Brain Research Institute, University of Zurich, CH-8057 Zurich, Switzerland
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ABSTRACT |
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Tanabe, Mitsuo, Masahiro Mori, Beat H. Gähwiler, and Urs Gerber. Apamin-Sensitive Conductance Mediates the K+ Current Response During Chemical Ischemia in CA3 Pyramidal Cells. J. Neurophysiol. 82: 2876-2882, 1999. Pyramidal cells typically respond to ischemia with initial transient hyperpolarization, which may represent a neuroprotective response. To identify the conductance underlying this hyperpolarization in CA3 pyramidal neurons of rat hippocampal organotypic slice cultures, recordings were obtained using the single-electrode voltage-clamp technique. Brief chemical ischemia (2 mM 2-deoxyglucose and 3 mM NaN3, for 4 min) induced a response mediated by an increase in K+ conductance. This current was blocked by intracellular application of the Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), reduced with low external [Ca2+], and inhibited by a selective L-type Ca2+ channel inhibitor, isradipine, consistent with the activation of a Ca2+-dependent K+ conductance. Experiments with charybdotoxin (10 nM) and tetraethylammonium (TEA; 1 mM), or with the protein kinase C activator, phorbol 12,13-diacetate (PDAc; 3 µM), ruled out an involvement of a large conductance-type or an apamin-insensitive small conductance, respectively. In the presence of apamin (1 µM), however, the outward current was significantly reduced. These results demonstrate that in rat hippocampal CA3 pyramidal neurons an apamin-sensitive Ca2+-dependent K+ conductance is activated in response to brief ischemia generating a pronounced outward current.
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INTRODUCTION |
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Considerable variation in the susceptibility to
ischemic damage is observed among neuronal cell types and regions in
the CNS (Pulsinelli 1985). The elucidation of the
mechanisms conferring partial resistance to specific classes of neurons
during energy deprived states would advance our understanding of
cellular homeostatic processes and may provide new leads for drug
development. A majority of studies addressing these questions have
focused on the hippocampus, which exhibits a characteristic pattern of
neuronal damage. CA1 pyramidal cells show the greatest sensitivity to
ischemic insults, whereas CA3 pyramidal cells remain relatively
resistant. This pattern is observed both in the hippocampus of stroke
patients as well as in hippocampal in vitro models of ischemia
(Schmidt-Kastner and Freund 1991
).
Differences in functional properties between CA1 and CA3 pyramidal
cells after ischemia have also been noted. Thus CA1 cells exhibit a
decrease in excitability during hypoxia (Fujiwara et al.
1987; Hansen et al. 1982
; Leblond and
Krnjevi
1989
; Nowicky and Duchen 1998
;
Urban et al. 1989
), which is not observed in CA3 neurons
(Howard et al. 1998
), long-term potentiation
(LTP) can no longer be induced in CA1 versus CA3 cells after
ischemia (Kirino et al. 1992
), and CA1 cells are more
prone to hypoxic seizures than CA3 cells (Kawasaki et al.
1990
). The decreased excitability of CA1 cells is largely due
to a transient hyperpolarization mediated by an increase in potassium
conductance (Erdemli et al. 1998
; Fujiwara et al.
1987
; Hansen et al. 1982
; Leblond and
Krnjevi
1989
). Recent studies have shown that this
conductance corresponds to the calcium-dependent and
charybdotoxin-sensitive K+ current
IC, conducted by large conductance
(BK) channels (Harata et al. 1997
;
Nowicky and Duchen 1998
). A brief period of ischemia similarly induces hyperpolarization or a transient outward current in
CA3 pyramidal cells. (Ben-Ari 1990
; Takata and
Okada 1995
; Tanabe et al. 1998
). The aim of the
present investigation was to characterize this current and to determine
whether its properties differ from the ischemic current described in
CA1 neurons.
