Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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Chi, Xian Xuan and Zao C. Xu. Differential Changes of Potassium Currents in CA1 Pyramidal Neurons After Transient Forebrain Ischemia. J. Neurophysiol. 84: 2834-2843, 2000. CA1 pyramidal neurons are highly vulnerable to transient cerebral ischemia. In vivo studies have shown that the excitability of CA1 neurons progressively decreased following reperfusion. To reveal the mechanisms underlying the postischemic excitability change, total potassium current, transient potassium current, and delayed rectifier potassium current in CA1 neurons were studied in hippocampal slices prepared before ischemia and at different time points following reperfusion. Consistent with previous in vivo studies, the excitability of CA1 neurons decreased in brain slices prepared at 14 h following transient forebrain ischemia. The amplitude of total potassium current in CA1 neurons increased ~30% following reperfusion. The steady-state activation curve of total potassium current progressively shifted in the hyperpolarizing direction with a transient recovery at 18 h after ischemia. For transient potassium current, the amplitude was transiently increased ~30% at ~12 h after reperfusion and returned to control levels at later time points. The steady-state activation curve also shifted ~20 mV in the hyperpolarizing direction, and the time constant of removal of inactivation markedly increased at 12 h after reperfusion. For delayed rectifier potassium current, the amplitude significantly increased and the steady-state activation curve shifted in the hyperpolarizing direction at 36 h after reperfusion. No significant change in inactivation kinetics was observed in the above potassium currents following reperfusion. The present study demonstrates the differential changes of potassium currents in CA1 neurons after reperfusion. The increase of transient potassium current in the early phase of reperfusion may be responsible for the decrease of excitability, while the increase of delayed rectifier potassium current in the late phase of reperfusion may be associated with the postischemic cell death.
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INTRODUCTION |
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CA1 pyramidal neurons in
hippocampus start to degenerate 2-3 days after transient cerebral
ischemia and 90% of them die in 1 wk (Kirino 1982;
Pulsinelli et al. 1982
). The mechanisms of this delayed
cell death are under active investigation. It has been postulated that
the neuronal hyperactivity due to an elevation of extracellular
excitatory amino acids during ischemia leads to a massive increase in
intracellular-free Ca2+, which may trigger the
process of neuronal degeneration (Choi and Rothman 1990
;
Rothman and Olney 1986
). To test the excitotoxic hypothesis, many investigators examine the neuronal activity during hypoxia/ischemia and after reperfusion. It has been shown that the
neuronal activity was reduced and the excitability was depressed during
hypoxia in vitro (Boissard and Gribkoff 1993
;
Fujiwara et al. 1987
; Hansen et al. 1982
;
Leblond and Krenjevic 1989
) and during ischemia in vivo
(Xu and Pulsinelli 1994
, 1996
). Because the elevated
extracellular concentration of glutamate and aspartate returns to
control levels 30 min after reperfusion (Benveniste et al.
1984
; Mitani et al. 1992
) and the CA1 neurons
begin to die in 2-3 days, the changes of neuronal activity after
reperfusion may be more important in the process of cell death than
those during ischemia. Due to the technical limitations, it is
difficult to study the electrophysiological changes after reoxygenation in an in vitro preparation. Using brain slices prepared at different intervals following ischemia in vivo, Urban et al.
(1989)
have shown that the population spike was unchanged or
reduced 5-10 h after reperfusion and was markedly depressed by 24 h reperfusion, suggesting the depression of neuronal excitability
following reperfusion. Using extracellular recording techniques in
vivo, early studies have shown that the neuronal firing rate increases
in CA1 region following reperfusion (Chang et al. 1989
;
Suzuki et al. 1983
). More studies, on the other hand,
report a decrease of neuronal firing rate in CA1 region after
reperfusion (Buzsáki et al. 1989
; Furukawa
et al. 1990
; Mitani et al. 1990
). Using
intracellular recording and staining techniques in vivo, recent studies
have clearly demonstrated that the spontaneous neuronal activity and excitability of CA1 pyramidal neurons significantly decreased
48 h
following transient forebrain ischemia (Gao et al. 1998
, 1999
).
