 µM
fura-2 was added. The intracellular fluorescence ratios were calculated
according to Eq. 1. K was calculated as K = 300 nM (Rmax
R)/(R
Rmin).
Data analysis
Electrophysiological and microfluorometrical signals were sampled into a Macintosh PowerPC (Apple Computers, Cupertino, CA) using Pulse/Pulsefit and X-chart/Fura extension from HEKA. Current and voltage signals were also digitized (Instrutech VR-100A, Elmont, NY) and recorded on a VCR. Values are means ± SE.
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RESULTS |
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Response to anoxia
In an initial series of experiments, the effects of tissue anoxia
were investigated on Vm and
[Ca2+]i. Spontaneous
spike discharge at a frequency of 0.5-5 Hz was observed in 11 of 14 DVN in which [Ca2+]i
baseline ranged between 38 and 156 nM. In 10 of these cells, anoxia
produced a stable hyperpolarization by up to 20 mV. The anoxia-evoked
hyperpolarization to a maximal of 75 mV was accompanied by a modest
rise of [Ca2+]i by <100
nM (Fig. 1A). In four cells
with spike frequencies >2 Hz and therefore a higher
[Ca2+]i baseline, the
anoxia-evoked [Ca2+]i
rise was preceded by a 15- to 35-nM reduction of
[Ca2+]i. In these DVN,
reoxygenation first led to a rapid fall of
[Ca2+]i that was followed
by a reincrease associated with recovery of spike activity (Fig.
1A). In neurons with a spike frequency <2 Hz, no biphasic
Ca2+ response was observed (not shown).
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To exclude effects of voltage-activated Ca2+
channels, the anoxia response was analyzed under voltage clamp. A 5-min
period of anoxia led to a stable increase of
[Ca2+]i by 39 ± 3 nM (mean ± SE, n = 43; Fig. 1, B and
C). In 27 of these cells, the
[Ca2+]i rise was
accompanied by a sustained outward current of 59 ± 7 pA and a
gm increase by up to 500% (Figs.
1B and 2). The anoxic [Ca2+]i rise was not
affected by block of the outward current by the sulfonylureas
tolbutamide (50-200 µM; n = 9; Fig. 1B)
or gliquidone (1 µM; n = 3; not shown). In eight of
the cells, the sulfonylurea-induced block of the anoxic outward current
unmasked a persistent inward current of between 10 and
40 pA and a
decrease of gm down to 20% of control
(Fig. 1B). This inward current was not accompanied by a
significant increase in spontaneous subthreshold synaptic activity (not
shown). Both the anoxic
[Ca2+]i increase and the
outward current were not changed by a solution that contained
tetrodotoxin (TTX, 0.5 µM), CNQX (50 µM), and APV (100 µM) to
block synaptic transmission as well as ionotropic glutamate receptors
(n = 5; Fig. 2A). The anoxic outward current and [Ca2+]i elevation
were also unaffected by Ca2+-free solution, which
abolished depolarization-induced
[Ca2+]i increases
(n = 3; Fig. 2B). The unspecific
metabotropic glutamate receptor agonist [1S,3R]-ACPD (100 µM) did
not change Im,
gm, or
[Ca2+]i in cells
that responded to anoxia with both a moderate
[Ca2+]i rise and an
outward current (n = 6; not shown). The results suggest
that glutamate receptors are not involved in the anoxia response.
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Response to ischemia
The effects of in vitro ischemia on [Ca2+]i and Vm of DVN were tested by anoxic exposure of the slices after preincubation in glucose-free solution for at least 30 min. The cell illustrated in Fig. 3A hyperpolarized with a concomitant gm increase after 20 min of glucose depletion. The hyperpolarization-related suppression of spontaneous spiking caused a fall of [Ca2+]i by ~80 nM. Anoxic exposure during glucose depletion evoked a progressive depolarization by ~30 mV, which was accompanied by a fourfold increase of gm, and a rise of [Ca2+]i by >400 nM. The cell recovered from both the depolarization and the prominent [Ca2+]i rise on reoxygenation of the glucose-free solution. After readdition of glucose, [Ca2+]i and Vm returned to control levels, and spiking reappeared. A similar response was revealed in three further DVN during ischemia.