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METHODS |
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Experiments were performed with organotypic hippocampal slice
cultures. Hippocampi were removed aseptically from 6-day-old Wistar
rats that had been killed by cervical dislocation. Tissue slices of
400-µm thickness were prepared and cultured by means of the roller
tube technique as described previously (Gähwiler 1981). After 15-30 days in vitro, the cultures were
transferred to a recording chamber mounted onto the stage of an
inverted microscope (Axiovert 35 M, Zeiss, Jena, Germany), and
superfused with an external solution (at 32°C, pH 7.4) containing (in
mM) 148.9 Na+, 2.7 K+,
146.2 Cl
, 2.8 Ca2+, 0.5 Mg2+, 11.6 HCO3
, 0.4 H2PO4
, and 5.6 D-glucose. Single-electrode voltage-clamp recordings were
made from CA3 pyramidal neurons (Axoclamp 2 amplifier, Axon Instruments, Foster City, CA) with microelectrodes having tip resistances of 50-80 M
and filled with 2 M potassium
methylsulphate. Most experiments were performed in the presence of
tetrodotoxin (TTX, 0.5 µM) at a holding potential
(VH) between
50 and
60 mV. Slow
afterhyperpolarization currents
(sIAHP) were evoked with a brief
depolarizing voltage jump (to 0 mV for 100-200 ms, 0.04 Hz). Input
resistance was assessed with 500-ms hyperpolarizing voltage commands of
10 mV. Chemical ischemia was induced by bath application of sodium
azide (NaN3, 3 mM) and 2-deoxyglucose (2-DG, 2 mM) to block the cytochrome oxidase system and glycolysis,
respectively. The duration of chemical ischemia is indicated by a
horizontal bar in the figures (normally 4 min). In experiments to
assess the role of extracellular Ca2+, the normal
superfusing solution was replaced with one containing low
Ca2+ (0.5 mM) high Mg2+ (10 mM). Completely removing extracellular Ca2+
resulted in unstable recordings. When working with large polypeptides such as apamin, variability in responses can arise, which may reflect
problems with tissue penetration (Lancaster and Adams 1986
). For these experiments, we therefore preincubated tissue with apamin (1 µM) for 15 min.
NaN3, 2-DG, tolbutamide, and tetraethylammonium chloride (TEA) were purchased from Sigma (St. Louis, MO). Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) was from Molecular Probes (Eugene, OR). Phorbol 12,13-diacetate (PDAc) was from LC Laboratories Europe (Läufelfingen, Switzerland). TTX was from Sankyo (Tokyo). Isradipine was a gift from Novartis (Basel). All drugs except BAPTA were directly applied via the superfusing solution.
Numerical data are expressed as means ± SE. Student's t-test was used to compare means when appropriate. P < 0.05 was considered significant.
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RESULTS |
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Brief chemical ischemia induces outward current in CA3 cells
Transient chemical ischemia (2 mM 2-DG and 3 mM
NaN3 for 4 min) induced an outward current
(348 ± 23 pA, mean ± SE, n = 64) associated
with a conductance increase (28 ± 3%, n = 64) in
CA3 hippocampal pyramidal cells in the presence of TTX (Fig.
1A). Reoxygenation often induced an additional outward current, reflecting reactivation of the electrogenic membrane
Na+-K+ pump
(Fujiwara et al. 1987). This transporter response was
not further examined in this study. The current-voltage relationship of
the outward current induced by ischemia was determined in the range
from
130 to
50 mV using step pulse commands (500 ms duration) with
10-mV increments from a VH of
50 mV.
The reversal potential was
96 ± 6 mV (n = 4),
which is close to the theoretical equilibrium potential for
K+ (
100 mV) with 2.7 mM
K+ in the external solution, assuming an
intracellular K+ concentration of 120 mM for CA3
pyramidal cells (Lüthi et al. 1996
) (Fig.
1B). With 8.0 mM K+ in the external
solution the reversal potential shifted to
70 ± 4 mV, which is
close to the predicted value of
72 mV according to the Nernst
equation (n = 4; Fig. 1C).
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In subsequent experiments, the nature of the K+
conductance underlying the outward current was investigated. During
transient energy deprivation the intracellular
[Ca2+] in neurons increases (Szatkowski
and Attwell 1994). To determine whether the
K+ conductance activated during ischemia is
sensitive to changes in intracellular [Ca2+],
responses were examined in cells filled with BAPTA, a
Ca2+ chelator. BAPTA (150 mM in the
microelectrode), applied by ionophoretic injection for 20 min,
effectively chelated intracellular Ca2+, as shown
by the complete blockade of the Ca2+-dependent
IAHP (Fig.