Potassium currents are important for the regulation of neuronal
excitability and the maintenance of baseline membrane potential (Brown et al. 1990; Hille 1992
;
Storm 1990
). The regulation of potassium channel
activity is believed to have a major impact on the overall neuronal
response and adaptation to O2 deprivation (Haddad and Jiang 1993
). During hypoxia in vitro, the
extracellular potassium markedly increases (Haddad and Donnelly
1990
). Activation of potassium channels induces
hyperpolarization, decreases membrane excitability, and reduces
O2 consumption (Belousov et al.
1995
; Croning et al. 1995
; Fujimura et
al. 1997
; Haddad and Jiang 1993
; Yamamoto
et al. 1997
). The alteration of potassium conductance during
hypoxia has been shown to be responsible for the prevailing hyperpolarization, possible through several different types of conductance including Ca2+-sensitive potassium
current (Krnjevic 1993
), ATP-sensitive potassium current
(Ashcroft 1988
), and adenosine activated
G-protein-dependent potassium conductance (Berne et al.
1974
). Voltage-dependent potassium currents are also involved
in neuronal response during hypoxia (Cummins et al.
1991
; Gebhardt 1999
; Hyllienmark and
Brismar 1996
; Krnjevic and Leblond 1989
). The
preceding studies indicate that the increase of potassium conductance
contributes to the decrease of excitability during hypoxia/ischemia.
Little is known about the ionic mechanisms underlying the decrease of
excitability following reperfusion. A recent report has indicated the
increase of total potassium current in CA1 pyramidal neurons 6-8 h
after reperfusion (Chi and Xu 2000
).
In the present study, the hippocampal slices were prepared at different
intervals after ischemia in vivo. The membrane properties of CA1
neurons after reperfusion were studied to confirm the postischemic changes in our in vitro preparation are comparable to those observed in
in vivo preparation. Then the temporal profiles of amplitude and
kinetics of different voltage-dependent potassium currents were
examined 36 h following reperfusion to reveal their roles in
excitability changes in CA1 pyramidal neurons following transient forebrain ischemia.
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METHODS |
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Transient forebrain ischemia
Male adult Wistar rats (150-200 g) were used in the present
study. The National Institutes of Health guides for the care and use of
laboratory animals were strictly followed. Transient forebrain ischemia
was induced using the four-vessel occlusion method (Pulsinelli and Brierley 1979) with modifications (Xu et al.
1999
). Briefly, the animals were fasted overnight and
anesthetized with 1-2% halothane mixed with 33%
O2 and 66% N2. The
vertebral arteries were electrocauterized. The common carotid arteries
were isolated after which an occluding device was placed loosely around
each carotid artery to allow subsequent occlusion of these vessels. The
animal was then placed on a stereotaxic frame and a temperature probe
(0.025-in diam) was placed beneath the skull in the extradural space,
after which brain temperature was maintained at 37°C with a heating
lamp through a temperature control unit (BAT-10, Physitemp). Severe
forebrain ischemia was produced by occluding both common carotid
arteries to induce ischemic depolarization for ~14 min. Animals were
returned to the cage after recovering from ischemia and allowed free
access to water and food. The animals were then re-anesthetized and
prepared for brain slices at different time points after reperfusion.
Electrophysiology
Brain slices were prepared from animals before ischemia and at
6-8, 12-14, 18-20, and 36-38 h following reperfusion as described in previous publications (Chi and Xu 2000). The animals
were anesthetized with ketamine-HCl (13 mg/kg) and decapitated. The
brains were quickly removed and immersed in ice-cold artificial
cerebrospinal fluid (ACSF), which was composed of the following (in
mM): 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. Hippocampal slices of 300 µm thickness were cut using a vibroslice (Campden 752 M), and were
incubated in ACSF for
1 h in room temperature before transferring to
recording chamber. The slice was submerged beneath the fluid surface
and superfused continuously with oxygenated ACSF. The flow rate was
adjusted to 2-3 ml/min. Recordings were carried out at room
temperature (~24°C). Tetrodotoxin (1 µM) and cadmium chloride
(0.3 mM) were added into the solution to block
Na+ and Ca2+ channels.