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The mechanism of the ischemia-induced depolarization and [Ca2+]i rise was analyzed under voltage clamp. Eighteen of 29 DVN responded to glucose removal with an outward current of 47 ± 5 pA and a gm increase by 100-500% after a delay of 8-20 min. Im and gm of the remaining cells did not change. Thirty minutes of glucose removal induced a minor (23 ± 3 nM, n = 29) rise in [Ca2+]i baseline. The cell of Fig. 3B responded to anoxia with a moderate increase of [Ca2+]i in the presence of glucose. An ischemia period that was induced after 40 min of glucose depletion led initially to a slow, moderate increase of [Ca2+]i. However, after ~5 min, a strong inward current developed that was accompanied by a rapid increase of [Ca2+]i to >600 nM. Reoxygenation of the glucose-free solution led to complete recovery of both [Ca2+]i and Im. In 9 of 27 DVN, the inward current, which was accompanied by an up to sixfold increase of gm, was larger than 1 nA and [Ca2+]i increased to >1 µM (Fig. 3C). Complete recovery from the ischemic [Ca2+]i rise was critically dependent on immediate reoxygenation after onset of the ischemic current.
Pharmacology of the ischemia response
We next explored the basis for the ischemic depolarization and fast [Ca2+]i rise. A mixture of TTX (0.5 µM) and both CNQX (50 µM) and APV (100 µM) reversibly abolished the ischemia-induced inward current as well as the secondary large [Ca2+]i rise (n = 9; Fig. 4A), whereas TTX plus APV, or TTX plus CNQX only attenuated the ischemic current and Ca2+ rise (not shown). The protection by TTX plus CNQX and APV was also observed when ischemic exposure was prolonged for several minutes compared with control ischemia (Fig. 4A). In >50% of cells, membrane noise increased within 1-2 min before onset and during the initial phase of the ischemic inward current due to increased spontaneous synaptic activity. Such ischemia-induced synaptic activity did not develop on ischemia in the presence of CNQX/APV (not shown, but see Fig. 4, B and C).
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Quantitative analysis of the effects of glutamate receptor blockers on
the ischemia response was hampered by the observation that the onset of
the progressive inward current and
[Ca2+]i rise varied
between 3 and >10 min in different preparations. It turned out that
the delay of onset as well as the magnitude of the effects were
dependent on both the age of the animal and the time of in vitro
storage of the slices. Thus for quantitative pharmacological analysis
of the ischemia response, animals of an age between 18 and 20 days were
chosen, slices were investigated within 1 to 4 h after isolation,
and chemical anoxia was induced by CN. Under
these experimental conditions, 5 min application of 1 mM
CN
30 min after preincubation in glucose-free
saline evoked a peak inward current of
1.75 ± 0.27 nA and an
average [Ca2+]i rise of
0.912 ± 76 µM after a delay of 2.88 ± 0.12 min
(n = 12; Figs. 4B and
5, C and D). In
>60% of these recordings, subthreshold synaptic activity increased
considerably before development of the fast phase of the ischemic
inward current (Fig. 4, B and C). This, as well
as the inward current and fast
[Ca2+]i rise, were
blocked by CNQX and APV (n = 5; Figs. 4, B
and C, and 5D). As tested during 5-20 min
exposure to CN
in glucose-free solution, either
of the drugs alone only attenuated both the inward current and
[Ca2+]i rise by <40%
(Fig. 5, A and D). CNQX and APV also increased further the time lag (tonset) from the
beginning of ischemia until an inward current of
150 pA was reached
(Fig. 5, A and C).
