Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Müller, Michael and
George
G. Somjen.
Na+ Dependence and the Role of Glutamate Receptors
and Na+ Channels in Ion Fluxes During Hypoxia of Rat
Hippocampal Slices.
J. Neurophysiol. 84: 1869-1880, 2000.
Spreading depression (SD) as well as
hypoxia-induced SD-like depolarization in forebrain gray matter are
characterized by near complete depolarization of neurons. The
biophysical mechanism of the depolarization is not known. Earlier we
reported that simultaneous pharmacological blockade of all known major
Na+ and Ca2+ channels prevents hypoxic SD. We
now recorded extracellular voltage, Na+, and K+
concentrations and the intracellular potential of individual CA1
pyramidal neurons during hypoxia of rat hippocampal tissue slices after
substituting Na+ in the bath by an impermeant cation, or in
the presence of channel blocking drugs applied individually and in
combination. Reducing extracellular Na+ concentration
[Na+]o to 90 mM postponed the hypoxia-induced
extracellular DC-potential deflection
(Vo) and reduced its amplitude, and it
also postponed the SD-like depolarization of neurons. After lowering
[Na+]o to 25 mM, SD-like
Vo became very small, indicating that an influx of Na+ is required for SD; influx of
Ca2+ ions alone is not sufficient. We then asked whether
the SD-related Na+ current flows through
glutamate-controlled and/or through voltage-gated Na+
channels. Administration of either the
non-N-methyl-D-aspartate (NMDA) receptor
antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the NMDA
receptor antagonist
(±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP)
postponed the hypoxic
Vo and depressed
its amplitude but the effect of the combined administration of these
two drugs was not greater than that of either alone. During the early
phase of hypoxia, before SD onset, [K+]o
increased faster and reached a much higher level in the presence of
glutamate antagonists than in their absence. The
[K+]o level reached at the height of hypoxic
SD was, however, not affected. When TTX was added to DNQX and CPP, SD
was prevented in half the trials. When SD did occur, it was greatly
delayed, yet eventually neurons depolarized to the same extent as in
normal solution. The SD-related sudden drop in
[Na+]o was depressed by only 19% in the
presence of the three drugs, indicating that Na+ can flow
into cells through pathways other than ionotropic glutamate receptors
and TTX-sensitive Na+ channels. We conclude that, when they
are functional, glutamate-receptor-mediated and voltage-gated
Na+ currents are the major generators of the
self-regenerative rapid depolarization, but in their absence other
pathways can sometimes take their place. The final level of SD-like
depolarization is determined by positive feedback and not by the number
of channels available. A schematic flow chart of the events generating
hypoxic SD is discussed.
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INTRODUCTION |
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In forebrain gray matter,
spreading depression (SD) is associated with nearly complete
depolarization of neurons and glial cells as well as a massive
disturbance of extracellular ion concentrations. Ischemia and severe
hypoxia trigger a similar depolarization (Bure et al.
1974
; Leão 1947
; Marshall
1959
; Müller and Somjen 2000
; Nicholson 1984
; Somjen et al. 1992
).
Despite their magnitude, all these changes are reversible, and up to
four repetitive hypoxic SD-like episodes can be induced in hippocampal
slices in vitro without necessarily reducing electrophysiological
responsiveness, i.e., causing acute neuronal damage
(Müller and Somjen 1998
).
The first to attempt to explain the nature of SD was Grafstein
(1956), who proposed that K+ released by
a group of neurons can raise extracellular K+ and
thus depolarize nearby cells, providing the feedback responsible for
the propagation of SD. Subsequent research has amply confirmed the
massive release of K+ during SD
(Bure
et al. 1974
), but other observations
suggested that K+ increase alone cannot
satisfactorily explain the complicated nature of SD, although it
undoubtedly does play a crucial role (Somjen et al.
1992
; van Harreveld 1959
). As of today, SD is
understood as a cascade of various events, but the precise mechanism of
its self-regenerative nature remains obscure.