For intracellular recording, the recording electrodes were pulled from
glass capillaries with filaments to a tip resistance of 50-80 M
when filled with a solution of 3% neurobiotin (Vector) in 2 M
potassium acetate. The microelectrode was advanced slowly into the CA1
pyramidal cell layer. After impalement, the neurons with a stable
membrane potential of
60 mV or greater were selected for further study.
For whole cell recording, patch electrodes were prepared from
borosilicate glass (Warner Instrument) to produce tip openings of 1-2
µm (3-5 M). Electrodes were filled with a intracellular solution
containing (in mM)145 KCl, 1 MgCl2, 10 EGTA, 0.2 CaCl2, and 10 HEPES buffer (Sigma) and 3%
neurobiotin (Vector). CA1 neurons were visualized with an infrared-DIC
microscope (Olympus BX 50 WL) and a CCD camera. Positive pressure was
applied to recording pipette as it was lowered into the medium and
approached the cell membrane. Constant negative pressure was applied to
form the seal (>1 G
) when the recording pipette attached the
membrane. A sharp pulse of negative pressure was applied to open the
cell membrane for whole cell recording.
Voltage-clamp recording was performed with an Axopatch 200 B amplifier
(Axon Instruments). Pipette tip junction potentials were continuously
monitored and compensated as necessary before breakage of the membrane.
No leak current subtraction was performed because we found that the
leak current was much smaller than the currents activated by
depolarization. Cells were voltage-clamped and held near resting
membrane potential (about 60 mV). In brain slice preparation, CA1
pyramidal neurons are not fully space-clamped due to the extensive
dendritic trees. However, it has been shown that the lack of voltage
control in the dendrites did not dramatically alter the kinetics of
potassium current (Surmeier et al. 1994
). Furthermore
the poor space-clamp will not invalidate the outcome of the present
study because the comparison was based on the data collected under the
same preparation before and after ischemia. Signals were filtered at 5 kHz and digitized at a sampling rate of 2 kHz using data-acquisition
program (Axodata, Axon Instruments).
After each successful recording, neurobiotin was iontrophoresed into the cell by passing depolarizing pulses. The slice was then fixed in 4% paraformaldehyde overnight and incubated in 0.1% horseradish peroxidase conjugated avidin D (Vector) in 0.01 M potassium phosphate-buffered saline (pH 7.4) with 0.5% Triton X-100 for 24 h at room temperature. After detection of peroxidase activity with 3,3'-diaminobenzidine, slices were examined in potassium phosphate buffered saline. Slices containing labeled neurons were mounted on gelatin-coated slides and processed for light microscopy.
Data analysis
To establish steady-state activation or inactivation curves, the
peak current (I) was measured at each potential and the
corresponding conductance (G) was calculated with the use of
the following equation
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The values were presented as means ± SE. The Student's t-test (for 2 groups) or ANOVA (for more than 2 groups) was used for statistical analysis (Statview, Abacus Concepts).
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RESULTS |
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Experiments were performed on control rats (n = 25) and rats subjected to forebrain ischemia with ischemic
depolarization of 13.8 ± 0.9 min (n = 45). Such
degree and duration of ischemia consistently produced 90% cell death
in the CA1 region 1 wk after reperfusion (Xu et al.
1999). A total of 81 neurons were analyzed in the present study
of which 60 were successfully stained and identified as CA1 pyramidal
cells. No overt degeneration signs, such as swelling or shrinkage of
cell body and dendritic fragmentation, were observed in these neurons.
However, small beaded dendrites were observed in apical dendrites of
some neurons recorded at 36 h following reperfusion, suggesting
the beginning of degeneration at this time (Fig.
1). Two glial cells and five interneurons
were morphologically identified and were excluded from the analysis. Whole cell recording of CA1 neurons became very difficult at 2 days
after reperfusion, especially in the medial portion in of the CA1 zone.