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In addition, the effects of the Ca2+ channel blocker D-600 (200 µM) were tested. D-600 attenuated the ischemic [Ca2+]i increase by ~35% and increased tonset by further 4.8 min, but induced only a minor reduction of the ischemic inward current (Fig. 5D). In contrast, the drug depressed depolarization-induced [Ca2+]i rises by ~90% (n = 6; not shown). In the presence of CNQX plus APV, or CNQX, APV, plus D-600, tonset was increased to 13.3 and 18.0 min, respectively (Fig. 5, B and C). In the presence of these drugs, 20 min of ischemia led to a [Ca2+]i increase that was not significantly larger than during anoxia in glucose-containing solution (Fig. 5D).
Effects of glutamate application
Because the latter results indicated involvement of glutamate in
the ischemia response, the effects of exogenous glutamate and glutamate
receptor antagonists were tested. Bath application of 0.5 mM glutamate
(20 s) led to a rise of
[Ca2+]i by
0.30 ± 0.05 µM (n = 16) and an inward current
of 0.70 ± 0.06 nA accompanied by a severalfold increase in
gm. Glutamate (0.1 mM) evoked an
inward current of less than
0.1 nA, a rise of
gm by up to 300%, and a
[Ca2+]i elevation by
~50 nM. A mean inward current of
1.24 ± 0.2 nA or
1.95 ± 0.1 nA, and a concomitant
[Ca2+]i rise of 0.64 ± 0.05 µM or 0.71 ± 0.03 µM were evoked in four DVN in
response to 1 and 2 mM glutamate, respectively (not shown). Coapplication of CNQX and APV abolished both the current and the [Ca2+]i transients (Fig.
6, A and B). CNQX
alone blocked the glutamate-activated inward current and rise of
[Ca2+]i by 80 and 67%,
respectively, whereas APV reduced the glutamate-induced current by 40%
and the elevation of
[Ca2+]i by 50% (Fig.
6B). The blocker of Ca2+-permeable
AMPA/KA receptors NHPPS (1 µM) did neither attenuate the inward
current nor the [Ca2+]i
rise in response to 0.5 mM glutamate in four DVN (Fig. 6B).
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The lack of effects of NHPPS suggests that a considerable portion of
the [Ca2+]i rise in
response to glutamate is caused by imperfect voltage control of
(remote) dendritic regions resulting in activation of voltage-activated
Ca2+ channels. To test for this possibility,
cells were exposed to glutamate under current and voltage clamp.
Glutamate (0.5 mM; 20 s) evoked a mean membrane depolarization of
30 ± 0.7 mV (Fig. 6, C and D) that was
associated with an average
[Ca2+]i increase of
0.5 ± 0.03 µM (n = 6). In these cells, the
glutamate-induced [Ca2+]i
rise was reduced by ~50% after switching to voltage clamp (Fig. 6,
C and D). In individual voltage-clamped cells,
the magnitude of the
[Ca2+]i transients in
response to 0.5 mM glutamate varied considerably (not shown). If
insufficient space clamp was responsible for the [Ca2+]i rises in some
cells, an attenuated response would be expected at more negative
Vh. In contrast, if the
Ca2+ transient was due to influx through
glutamate receptors, an increased response would be expected because
the driving force is increased. Changing
Vh from 50 to
80 mV reduced the
mean glutamate-induced [Ca2+]i rise by 64%,
whereas the accompanying inward current slightly increased
(n = 3; Fig. 6, E and F).