Against the potassium hypothesis, van Harreveld
(1959, 1978
) proposed that glutamate is the agent, or one of
two agents, mediating the generation and the propagation of SD.
van Harrelveld (1959)
demonstrated the release of
glutamate during SD and also that cortical application of glutamate is
capable of inducing SD. Indeed, in some models, glutamate receptors
seem critically involved in neuronal loss as a consequence of
excitotoxic, ischemic, or hypoxic insults (Choi 1987
;
Garthwaite et al. 1986
; Tanaka et al.
1997
; Yamamoto et al. 1997
). The contribution of
glutamate-mediated inward currents to the immediate
electrophysiological consequences of oxygen and glucose deprivation is,
however, not yet clear. Glutamate's postulated role in the generation
and propagation of SD could be important, since recurrent waves of SD
have been blamed for the extension of cerebral infarcts into marginal
areas (Busch et al. 1996
), and during hypoxia, glutamate
is apparently released not only by synaptic but also extrasynaptic
mechanisms (Drejer et al. 1985
). Among others, its
release can be induced by elevated
[K+]o (Fujikawa et
al. 1996
; Szerb 1991
) and reversal of
Na+-dependent glutamate uptake as a consequence
of a steadily increasing intracellular Na+
concentration ([Na+]i)
(Attwell et al. 1993
). Glutamate is also released
from glial cells via swelling-activated anion channels (Basarsky
et al. 1999
; Kimelberg et al. 1990
). Earlier
tests of the ability of glutamate antagonists to suppress SD unveiled
an important difference between normoxic SD and hypoxic SD-like
depolarization. Normoxic SD can be successfully depressed by the
application of N-methyl-D-aspartate (NMDA)
antagonists; hypoxia-induced SD cannot. Hypoxic SD was sometimes
delayed, but in some tests its onset was actually accelerated and the
associated
Vo was also only in some
cases reduced (Aitken et al. 1991
;
Hernández-Cáceres et al. 1987
;
Jing et al. 1993
; Lauritzen and Hansen
1992
; Marrannes et al. 1988
).
Recently we examined in a computer model the conditions for the
generation of SD-like depolarization (Kager et al. 2000;
Somjen et al. 2000
). In the model either a simulated
NMDA-mediated current or a persistent voltage-gated
Na+ current could generate this phenomenon. The
results validate the dual hypothesis of van Harreveld
(1978)
, who suggested that in the retina two kinds of SD can
occur, one mediated by glutamate and the other by
K+.
In the study presented here we first tested whether Na+ is a major component of the inward current causing SD-like depolarization. We found that hypoxic SD is indeed Na+ dependent. Then, to define more precisely the channels generating the SD-like depolarization, the effects of glutamate antagonists and TTX were tested on membrane potential and input resistance changes of single pyramidal neurons, and on extracellular voltage and Na+ and K+ concentrations during severe hypoxia. We confirmed that hypoxic SD does occur when both NMDA and non-NMDA glutamate receptors are blocked, but its onset is much delayed. Adding TTX to the glutamate antagonists halved the incidence of SD, but did not completely prevent its occurrence. We conclude that besides channels controlled by ionotropic glutamate receptors and TTX-sensitive Na+ channels, other yet to be identified pathways can mediate SD-like depolarization. At the end of the paper we present a schematic flow chart of the cascade of events generating hypoxic SD.
Some of the results are being published as an abstract (Somjen
and Müller 2000).
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METHODS |
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Preparation
Hippocampal tissue slices were prepared from ether anesthetized, male Sprague-Dawley rats of 120-260 g body wt (4-7 wk old). Following decapitation, the brain was rapidly removed from the skull and placed in chilled artificial cerebrospinal fluid (ACSF) for 1-2 min. The two hemispheres were separated, one hippocampus was isolated, and transverse slices of 400-µm thickness were cut using a tissue chopper. Slices were transferred to an interface recording chamber of the Oslo style and were left undisturbed for at least 90 min. The recording chamber was kept at a temperature of 34.5-35.5°C. It was continuously aerated with 95% O2-5% CO2 (400 ml/min) and perfused with oxygenated ACSF (1.5 ml/min). Hypoxia was induced by switching the chamber's gas supply to 95% N2-5% CO2. Exchange of the bathing solution took about 5 min and diffusion of applied drugs into the slice was completed in about 30 min.