Hematoxylin-eosin staining was performed in four animals at 48 h
after reperfusion, signs of neuronal death were evident in the medial
portion of CA1 region.
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Membrane properties of CA1 neurons in brain slice following reperfusion
The membrane properties of CA1 pyramidal neurons were compared
before ischemia and at 14-16 h following reperfusion. As shown in
Table 1, the resting membrane potential
of CA1 neurons depolarized from 68 ± 1.62 mV of control value
to
62 ± 1.93 mV after reperfusion (P < 0.05).
The spike width was decreased from 1.11 ± 0.06 to 0.95 ± 0.05 ms (P < 0.05). No significant difference in spike height was observed after reperfusion.
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The changes in neuronal excitability were evaluated by comparing
the spike threshold and rheobase of CA1 neurons before ischemia and
after reperfusion. The spike threshold (54 ± 0.68 vs.
51 ± 1.15 mV, P < 0.05), and rheobase (0.3 ± 0.03 vs. 0.7 ± 0.09 nA, P < 0.05) of CA1 neurons
increased after reperfusion, indicating the decrease of excitability.
To compare the current-voltage relationship of CA1 neurons before and
after reperfusion, constant-current pulses (200 ms, 1.0 to +0.5 nA,
0.1-nA increment) were delivered. The voltage values were measured from
the averages of four recordings at the steady state of the transients
(average between 160 and 180 ms from the beginning of the pulse). The
slope of I-V curve in CA1 neurons significantly decreased
after reperfusion indicating the decrease of input resistant (Fig.
2).
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The postischemic change of fast afterhyperpolarization (fAHP) in CA1 neurons was also examined. The amplitude of fAHP was measured from the beginning of the upstroke of an action potential to the most hyperpolarizing point within 5 ms after the peak of the action potential. The amplitude of fAHP increased from 4.7 ± 0.37 mV of control level to 9.4 ± 0.48 mV at 14-16 h after reperfusion (P < 0.05).
Total potassium current in CA1 neurons after reperfusion
To activate outward currents, membrane potential was held at 60
mV and stepped voltage command pulses (
80 ~ +70 mV, 10 mV/step, 400 ms) were applied following a conditioning voltage step of 300 ms at
120 mV. Outward currents became apparent at approximately
55 mV, and their amplitude increased at more depolarizing potentials (Fig. 3A). The amplitude of
total potassium current was measured at 20 ms after the onset of
command pulses. The peak amplitude of total potassium current
significantly increased from 2.31 ± 0.24 nA of control to
3.08 ± 0.22 nA at 12 h after reperfusion (measured at +70
mV, P < 0.05) and slightly reduced at later time points (Fig. 3B).
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The activation curve was obtained by fitting the data points with a Boltzmann equation. Figure 3C shows that the steady-state activation curve of total potassium current progressively shifted in hyperpolarizing direction with a transient recovery at 18 h after ischemia. The slope of the activation curves did no change after reperfusion (Table 2).
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Steady-state inactivation of total potassium current was studied by
holding the cell for 2 s at potentials between 150 and
10 mV
prior to delivering a testing pulse to +30 mV. Normalized peak current
amplitude as a function of conditioning potential was fitted by a
Boltzmann equation. No significant difference in
V1/2 and
Vc was found in CA1 neurons after
reperfusion (Fig. 3D, Table 2).
Pharmacological intervention allows us to dissect the different
components of potassium current and to investigate their gating properties respectively. The total potassium current was reduced ~60% (1.52 ± 0.20 vs. 0.66 ± 0.08 nA, n = 12, measure at +30 mV, P < 0.01) after bath
application of tetraethylammonium (TEA, 30 mM, Fig.
4A). A slow inactivating
component was evident after subtracting the current following TEA
application from the control one (Fig. 4B). This component
showed little inactivation and resembles delayed rectifier current
(Storm 1990) and indicated as
IKd. After TEA application, a
transient component was also identified in the total outward current.