Origin of ischemic glutamate release
The facts that 1) the ischemic response of DVN can be blocked by glutamate receptor antagonists and 2) exogenous glutamate mimics the effects of ischemia suggest that ischemia but not anoxia evokes a rise of extracellular glutamate. To investigate whether the glutamate accumulation is caused by Ca2+-dependent vesicular release, slices were exposed to Ca2+-free solution 25 min after removal of glucose. This led to a fall of [Ca2+]i baseline by between 20 and 50 nM (Fig. 7A). Subsequent ischemia evoked the initial moderate rise of [Ca2+]i, whereas the profound [Ca2+]i transient was blocked (Fig. 7A). The Ca2+-free solution doubled tonset from 2.9 min under control (Fig. 5C) to ~6 min (Fig. 7B). Addition of the endoplasmatic reticulum Ca2+ pump blocker CPA (30 µM) to the Ca2+- and glucose-free superfusate further delayed tonset by ~2 min without an effect on the magnitude of the ischemic current (n = 6; Fig. 7, B and D). In contrast to this effect, addition of CPA neither induced spontaneous synaptic activity, nor did it potentiate spontaneous synaptic activity that developed in the initial phase of ischemia (compare Fig. 4, B and C).
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In another set of experiments, it was studied whether the ischemic
glutamate rise is caused by reversed glutamate uptake. Arachidonic acid
(200 µM), which inhibits glutamate transporters (Barbour et
al. 1989), increased tonset in
both Ca2+-containing (4.3 vs. 2.9 min in control)
and in Ca2+-free (9 vs. 6 min) solution, but had
no significant effect on the peak ischemic current (Fig.
7D). In contrast to arachidonic acid, addition of the
glutamate transport blockers THA or PDC (1 mM each) to the standard
solution for 1 min evoked an inward current of
1.4 ± 0.3 nA and
1.5 ± 0.2 nA and a
[Ca2+]i increase by
1.3 ± 0.4 µM and 1.3 ± 0.1 µM, respectively
(n = 5; Fig. 7C). Both the THA-induced
inward current and
[Ca2+]i rise were reduced
by >80% after preincubation with 50 µM CNQX (n = 3;
not shown). An inward current of
0.2 ± 0.17 nA and a [Ca2+]i increase of
0.04 ± 0.02 µM were evoked by 1 min exposure to DHK (1 mM),
another blocker of glutamate transporters (Fig. 7C). It has
been described previously that the transport blockers need to be loaded
intracellularly before ischemia (Madl and Burgesser 1993
; Roettger and Lipton 1996
). Thus slices
were incubated for 1 h with THA, PDC, and DHK (1 mM each). CNQX
(50 µM) and APV (100 µM) were also added to avoid excessive
intracellular Ca2+ accumulation in the presence
of THA and PDC. After loading, slices were kept in standard solution
for 1 h before use for intracellular recording. There was no
apparent difference in the ischemia effects between control and treated
slices (compare Fig. 5, C and D, and Fig. 7,
B and D). The "cocktail" of glutamate
transport blockers was also ineffective in
Ca2+-free solution (Fig. 7, B and
D). NPPB (200 µM), a blocker of glial volume-sensitive
Cl
channels that permit efflux of glutamate
(Basarsky et al. 1999
), did not change
tonset or the magnitude of the
ischemic current in Ca2+-free solution
(n = 4; Fig. 7, B and D).
Effects of ouabain and iodoacetate
In a final experimental approach, it was tested whether direct
block of the Na+/K+ pump or
block of glycolysis with iodoacetate (Haddad and Jiang 1993a; Hansen 1985
) mimic the effects of
ischemia. Ouabain (50 µM), the blocker of the
Na+/K+ pump, induced an
immediate and stable inward current of between
20 and
100 pA (Fig.
8). After a delay of 6.9 ± 0.5 min
(n = 4), a strong inward current (
1.1 ± 0.2 nA)
and [Ca2+]i rise
(1.6 ± 0.1 µM) developed that were very similar to those seen
under ischemia (not shown). Also similar to ischemia, the inward
current was preceded by increased synaptic activity (Fig. 8). This as
well as the progressive inward current and
[Ca2+]i increase were
suppressed by preincubation with CNQX plus APV (n = 4).