2A2). In
BAPTA-loaded neurons, the outward K+ current was
no longer observed during ischemia, but rather an inward current became
apparent (278 ± 138 pA, n = 5; Fig.
2A1). A similar inward current also occurred when
K+ conductance was blocked with 2 M CsCl in the
electrode and 10 mM TEA in the extracellular solution (185 ± 62 pA, n = 5, not shown).
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Previous studies have shown that an ATP-sensitive
K+ conductance (Fujimura et al.
1997; Fujiwara et al. 1987
; Jiang et al. 1994
) contributes to hyperpolarization observed in response to hypoxia. In CA3 cells, however, tolbutamide (500 µM), a sulfonylurea that specifically blocks ATP-sensitive K+
conductance (Ashcroft 1988
), did not significantly
change the amplitude of the ischemic K+ current
(control 473 ± 123 pA vs. tolbutamide 603 ± 153 pA,
n = 5; Fig. 2B). These results indicate that
in CA3 pyramidal cells the outward current induced by ischemia is
primarily due to the activation of a
Ca2+-activated K+ conductance.
Activation of the ischemic K+ current depends on Ca2+ influx
The amplitude of the ischemic K+ current was
reduced with low Ca2+ (0.5 mM) high
Mg2+ (10 mM) in the external solution (control
338 ± 65 pA vs. low Ca2+ 71 ± 8 pA,
P < 0.05, n = 5, Fig.
3A). This
suggests that the activation of ischemic K+
current is mediated by Ca2+ influx from the
extracellular compartment. A recent report has shown that in CA1
pyramidal cells, Ca2+ influx through L-type
Ca2+ channels is required to induce the outward
K+ current in response to chemical ischemia
(Nowicky and Duchen 1998). To examine whether a similar
mechanism operates in CA3 pyramidal cells, the ischemic
K+ current was recorded in the presence of a
dihydropyridine derivative, isradipine. For these experiments, cells
were clamped at a depolarized holding potential of
40 mV, because the
blockade of L-type Ca2+ current by
dihydropyridines is voltage and use dependent (Gähwiler and Brown 1987
). In cells exposed to isradipine (2 µM for 7 min) the ischemic K+ current was strongly
attenuated (control 338 ± 58 pA, isradipine 58 ± 16 pA,
n = 5; P < 0.05, Fig.
3B).
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Ca2+-dependent K+ conductance mediating the outward current is apamin sensitive
Three types of Ca2+-dependant
K+ currents have been characterized in
hippocampal pyramidal neurons, the fast afterhyperpolarization current
IC, and two types of slow
afterhyperpolarization currents IAHP
and sIAHP (Sah and Clements
1999; Storm 1993
).
IC is a large conductance
Ca2+-dependent K+ current
that underlies the fast components of the AHP. Submillimolar concentrations of extracellular TEA block
IC, thereby prolonging action
potential duration (Lancaster and Nicoll 1987
;
Storm 1989
). Experiments were performed to evaluate the
possible contribution of IC to the
ischemic outward current. Although TEA (1 mM) increased the duration of
action potentials, the ischemic response was not modified (Fig.
4A, n = 3). IC is conducted
by BK channels, which are selectively blocked by charybdotoxin
(Miller et al. 1985
). To confirm the finding with TEA,
the effects of charybdotoxin on the outward current were examined.
Slice cultures were preincubated with charybdotoxin (10 nM) for 15 min
to prevent activation of BK channels. As previously shown
(Lancaster and Nicoll 1987
; Storm 1989
),
charybdotoxin under control conditions prolonged the repolarization phase of the action potential (Fig. 4B2, n = 4). The
ischemic K+ current response was, however, not
affected (Fig. 4B1, n = 4). Even at a higher
concentration of charybdotoxin (300 nM), the ischemic
K+ current was not inhibited (n = 5, data not shown), ruling out an involvement of
IC.
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The possibility that sIAHP corresponds
to the ischemic K+ current was then tested. This
current is insensitive to apamin (Lancaster and Nicoll
1987), but is blocked by protein kinase C activators (Baraban et al. 1985
).
sIAHP was activated by clamping cells
at potentials between
50 and
60 mV and applying voltage step
commands to 0 mV for 200 ms every 25 s. Although
sIAHP was completely suppressed by
PDAc (3 µM), an activator of protein kinase C, this treatment did not
reduce the ischemic K+ current (Fig.