Bath application of 4-aminopyradine (4-AP, 10 mM) completely blocked
this component (Fig. 4A). The transient component was
isolated by subtracting the current following TEA and 4-AP application
from the control one (Fig. 4C). This transient component
reached its peak within 20 ms following the onset of membrane
depolarization and rapidly inactivated despite the continued membrane
depolarization. The inactivation profile was best fitted by a
monoexponential function. The time constant for the inactivation was
10.03 ± 1.49 ms (n = 12). This current resembles
a transient potassium current and refereed as
IA (Storm 1990
). No
attempt was made to distinguish the slow and fast components of
IA in the present study.
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Transient potassium current in CA1 neurons after reperfusion
IA was isolated by subtracting
slow component from the total potassium current (Klee et al.
1997; Numann et al. 1987
). Membrane potential
was held at
60 mV. Depolarizing potential steps were preceded by 300 ms hyperpolarizing pulse at
120 mV (Fig.
5A, voltage protocol
P1) to evoked outward currents including
IA. IA was inactivated by a 50-ms prepulse
at +10 mV (Fig. 5A, voltage protocol
P2). IA was
obtained by subtracting current evoked from P2
from that evoked from P1. The amplitude of
IA was measured at the peak of the
current (at ~20 ms after the onset of testing pulses). The threshold
for activating IA was around
55 mV.
The amplitude of IA progressively
increased from 0.42 ± 0.06 nA of control to 0.70 ± 0.09 nA
(measured at +30 mV, P < 0.05) at 12 h after
reperfusion. The amplitude returned to control level 18-36 h after
reperfusion (Fig. 5B).
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In whole cell patch-clamp study, the current amplitude may vary due to the variation of cell body volume. In an attempt to eliminate the changes of current amplitude resulting from the changes of cell volume, which may occur after ischemic insult, the current density of IA was compared before ischemia and after reperfusion. The membrane capacitance, which indirectly represents the cell volume, was measured. The current amplitude of IA measured at command pulses of +30 mV was divided by the membrane capacitance of each individual neuron yielding measurement of current density (expressed as pA/pF). The average current density was 32.9 ± 4.2 pA/pF for control and 54.6 ± 7.7 pA/pF for neurons at 12-14 h after reperfusion (n = 5, P < 0.05, Fig. 5C).
Activation curves of IA shifted in
hyperpolarizing direction within 12 h after reperfusion and
returned to the control level at 18-36 h reperfusion. The
V1/2 shifted from 6.31 ± 1.48 mV of
control value to
27.03 ± 1.24 mV at 12 h after reperfusion (P < 0.05, Fig.
6A). Mean
Vc values were similar across
different time points following reperfusion (Table
3). The steady-state inactivation
properties of IA were determined by
measuring current availability following 400-ms prepulses steps to
voltage between
120 and 0 mV with a test pulse to +30 mV. No
significant change in V1/2 and
Vc of inactivation curve was detected
in CA1 neurons after reperfusion (Table 3, Fig. 6B).
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To determine the removal of inactivation of
IA, the cells were held at 20 mV to
inactivate IA, and the membrane
potential was then stepped to
120 mV for periods between 0 and 200 ms
to remove inactivation of IA prior to
a test pulse of +70 mV. The inset in Fig.
7A shows a representative
recording of the time-dependent recovery from inactivation of
IA. Normalized peak
IA was plotted against prepulse
duration to reveal the time course of recovery from inactivation. The
curve was fitted by a single exponential function with the time
constant of 42.39 ± 6.67 ms for control neurons and increased to
96.76 ± 16.52 ms for neurons at 12 h after reperfusion
(P < 0.05). The time constant returned to control level at 36 h after reperfusion (Fig. 7B).
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Delayed rectifier potassium current in CA1 neurons after reperfusion
In the present study, the IKd was
isolated by inactivating the fast transient components of the total
potassium current (Klee et al. 1997; Numann et
al. 1987
). With holding potential at
60 mV,
IKd were elicited by a protocol where
hyperpolarizing or depolarization were separated by a 50-ms prepulse at
+10 mV, which inactivated the transient potassium current (Fig.