However, within several minutes after washout of CNQX/APV in the
presence of ouabain, the inward current and
[Ca2+]i rise developed
(Fig. 8).
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In 12 of 19 DVN, iodoacetate evoked a sustained outward current
(0.1 ± 0.04 nA) and a minor rise of
[Ca2+]i (45 ± 15 nM) after 5 min. As exemplified for the cell of Fig. 9A that did not show an
initial iodoacetate-evoked outward current, a progressive inward
current of 1.6 ± 0.2 nA and a concomitant [Ca2+]i rise by 1.3 ± 0.1 µM developed after further 17.5 ± 1.2 min. Addition of
CNQX (50 µM) and APV (100 µM) 10 min after start of exposure to
iodoacetate prevented both the inward current and rise of
[Ca2+]i
(n = 5; Fig. 9B).
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DISCUSSION |
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Anoxia response
As established previously, the anoxic hyperpolarization of DVN is
mediated by ATP-sensitive K+ channels
(Ballanyi et al. 1996; Karschin et al.
1998
; Trapp and Ballanyi 1995
).
Sulfonylurea-induced block of the underlying anoxic outward current
revealed a moderate and sustained inward current (Ballanyi and
Kulik 1998
; Trapp and Ballanyi 1995
). This
anoxic inward current was accompanied by a conductance decrease and not an increase as seen on bath application of glutamate. Also spontaneous synaptic activity was not increased under anoxia. Thus it is unlikely that the inward current is caused by elevated levels of interstitial glutamate (see also Haddad and Jiang 1993b
). Probably,
the inward current is due to the anoxia-induced stable rise of
extracellular K+ by 1 mM (Ballanyi et al.
1996
). Also the minor stable increase in
[Ca2+]i in response to
block of aerobic metabolism is not caused by activation of ionotropic
glutamate receptors because it was not attenuated by CNQX and APV.
Similar observations were made for striatal interneurons (Pisani
et al. 1999
) and dopaminergic neurons (Guatteo et al.
1998
) of brain slices. The finding that CNQX plus APV abolishes
the glutamate-induced
[Ca2+]i transient
indicates that glutamate does not promote Ca2+
release from intracellular stores via metabotropic receptors (Hollmann and Heinemann 1994
; Metzger et al.
2000
; Schoepp et al. 1990
) in DVN. This is
substantiated by our observation that [1S,3R]-ACPD does not mimic
effects of anoxia (Bickler and Hansen 1994
). The
anoxia-related [Ca2+]i
rise persists in Ca2+-free solution (present
study) and is not changed by depletion of CPA-sensitive
Ca2+ stores (Kulik and Ballanyi
1998
). This suggests that inhibition of
Ca2+ uptake into or release from mitochondria
(Budd and Nicholls 1996
; Schinder et al.
1996
) causes the anoxic
[Ca2+]i rise in DVN.
However, because CPA might not deplete all types of nonmitochondrial
Ca2+ stores (Golovina and Blaustein
1997
; Meldolesi and Pozzan 1998
), drugs such as
dantrolene, ryanodine, or caffeine should be used in future studies to
address this issue in more detail.
Role of glutamate in the ischemia response
In contrast to anoxia, ischemia leads to a prominent
depolarization of DVN, which is very similar to that observed during anoxia alone in cortical neurons (Ben-Ari 1990;
Bures and Buresova 1957
; Silver and Erecinska
1990
). We found that the underlying progressive inward current
and conductance increase are abolished by blockers of ionotropic
glutamate receptors. This indicates that accumulation of extracellular
glutamate (Benveniste et al. 1984
; Choi
1988
; Roettger and Lipton 1996
) is responsible
for the ischemia-induced depolarization. Glutamate can accumulate to
millimolar concentrations in the synaptic cleft during physiological activity (Clements 1996
). Thus the postsynaptic
glutamate receptors might be exposed to even higher concentrations
during impairment of metabolism, which causes massive release of this
neurotransmitter, often associated with impaired glutamate uptake
(Hansen 1985
; Szatkowski and Attwell
1994
). Accordingly, we have shown here that millimolar
concentrations of bath-applied glutamate mimic the ischemia-induced
inward current, conductance increase, and [Ca2+]i elevation. The
ischemic (and the glutamate-induced) inward current and the prominent
Ca2+ rise are only blocked by combined
application of CNQX and APV. Thus AMPA/KA as well as NMDA receptors
appear to contribute to the membrane depolarization of DVN on ischemia
as shown for the response to anoxia of cortical neurons
(Müller and Somjen 1998
; Silver and
Erecinska 1990
; Tanaka et al. 1997
).