Solutions
The ACSF had the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 1.2 CaCl2, 1.2 MgSO4, and 10 dextrose; aerated with 95% O2-5% CO2 to adjust pH to 7.4. In reduced Na+ solutions, Na+ was substituted by NMDG+ (N-methyl-D-glucamine, Sigma). TTX (citrate-buffered; Sigma) was prepared as 1-mM stock solution in distilled water and kept frozen. DNQX (6,7-dinitroquinoxaline-2,3-dione, Research Biochemicals International) and CPP [(±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; RBI and Tocris] were dissolved in ACSF and kept frozen as 1 and 2 mM stock solutions, respectively.
Microelectrodes
Single-barreled glass microelectrodes for extracellular
recordings were pulled from thin-walled borosilicate glass [TW150F-4, World Precision Instruments (WPI)] using a horizontal puller (P-80/PC, Flaming Brown). They were filled with ACSF and their tips were broken
to a final resistance of 5-10 M. Sharp microelectrodes for
current-clamp recordings were made from thick-walled borosilicate glass
(1B150F-4, WPI) and filled with 2 M K-Acetate + 5 mM KCl + 10 mM HEPES (Sigma); pH 7.4; their resistances were 60-80 M
.
Extracellular Na+ and K+
concentrations were simultaneously measured using triple-barreled
Na+/K+-sensitive
microelectrodes of the twisted type, as has been described in detail
earlier (Müller and Somjen 2000). In brief, a
single-barreled capillary (1B150F-4, WPI) was glued to the
double-barreled theta-type capillary (GCT 200-10, Clark Electromedical
Instruments), and this capillary assembly was then pulled on a vertical
puller (Narishige PE-2). In a first step, it was pulled by only 2-4 mm
simultaneously twisting the attached reference barrel by 180° around
the centered theta capillary. After cooling, the capillaries were
pulled apart in a second pulling step. Both barrels of the theta
capillary were silanized by 60-min exposure to HMDS vapors
(hexamethyldisilazane, 98%, Fluka; vaporized at 40°C) and
subsequent baking in the oven (200°C, 2 h). Silanization
of the attached reference barrel was prevented by filling it with
distilled water.
The reference barrel contained 150 mM NaCl + 10 mM HEPES, pH 7.4, while
the tip of the K+-sensitive barrel was filled
with the valinomycin-based K+ ion neutral carrier
(Potassium Ionophore I, Cocktail A, Fluka 60031) and backfilled with
150 mM KCl + 10 mM HEPES, pH 7.4. The tip of the
Na+-sensitive barrel was filled with a
Na+ cocktail based on the
Na+ ionophore VI (Deitmer and Munsch
1995; Müller and Somjen 2000
) and
backfilled with 150 mM NaCl + 10 mM HEPES, pH 7.4. Electrode resistances of the Na+-sensitive,
K+-sensitive, and reference barrel were 200, 130, and 40 M
, respectively. There was no noticeable interference between
adjacent barrels.
Ion-sensitive electrodes were calibrated before and after each experiment by detecting their response generated in standard solutions (0, 1, 5, 10, 50, and 100 mM K+; 150, 100, 50, 10, 5, 0 mM Na+). To maintain constant ionic strength similar to that in interstitial fluid, Na+ in calibration solutions was replaced by K+ and vice versa (reciprocal calibration method). The reciprocal adjustment of ion concentrations in the calibrating solutions discounts the small co-ion interference and further correction of the data were not required. Average slopes of the K+- and Na+-sensitive barrels were 53.1 ± 1.9 mV/decade K+ and 54.2 ± 2.0 mV/decade Na+; their detection limits were 1.0 ± 0.4 mM K+ and 4.4 ± 0.5 mM Na+ (n = 9).