8A). The amplitude of the IKd gradually increased and reached
the peak at 36 h after reperfusion (P < 0.05, Fig. 8B). No significant difference in membrane capacitance was found in these neurons (12.85 ± 0.33 pF for control and
12.68 ± 0.18 pF at 36 h after reperfusion), suggesting that
the increase of IKd current after
reperfusion was not due to the increase of cell volume. The activation
curve shifted ~12 mV in the hyperpolarizing direction
(V1/2:
4.95 ± 1.92 mV for control
and
16.36 ± 1.64 mV at 36 h after reperfusion;
P < 0.05, Fig. 8C). No significant change
in V1/2 and
Vc of inactivation curves was found
after reperfusion (Fig. 8D, Table
4).
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DISCUSSION |
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The present study has shown that the excitability of CA1 neurons decreases in brain slices 14-16 h after reperfusion, which is coincide with results from previous studies using in vivo preparation. Our results also demonstrate a differential change of potassium currents to ischemia insult. Transient potassium current (IA) was transiently enhanced at ~12 h reperfusion whereas the delayed rectifier potassium current (IKd) was enhanced at ~36 h reperfusion. The increase of IA at the early phase of reperfusion may be responsible for the decrease of excitability during this period, and the increase of IKd at the late phase of reperfusion may be associated with postischemic cell death in hippocampus.
Alteration of transient potassium current
The enhancement of IA in CA1
neurons following reperfusion as demonstrated in the present study is
different from previous studies on IA
during hypoxia. Cummins et al. (1991) reported that no
consistent change of voltage-dependent potassium current was observed
in isolated CA1 neurons during hypoxia, and others have shown the
decrease of IA in CA1 neurons during
hypoxia (Gebhardt and Heinemann 1999
; Hyllienmark
and Brismar 1996
). The conflicting results between previous
reports and our observation may reflect the difference between changes
during hypoxia and after reperfusion. It may also stem from the
difference in preparation [dissociated or cultured neurons
(Cummins et al. 1991
; Gebhardt and Heinemann 1999
) vs. brain slice] and the age of the animal [early
postnatal (Hyllienmark and Brismar 1996
) vs. adult].
Our results, on the other hand, are in line with many studies showing
the increase of potassium currents during hypoxia (Boissard and
Gribkoff 1993
; Fujiwara et al. 1987
;
Hansen et al. 1982
; Leblond and Krenjevic 1989
). It is conceivable that the decrease of excitability in CA1 neurons during hypoxia and after reperfusion may be governed by the
same mechanism (i.e., increase of potassium conductance).
One possible mechanism underlying the enhancement of potassium currents
is that the number of K+ channels in CA1 neurons
increases after reperfusion. It has been shown that the
Na+ channel mRNA and protein levels increase in
immature brain but decrease in adult animals, suggesting that the
number of ion channel could be altered by hypoxia or ischemia
(Xia and Haddad 1999). However, considering the fact
that protein synthesis in vulnerable neurons was inhibited after
ischemia, especially during early reperfusion (Lipton
1999
; Schmidt-Kastner and Freund 1990
), it is
unlikely the enhancement of IA at the
early phase of reperfusion is due to the increase number of potassium
channels. The other possibility is that the channel properties, such as
the open probability and opening time, have been altered after
ischemia. It has been shown that membrane proteins including ion
channels are responsive to their redox state (Bertl and Slayman
1990
). Changes in the redox state of amino acid residues in
channel proteins may lead to a conformational change and hence alters
the channel activity (Ruppersberg et al. 1991
). Ischemia
could induce the change of redox state and subsequently alter the
channel activity (Gozlan et al. 1994
). It is possible
that the channel open probability and opening time increase after
ischemia and result in the enhancement of
IA current after reperfusion.
The transient potassium current is important to determine the spike
threshold because it is activated near the resting membrane potential
range and affects the latency of first spike (Brown et al.
1990; Hille 1992
; Segal et al.
1984
; Storm 1990
). Increase of
IA will affect the spike threshold and
therefore decrease the excitability of the neuron.