The established Ca2+ permeability of the NMDA
receptor (MacDermott et al. 1986) makes it plausible
that a considerable component of the ischemic
[Ca2+]i rise in the DVN
is caused by these receptors. However, a major portion of the ischemic
elevation of [Ca2+]i
persisted during blockade of NMDA receptors.
Ca2+-permeable AMPA/KA receptors (Hollmann
and Heinemann 1994
; Metzger et al. 2000
;
Pellegrini-Giampietro et al. 1997
) do not appear to be
responsible for the
[Ca2+]i rise remaining
after block of NMDA receptors because NHPPS, an inhibitor of such
channels (Weigand and Keller 1998
), does not affect the
glutamate-evoked [Ca2+]i
rise. This suggests that insufficient space clamp during AMPA/KA receptor-mediated currents results in activation of voltage-activated Ca2+ channels that promote considerable
Ca2+ influx into the DVN (Ballanyi and
Kulik 1998
). A significant contribution of
Ca2+ channels is also indicated by our finding
that the glutamate-induced [Ca2+]i transient is
greatly reduced under voltage clamp and diminished further at more
negative holding potentials (Metzger et al. 2000
). Despite lack of direct Ca2+ influx via AMPA/KA
receptors, their activation has a major contribution to the
ischemia-induced [Ca2+]i
rise because the resulting rapid and prominent depolarization opens
voltage-activated Ca2+ channels and furthermore
removes the Mg2+ block of NMDA
receptors (Hollmann and Heinemann 1994
). In addition to
such channel-mediated Ca2+ influx, inhibition or
reversed action of plasmalemmal Ca2+ transporters
such as Na+/Ca2+-exchanger
or Ca2+/H+-ATPase might
constitute a further source of cellular Ca2+
accumulation during ischemia (Blaustein and Lederer
1999
; Trapp et al. 1996
).
Source of ischemic glutamate release
Removal of extracellular Ca2+ delays the
onset of the ischemia-related inward current approximately twofold.
This, and also the occurrence of spontaneous CNQX/APV-sensitive
EPSCs in the DVN, suggests that the early phase of ischemic glutamate
release is caused by Ca2+-dependent exocytosis as
also proposed for other neuronal structures (Drejer et al.
1988; Katayama et al. 1991
).