Electrical recordings
Ion-sensitive electrode signals were referred to an
Ag/AgCl bridge electrode embedded in 2% agar in 3 M KCl. They were
recorded by a DC amplifier (constructed locally) and digitized by a
TL-1/Lab Master acquisition system at sampling rates of 25 Hz. Since
electrodes were calibrated to Na+ and
K+ concentrations and the activity coefficient of
the measured ion was held constant, changes in
[Na+]o and
[K+]o could directly be
calculated from the electrode responses using the electrodes'
averaged slope of pre- and postexperiment calibration. All signal
amplitudes were measured between the prehypoxia baseline and the
maximal change. Only rapid negative extracellular DC potential changes
(Vo) of at least 10 mV amplitude
were considered as SD. SD onset was defined as occurrence of the sudden
Vo.
Current-clamp recordings from CA1 pyramidal neurons were
performed with an intracellular recording amplifier (Neuro Data, IR-283) as described earlier (Müller and Somjen
2000). CA1 pyramidal neurons were identified by their location
in st. pyramidale, membrane potential, spontaneous activity,
action-potential shape, and input resistance (Morin et al.
1996
). Only pyramidal neurons with a stable membrane potential
of at least
55 mV were accepted. Their input resistance was
determined every 10 s by injecting a hyperpolarizing current of
400-pA amplitude and 200-ms duration. Data were sampled at 1 kHz using
the TL-1/Lab Master acquisition system and the Axotape V2 software
(Axon Instruments). Input resistances were measured at the steady-state
level of the voltage deflections and changes in input resistance were
expressed as a percent of pretreatment value.
Antidromically and orthodromically evoked responses were elicited
by delivering 10-150 µA stimuli of 0.1-ms duration via monopolar stimulation electrodes (for details see Müller and Somjen
1998).
Statistics
The data were obtained from 30 rats, and since most experiments
did not last longer than 2 h, up to four slices could be used from
each brain, but, to insure independence of treatments, each group of
experiments was performed on three to six different rats. In 17 of the
30 rats, two slices were used from the same brain for the same
experimental treatment (low external Na+ or
drug), and only one slice was used for a given experimental condition
from the 13 other brains. Each slice was used for only one experimental
condition. For the extracellular experiments with drugs, to reduce
variability of data, two hypoxic SD episodes were induced in every
slice, first a control SD, and, after at least 30 min, a second SD in
the treated condition. For the low-Na+
experiments, there were four hypoxic episodes, one in control ACSF, by
90 mM and 25 mM Na+, and finally a repeat to
check recovery in normal ACSF. The changes in SD parameters (amplitude,
onset, ion changes) observed during low Na+ and
drug treatments were normalized to the control SD recorded in a given
slice; each experiment therefore had its own control. Significance of
these averaged changes was tested in a one-sample t-test,
comparing the mean of the observed changes to the known standard, i.e.,
the control conditions defined either as unity or as 100%
(Bailey 1992; Sachs 1999
).
Technical reasons required that, for intracellular recordings from individual neurons, control and drug effects be recorded in different slices. Significance of these unpaired observations was tested in two-tailed, unpaired Student's t-tests at a significance level of 5%. Multiple comparisons were not done, since each group of drug-treated slices was only compared with the control group of slices. None of the drug-treated groups was compared with any other group than control and none of the slices was treated with more than a single drug or drug combination. All numerical values are represented as mean ± standard deviation (StD) and the number of experiments (n) refers to the number of slices investigated. In the diagrams, significant changes are marked by asterisks (* P < 0.05; ** P < 0.01).
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RESULTS |
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Na+ dependence of hypoxic SD
To assess the effect of a reduced extracellular
Na+ concentration
([Na+]o) on
hypoxia-induced SD, either 65 or 130 mM of extracellular Na+ were substituted by the membrane impermeable
NMDG+, thereby reducing
[Na+]o to 90 and 25 mM,
respectively. First, a control SD was recorded in every slice followed
by two hypoxic SD episodes in low
[Na+]o and one more
during recovery in normal solution (Fig.