IA also partially contributes to the repolarization of the action potential (Ficker and Heinemann
1992
). The increase of IA will
shorten the duration of action potential, reduce sodium and calcium
influx, hence decrease the excitability of the neuron. In the present
study, the duration of action potential significantly reduced after
reperfusion; this is coincide with the increase of
IA conductance. The increase of
transient potassium current therefore may be a major contributor to the
decrease of excitability in CA1 neurons during the early period of reperfusion.
The increase of potassium conductance during early reperfusion may be
an attempt to protect the neurons from ischemic insult. Some studies
have showed that the activation of ATP dependent K+ channel (KATP) during
anoxia may be of importance in the response and adaptation of neurons
(Ben Ari 1990; Jiang and Haddad 1991
; Jiang et al. 1992
). Activation of
KATP channels on postsynaptic membrane of
hypoglossal neurons tends to hyperpolarize or limit the depolarization
of these neurons (Jiang and Haddad 1991
).
Hyperpolarization would reduce energy consumption and prevent
activation of several cation channels, reduce energy expenditure, and
have a protective effect when neurons are exposed to
O2 deprivation (Haddad and Jiang
1993
). It has been demonstrated that application of
KATP channel activator can protect neurons from
focal or global ischemia (Heurteaux et al. 1993
;
Takaba et al. 1997
). Because
IA is not a major contributor to
resting membrane potential, its protective effect on neurons following
reperfusion probably is by saving energy through increase spike
threshold and decrease excitability rather than hyperpolarizing the
resting membrane potential.
Alteration of delayed rectifier potassium current
The forebrain ischemia induced in the present study consistently
produces cell death in 90% of CA1 pyramidal neurons 1 wk after
reperfusion (Xu et al. 1999). In recent years,
accumulating evidence has indicated that apoptosis, in addition to
necrosis, is involved in neuronal damage after ischemia
(MacManua and Linnik 1997
; Schreider and Baudry
1995
; Zeng and Xu 2000
). It has been shown that
the enhancement of outward potassium current is associated with
neuronal apoptosis induced in different experimental settings including
those mimicking ischemia conditions (Yu et al. 1997
, 1998
,
1999b
). Using potassium channel blocker or raising
extracellular potassium concentration reduces such apoptotic cell
death. These studies suggest that potassium efflux is an important
mediator of neuronal apoptosis. Further studies have indicated that the enhancement of potassium efflux, perhaps in particular via the delayed
rectifier potassium current (IKd),
participates apoptotic cell death (Yu et al. 1998
,
1999a
). However, a study using single-channel recording has
shown that the amplitude, open probability, and mean opening time of
IKd channel decreased in cerebellar
granule cells 12 h after hydrogen peroxide application, which
induced apoptosis (Chi et al. 1998
). The preceding
studies indicate that IKd is involved
in apoptosis, but its role is complex. Its activity may increase or
decrease depending on many factors such as the difference in insult and
the time course of apoptosis.
Given the property of very slow inactivation,
IKd contributes to extracellular
K+ accumulation and the reduction of
intracellular potassium during ischemia/hypoxia (Yu et al.
1997). Potassium efflux leading to a reduction of intracellular
potassium has been suggested to mediate several forms of apoptosis
(Bortner et al. 1997
; Hughes et al. 1997
;
Yu et al. 1997
). Intracellular potassium at the normal
levels inhibits caspase-3-like protease activation and apoptotic DNA fragmentation while reduction of intracellular potassium activates caspases and nucleases that play a central role in apoptotic cell death
(Alnemri et al. 1996
; Hughes et al. 1997
;
Yu et al. 1998
; Yuan et al. 1993
).
Therefore the increase of delayed rectifier potassium current at
36 h reperfusion may be involved in apoptotic cell death after reperfusion.
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ACKNOWLEDGMENTS |
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We thank Dr. G. Nicol for technical help and constructive comments during the study.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-38053 and American Heart Association Grants 0070048 to Z. C. Xu and 9920468Z to X. X. Chi.
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FOOTNOTES |
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Address for reprint requests: Z. C. Xu, Dept. of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 (E-mail: zxu{at}anatomy.iupui.edu).
Received 5 June 2000; accepted in final form 16 August 2000.
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REFERENCES |
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