Ca2+ release from presynaptic stores seems to
contribute noticeably to ischemia-induced
Ca2+-dependent exocytosis as depletion of
Ca2+ stores with CPA before ischemia further
delayed the inward current by several minutes in the absence of
extracellular Ca2+. A reversed action of
glutamate transporters does not appear to play a major role during the
early phase of ischemic glutamate release because preincubation in a
solution containing DHK, THA, and PDC as uptake blockers
(Barbour et al. 1989
; Madl and Burgesser 1993
; Obrenovitch et al. 1998
; Roettger
and Lipton 1996
) does not delay the inward current. Because the
drugs are also ineffective in Ca2+-free saline,
glutamate transport apparently has only a minor overall contribution to
ischemic glutamate release in the dorsal vagal complex as was also
hypothesized for other brain regions (Bickler and Hansen
1994
; Drejer et al. 1985
; Obrenovitch et
al. 1998
; O'Reagan et al. 1995
). In contrast to
DHK, THA, and PDC, arachidonic acid significantly delayed the ischemic
inward current in both Ca2+-containing and
Ca2+-free solution. Further analysis is required
to elucidate whether the protective action of arachidonic acid is due
to its inhibitory effect on glutamate transporters (Barbour et
al. 1989
; Szatkowski and Attwell 1994
). As an
alternative explanation, arachidonic acid might block vesicular uptake
(Roseth et al. 1998
), thereby decreasing the
compartmentalized presynaptic pool of releasable glutamate. Finally,
arachidonic acid might be converted in the slices, for example, into
prostaglandins that mediate its protective effect (Barbour et
al. 1989
). Recently, it was described that glutamate release in
response to ischemia (Phillis et al. 1997
) or spreading
depression (Basarsky et al. 1999
) can occur via efflux through anion channels that are activated by cellular swelling secondary to disturbance of ion homeostasis. In the latter reports, in
particular NPPB was effective to suppress this source of pathological glutamate release, whereas the drug had no effect in the present study.
Several groups have reported that ischemia-induced glutamate release is
associated with a breakdown of membrane function (Bickler and
Hansen 1994; Katayama et al. 1991
;
O'Reagan et al. 1995
). One of these studies presented
evidence that anoxia- and ischemia-induced Ca2+
influx and ATP loss in neurons of cortical slices coincide with membrane damage (Bickler and Hansen 1994
). In glial
cells as well as in neuronal somata or presynaptic structures,
ischemia-induced membrane damage is likely to result in an increase of
extracellular K+ to several tens of millimolar
(Ballanyi et al. 1999
; Hansen 1985
; Völker et al. 1995
). However, no major inward
current resulting from such putative K+
accumulation (compare Fig. 6 in Trapp and Ballanyi 1995
)
is revealed during ischemia or block of glycolysis with iodoacetate
after preincubation with CNQX and APV. Therefore at least on a time scale of ~30 min, membrane damage does not seem to occur within the
dorsal vagal complex. For interpretation of the minor effects of
ischemia on the postsynaptic membrane properties and
[Ca2+]i of DVN in the
presence of CNQX and APV, it needs to be considered that the patch
electrodes contained 1 mM ATP. For anoxia, an almost identical membrane
response is observed in DVN on whole cell or sharp microelectrode
recording (Ballanyi et al. 1996
; Cowan and Martin 1992
). Furthermore, anoxia produces an almost identical moderate and stable
[Ca2+]i rise in intact
fura-2 esterloaded and fura-2 dialyzed whole cell recorded DVN
(Ballanyi and Kulik 1998
). Nevertheless, the effects of
long-term ischemia (after block of ionotropic glutamate receptors) on
postsynaptic membrane properties and
[Ca2+]i in DVN should be
studied in detail in future studies in cells that are not dialyzed with ATP.
Metabolism of the dorsal vagal nucleus
No major ischemic inward current as indication of interstitial
accumulation of either neuromodulators or K+
develops within >30 min in the DVN during block of ionotropic glutamate receptors. This shows that the cellular elements of the
dorsal vagal complex surrounding the recorded postsynaptic neuron are
not only extremely tolerant to anoxia, but also to complete metabolic
arrest. This can only be explained by a particularly low metabolic rate
that is a major constituent of the anoxia tolerance of the brain of
cold-blooded vertebrates and perinatal mammals (Ballanyi et al.
1999; Lutz 1992
; Völker et al.
1995
). A low rate of energy consumption could maintain cellular
ATP levels for extended time periods sufficiently high to maintain
basic cellular function. Resting
Na+/K+ pump activity within
the dorsal vagal complex appears to be low because no change in resting
current as indication of interstitial K+
accumulation due to perturbance of ion homeostasis was revealed in the
presence of CNQX/APV after treating of the slices with ouabain.