1). Under control conditions, the
extracellular DC potential deflection (Vo) signaling SD onset occurred
within 2.1 ± 0.6 min of hypoxia and its amplitude averaged
19.8 ± 3.9 mV (n = 30;
Vo amplitude referred to prehypoxic
baseline). Reducing extracellular Na+ to 90 and
25 mM increased the time of SD onset more than twofold and the
Vo amplitude decreased by 58 ± 9 and 85 ± 7%, respectively (n = 6 each;
Table 1). The residual
Vo seen with the lowest [Na+]o would, ordinarily,
by definition, be considered too small for the diagnosis of SD. The
changes measured in the somatic layer [stratum
(st.) pyramidale] did not noticeably differ from
those in the dendritic layer (st. radiatum). Restoring
normal extracellular Na+ completely reversed
these depressant effects (Fig. 1).
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As expected, synaptic transmission and axonal conductance were also depressed in low Na+ solution. In 90 mM Na+ solution, the amplitudes of orthodromically evoked focal excitatory postsynaptic potentials (fEPSPs) and antidromically evoked population spikes reversibly decreased by 48.8 ± 17.6 and 50.0 ± 7.2%, respectively (n = 6 each, measured at stimulus intensities of 100-150 µA), while they were completely suppressed in 25 mM Na+ solution (n = 6).
Sensitivity of [Na+]o and [K+]o changes to glutamate antagonists
To investigate the involvement of ionotropic glutamate receptors
in the massive extracellular Na+ and
K+ changes during SD, we simultaneously recorded
[Na+]o and
[K+]o during hypoxic SD
in control solutions and induced a second hypoxic SD after 35-45 min
treatment with the respective glutamate antagonists. The glutamate
inhibitors DNQX and CPP were applied one by one as well as
simultaneously and also in combination with TTX. In a previous study,
we already ascertained that hypoxic SD can repeatedly be induced in a
given slice kept in control solutions, without causing significant
changes in the characteristic SD parameters for the first three SD
episodes (Müller and Somjen 1998).
The extracellular Na+ and
K+ concentrations showed characteristic and
clearly different time courses (Fig. 2,
A and C). In control solutions, during the
initial phase of hypoxia, before SD onset, [K+]o already increased
slightly at an average linear rate of 3.4 ± 1.5 mM/min, reaching
a level of 8.4 ± 1.8 mM immediately before SD onset;
[Na+]o still remained
unchanged. As soon as the rapid Vo
occurred, [K+] rapidly increased to 77.3 ± 20.8 mM (n = 24; measured in st. radiatum). Simultaneously,
[Na+]o sharply dropped to
61.8 ± 14.3 mM and then immediately started to increase again,
reaching a plateau of 92.5 ± 12.6 mM (n = 23). When reoxygenation was started 100 s after SD onset, the
extracellular ion concentrations recovered to their prehypoxia levels.
During the recovery phase,
[K+]o consistently
undershot its prehypoxic baseline (Heinemann and Lux
1975
; Pérez-Pinzón et al. 1995
),
reaching a minimum of 1.3 ± 0.4 mM (Figs. 2 and
3; see also Müller and
Somjen 2000
).
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As already reported by others, inhibition of glutamate receptors does
not prevent the occurrence of hypoxia-induced SD (Aitken et al.
1991; Hernández-Cáceres et al.
1987
; Jing et al. 1993
; Lauritzen
and Hansen 1992
; Marrannes et al. 1988
).
Application of either the non-NMDA antagonist DNQX (10 µM) or the
NMDA inhibitor CPP (10 µM) postponed the onset of hypoxic SD by
18 ± 18 and 25 ± 21% and reduced the
Vo amplitude by 24 ± 22 and
17 ± 9%, respectively (Table 1). In the presence of either DNQX
or CPP during the initial phase of hypoxia, before the onset of SD,
[K+]o increased by more
than twice the normal value, but at the height of SD, the maximal level
of [K+]o was about the
same as in normal solution (Fig. 2B). It could be thought
that the high [K+]o was
reached in the initial phase because more time has elapsed before SD
onset allowing more K+ to accumulate, but in fact
in the presence of the glutamate antagonists [K+]o increased by a
substantially higher rate than in normal solution (Table
2). By contrast, neither the
K+ peak at the height of hypoxic SD nor the
undershoot of the K+ baseline following
reoxygenation were significantly different from control (Fig.