5A).
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Hippocampal CA1 pyramidal cells exhibit an additional calcium-dependent
IAHP mediated by apamin-sensitive
small conductance (SK) channels (Sah and Clements
1999; Stocker et al. 1999
). Furthermore, autoradiographic studies provide evidence for the expression of apamin
binding sites in the hippocampal pyramidal cell layer (Gehlert and Gackenheimer 1993
; Mourre et al. 1986
). We
therefore tested whether an apamin-sensitive Ca2+-dependent
K+ current mediates the ischemic current in CA3 cells.
Slice cultures were preincubated with apamin (1 µM, 15 min). At this
concentration, apamin did not modify the resting membrane potential.
The outward K+ current induced by ischemia was, however,
significantly decreased in the presence of apamin (36 ± 44 pA,
n = 7, P < 0.05; Fig.
5B).
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DISCUSSION |
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Our results show that the transient outward current observed in
CA3 pyramidal cells in response to a brief period of energy deprivation
is mediated by a Ca2+-dependent
K+ conductance. As has been demonstrated in CA1
pyramidal cells (Nowicky and Duchen 1998), this response
is blocked by inhibitors of voltage-gated L-type
Ca2+ channels, indicating that an influx of
extracellular Ca2+ triggers the outward
K+ current. In CA1 cells, mainly BK channels
appear to conduct this Ca2+-dependent
K+ current (Harata et al. 1997
;
Nowicky and Duchen 1998
). In contrast, our experiments
demonstrate that in CA3 cells activation of apamin-sensitive SK
channels underlies the K+ current.
Ischemic outward current
Many types of central neurons respond to energy deprivation with
an initial transient hyperpolarization resulting from the activation of
a membrane K+ conductance (see Luhmann
1996, for review). Two main mechanisms have been put forward to
explain this increase in conductance. The first is the activation of
ATP-sensitive K+ channels that open in response
to decreased levels of cytosolic ATP under ischemic conditions
(Ashcroft 1988
). In the hippocampus, however,
experiments in which ATP-sensitive K+ channels were blocked
with sulfonylureas have provided contradictory results. Application of
the sulfonylurea tolbutamide significantly reduced hypoxic or ischemic
responses in CA1 cells (Fujimura et al. 1997
;
Godfraind and Krnjevi
1993
; Grigg and
Anderson 1989
), but was reported to have no effect in other
studies (Harata et al. 1997
; Nowicky and Duchen
1998
). Our results indicate that in CA3 cells ATP-sensitive
K+ channels do not contribute to the ischemic outward
current, as previously reported (Ben-Ari 1990
).
Our results are consistent with the second proposed mechanism involving
the activation of a K+ conductance sensitive to a rise in
intracellular [Ca2+]. The dependency of the
K+ conductance on an increase in cytosolic
[Ca2+] was shown in our experiments where the ischemic
outward current was prevented by chelation of intracellular
Ca2+ with BAPTA, as previously reported in CA1 cells
(Erdemli et al. 1998; Yamamoto et al.
1997
). Moreover, the ischemic hyperpolarization or outward
current occurs during the initial phase of ischemia, which is
associated with an increase in cytosolic [Ca2+] in
hippocampal neurons (Dubinsky and Rothman 1991
;
Duchen et al. 1990
; Kass and Lipton 1986
;
Mitani et al. 1993
; Nowicky and Duchen
1998
).
Ca2+ dependence of the current
The ischemic outward current was reduced by decreasing
extracellular [Ca2+] and abolished in the
presence of isradipine, a selective L-type Ca2+ channel antagonist, indicating that
Ca2+ influx is involved in generating the
response. A similar observation was reported in CA1 cells, where
Ca2+ influx through L-type channels appears to
account for most of the increase in intracellular
Ca2+ concentration (Nowicky and Duchen
1998). Furthermore, L-type channel blockers have been shown to
prevent the Ca2+ rise in cortical cells during
ischemia (Pisani et al. 1998
). The mechanism through
which voltage-gated Ca2+ channels are activated
during the ischemic hyperpolarization remains unresolved. One
possibility might be that energy deprivation causes a dysfunction in
the voltage-sensing elements of the channel proteins, which could shift
the voltage sensitivity to a more hyperpolarized level. In addition,
even a small influx of Ca2+ may suffice to induce
a large intracellular signal through a process of amplification
involving Ca2+ release from intracellular stores
(Belousov et al. 1995
; Yamamoto et al.