However, ouabain induced within several minutes interstitial accumulation of glutamate almost identical to that on ischemia. This is
consistent with the assumption that depolarization of presynaptic
structures due to block of the
Na+/K+ pump secondary to a
fall of ATP is a major factor of ischemic glutamate release
(Haddad and Jiang 1993a
; Madl and
Burgesser 1993
).
The delay of onset (18 min) of the iodoacetate-induced progressive
inward current and
[Ca2+]i rise of DVN was
very similar to that observed for the inward current and perturbation
of ion homeostasis of the in vitro respiratory network of newborn rats
(Ballanyi et al. 1999; Völker et al. 1995
). This supports the above view that the metabolic rate of DVN is similarly low as that of anoxia-tolerant immature brain structures (see also Ballanyi et al. 1996
;
Ballanyi and Kulik 1998
; Trapp and Ballanyi
1995
). A low metabolic rate is also suggested by the finding
that, in contrast to mature neurons of most brain regions
(Calabresi et al. 1997
; Haddad and Jiang
1993a
,b
; Hansen 1985
; Takata et al.
1995
), glucose-free superfusate neither causes a progressive
inward current nor a major rise of
[Ca2+]i in the DVN within
at least 30 min (see also Knöpfel et al. 1990
).
However, this tolerance to glucose depletion might also indicate that
(glial) glycogen stores allow for sustained glycolytic energy
production during anoxia (Tsacopoulos and Magistretti
1996
). Stimulation of anaerobic metabolism during anoxia
("Pasteur effect") in combination with a high glycolytic capability
of structures involved in homeostasis of glutamate within the dorsal
vagal complex is a possible explanation for the lack of a
"terminal" glutamate-induced depolarization during oxygen depletion
alone (Ballanyi et al. 1999
; Lutz
1992
; Völker et al. 1995
).
Conclusions
The present study investigated the contribution of glutamate
release to the response of the medullary dorsal vagal complex to anoxia
and ischemia. For this purpose, whole cell recorded DVN in brain stem
slices were dialyzed with fura-2, and their glutamate receptors and
[Ca2+]i transients were
used as potential sensors of extracellular glutamate. We found that
glutamate is not involved in the moderate response to anoxia. In
contrast, ischemia (as well as iodoacetate and ouabain) promote a
profound rise of interstitial glutamate concentration. The ischemic
glutamate increase evokes a prominent depolarization and a
[Ca2+]i increase caused
by Ca2+ influx through both the NMDA receptor and
voltage-activated Ca2+ channels. The occurrence
of EPSCs and the delay of the ischemic inward current by
Ca2+-free superfusate and block of
Ca2+ release show that the initial phase of the
ischemic glutamate rise is exocytotic. As glutamate transport blockers
and an inhibitor of volume-sensitive Cl
channels are not effective, the origin of the late phase of ischemic glutamate release in the dorsal vagal complex remains unclear. The
glycolytic blocker iodoacetate produced a similar CNQX/APV-sensitive inward current and
[Ca2+]i rise as ischemia.
Thus it appears that effective utilization of anaerobic metabolism by
structures involved in glutamate homeostasis constitutes an important
factor of the anoxia tolerance of dorsal vagal neurons.
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ACKNOWLEDGMENTS |
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We thank A.-A. Grützner for expert technical assistance, Dr. J. Brockhaus for critical reading of the manuscript, and Dr. B. U. Keller for the generous gift of NHPPS.
This study was supported by the Deutsche Forschungsgemeinschaft and the Hermann und Lilly Schilling-Stiftung.
Present address of S. Trapp: University Laboratory of Physiology, Parks Rd., Oxford OX1 3PT, UK.
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FOOTNOTES |
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Address for reprint requests: K. Ballanyi, II. Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Gottingen, Germany.
E-mail: kb{at}neuro-physiol.med.uni.goettingen.de
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 September 1999; accepted in final form 26 January 2000.
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REFERENCES |
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