2B). The maximal level of the hypoxia-induced [Na+]o decrease was
slightly reduced by 19 ± 14% in the presence of DNQX and by
10 ± 5% following administration of CPP, but the
Na+ plateau level was not significantly affected
(Fig. 2D).
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Combined application of DNQX and CPP did not prevent hypoxic SD. Remarkably, the inhibitory effects of DNQX and CPP did not sum up (Table 1). The initial pre-SD K+ level increased to threefold, and the initial rate of increase of [K+]o rose at a 116 ± 88% higher rate. The K+ peak at the height of SD was also slightly enhanced (Figs. 2A and 3, Table 2). However, no significant changes in the hypoxic SD-associated [Na+]o drop were observed (Fig. 2D). Unlike in normal solution, in the presence of the two glutamate antagonists, a small decrease in [Na+]o could be observed in some recordings even before SD occurred (Fig. 3).
Since our previous study (Müller and Somjen 2000)
suggested a role of voltage-gated Na+ channels in
the triggering of SD, we simultaneously applied DNQX (10 µM), CPP (10 µM), and TTX (1 µM). Under these conditions, there was no sign of
SD-like depolarization in three of six slices during severe 20-min
hypoxia; in the remaining three slices, the onset of hypoxic SD was
even more delayed than with the two glutamate antagonists without TTX,
and the
Vo amplitude decreased by
38 ± 18% (Table 1). Due to the small number of observations,
this amplitude reduction is, however, not significantly different from control. The initial, pre-SD
[K+]o increase became
more than threefold, slowly approaching a steady-state level of
16.7 ± 2.1 mM (n = 3, Fig.
4B). Once SD occurred,
however, the K+ peak did not differ from control
conditions (Fig. 2B). The Na+ peak was
reduced by 18 ± 5%, but the Na+ plateau
level was not significantly affected (Fig. 2D). In those slices which did not respond with a hypoxic SD during 20-min hypoxia, [K+]o reached a
steady-state level of 16.0 ± 5.7 mM that was maintained until
reoxygenation was started (Fig. 4C). Thus the
[K+]o increase amounted
to only 22.7 ± 10.6% of the change observed during the control
SD in the same slice (n = 3). If no SD occurred, [Na+]o decreased to
125.6 ± 1.9 mM, the decrease being 32.6 ± 1.5% of control.
After reoxygenation,
[K+]o decreased and,
following the characteristic undershoot which did not differ from
control conditions, it recovered to its prehypoxic baseline.
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Effects of glutamate antagonists on hypoxia-induced membrane and input resistance changes in single pyramidal neurons
In single CA1 pyramidal neurons, hypoxia causes first a
hyperpolarization, then a slow depolarization and eventually a
near-complete, self-regenerative depolarization coinciding with the
SD-like Vo, and it dramatically
decreases the input resistance (Hansen 1985
; Hansen et al. 1982
; Müller and Somjen 1998
,
2000
). To investigate the contribution of glutamate receptors
to the hypoxia-induced changes in pyramidal neurons, we performed
current-clamp experiments in control solution and following drug
administration of at least 30 min. Since stable cell impalement could
not be maintained for hours, and in most cells impalement was lost
during the recovery phase from hypoxia following reoxygenation, control
and drug effects had to be recorded in different cells. Each slice
underwent only one hypoxic episode.
In the control group of slices bathed in normal ACSF, CA1 pyramidal
neurons had an average membrane potential of 62.6 mV and an input
resistance of 39.0 M
(Table 3). The
detailed effects of hypoxia on the electrical properties of CA1
pyramidal neurons have been described in detail in our previous study
(Müller and Somjen 2000
). The initial
hyperpolarization was associated with a 37.8 ± 14.8% decrease in
input resistance, that reversed into a gradual, slow depolarization.