1997
).
Apamin-sensitive Ca2+-dependent K+ conductance is responsible for the ischemic K+ current
Three types of Ca2+-dependent
K+ conductances have been characterized in
central neurons, a charybdotoxin-sensitive BK conductance, an
apamin-insensitive SK conductance, and an apamin-sensitive SK
conductance (Sah 1996). In CA1 cells the increase in
intracellular Ca2+ during energy deprivation
activates a Ca2+-dependent
K+ current that is blocked by charybdotoxin
(Harata et al. 1997
; Nowicky and Duchen
1998
). The sensitivity of the response to charybdotoxin identifies the underlying current as
IC, which is carried by BK channels
(Miller et al. 1985
). In CA3 cells, however, blockade of
IC by TEA or charybdotoxin failed to
inhibit the ischemic K+ current. The
Ca2+-dependent K+ current,
sIAHP, underlying the slow
afterhyperpolarization, may also contribute to the hypoxic response
in CA1 cells, as muscarinic and
-adrenergic agonists suppress both
the hypoxic hyperpolarization as well as the
sIAHP (Erdemli et al.
1998
; Krnjevi
and Xu 1990
). Again, in CA3
cells under conditions where sIAHP was
blocked, we observed no change in the outward K+
current response. The third type of
Ca2+-dependent K+ current,
which is sensitive to apamin and is mediated by SK channels, has been
described in hippocampal interneurons (Zhang and McBain 1995
) and CA1 pyramidal cells (Sah and Clements
1999
; Stocker et al. 1999
), but not in CA3
pyramidal cells. In our experiments, apamin significantly inhibited the
Ca2+-dependent K+ current
induced by ischemia. Thus CA3 pyramidal cells also express an
apamin-sensitive K+ conductance. Furthermore,
although our results show that CA3 cells possess all three types of
known Ca2+-dependent K+
conductances, the increase in intracellular Ca2+
occurring during ischemia appears to preferentially activate the
apamin-sensitive channels. It is unclear why the other
Ca2+-activated K+ currents
are not activated by the increase in intracellular
Ca2+. The sIAHP
is blocked during ischemia (Tanabe and Gerber, unpublished results),
suggesting that an energy-dependent process is involved in the
activation of these channels. Alternatively,
sIAHP, which is strongly modulated by
various neurotransmitters through second-messenger systems
(Nicoll 1988
), may be inhibited as a result of an
intracellular disequilibrium during energy deprivation. In the case of
the fast IAHP mediated by BK channels,
further experiments will be required to explain the difference in the
response between CA1 and CA3 pyramidal cells.
In conclusion, we have identified a difference in the mechanism
underlying the transient hyperpolarization in response to energy
deprivation in two main types of hippocampal pyramidal cells. During
hypoxia or ischemia, an increase in extracellular glutamate depolarizes
neurons, a process that can ultimately end in excitotoxic cell death
through Ca2+ overloading (Szatkowski and
Attwell 1994). Hyperpolarization of CA3 pyramidal cells through
activation of the apamin-sensitive K+ conductance
would tend to prevent the membrane potential from attaining the
threshold for voltage-dependent Ca2+ influx, thus
protecting cells. Future work will aim to determine whether this can
account for the contrasting responses and susceptibility of CA1 versus
CA3 pyramidal cells to metabolic stress.
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ACKNOWLEDGMENTS |
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We thank Dr. Y. Fischer for valuable discussions and advice and Dr. R. Dürr, L. Heeb, R. Kägi, H. Kasper, L. Rietschin, and R. Schöb for excellent technical assistance.
This work was supported by Sankyo Ltd., the Ministry of Education, Science, and Culture of Japan, the Prof. Dr. Max Cloëtta Foundation, and the Swiss National Science Foundation (31-45547.95).
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FOOTNOTES |
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Address for reprint requests: U. Gerber, Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
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 26 April 1999; accepted in final form 7 July 1999.
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REFERENCES |
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