Within 1.8 ± 0.5 min of hypoxia, the rapid SD-like depolarization
occurred. It was triggered at an apparent threshold potential of
51.6 ± 4.3 mV and in most cells it was preceded by a
spontaneous discharge of action potentials. The rapid depolarization
brought the intracellular potential to
23.2 ± 4.9 mV and it
slowly continued to shift toward its peak of
7.8 ± 5.0 mV. The
input resistance decreased further, in total by 88.5 ± 11.9%
(Fig. 5A).
|
|
Reducing [Na+]o to 90 mM as well as application of the glutamate inhibitors and TTX resulted in somewhat more negative resting membrane potentials and a decreased resting input resistance (Table 3). In 90 mM Na+ solution, the onset of the hypoxia-induced SD-like depolarization was markedly delayed (Figs. 5B and 6C). The threshold potential at which the rapid depolarization was triggered, the amplitude of the rapid depolarization itself as well as the peak potential reached at the height of hypoxic SD were, however, not different from control conditions (Fig. 6; n = 7). DNQX acted in a similar way, postponing the onset of the SD-like depolarization but not affecting the amplitude of the rapid depolarization or its absolute peak (Fig. 6; n = 7). The NMDA-receptor antagonist CPP postponed the SD-like depolarization and shifted its apparent threshold by 15 mV to a more positive level. As a result, the amplitude of the rapid depolarization, the b-c segment of Fig. 6A, was reduced to 52% of the magnitude observed in control slices (Fig. 6, A and B). Surprisingly, the effects of CPP were less pronounced when DNQX was added to CPP (n = 9; Figs. 5C and 6).
|
When slices were pretreated with the triple combination of DNQX, CPP,
and TTX, 20 min of hypoxia still induced a SD-like depolarization in
five of eight pyramidal neurons, coincidentally with a characteristic Vo. The onset of the hypoxic SD was
in these cases delayed more than sixfold and the threshold potential of
the SD-like depolarization shifted to more positive potentials, again,
however, without significantly decreasing the final amplitude of the
depolarization (Figs. 5D and 6A). The combined
administration of DNQX, CPP, and TTX did diminish the reduction of the
input resistance during the rapid depolarization, which was 32%
smaller than control (n = 5; Fig. 6D).
Those three pyramidal neurons which did not respond with a SD-like
depolarization in the presence of DNQX, CPP, and TTX showed a slow and
incomplete depolarization, lacking the characteristic self-regenerative
character observed in normal solution. During 20-min hypoxia, their
intracellular potential slowly rose to a peak of 17.3 ± 21.6 mV, and the input resistance decreased by only 35.8 ± 10.6%
(n = 3). In these cases, the typical
Vo was also absent.
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DISCUSSION |
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Three main conclusions stand out. 1) Blocking NMDA receptors plus voltage-controlled channels reduces the likelihood and greatly delays the onset of hypoxic SD, but does not always prevent it. 2) Nonetheless, the depolarization associated with SD is dependent on external Na+. 3) Once the SD-like depolarization is started, it continues to completion, even if its onset has been delayed by low [Na+]o or by channel-blocking drugs.
Hypoxic SD-like depolarization is a Na+-dependent all-or-none process
The generation of hypoxic SD is clearly dependent on the
presence of extracellular Na+ because
substituting Na+ by an impermeant cation
dose-dependently reduced the SD-related Vo. With only 25 mM of
[Na+]o remaining, the
residual
Vo was so weak that it
could not be classified as SD. Yet, with 90 mM
[Na+]o, even though
Vo was reduced to 42 ± 9% of
normal after a prolonged delay, individual neurons eventually still
depolarized fully. Several factors could have contributed to the
discrepancy between extracellular and intracellular voltage changes. It
should be borne in mind that the extracellular voltage is an average
signal from a large population of units. In low
[Na+]o with the delay of
the onset of depolarization, very probably the individual responses
became more dispersed in time. Asynchrony among the responding units
depresses the extracellularly recorded signal. Another source for the
discrepancy could be if the depolarization would be distributed more
evenly over the surface of the cells than is usually the case.
Less-steep voltage gradients draw less extracellular current. It is
also possible that some neurons did and others did not respond with
SD-like depolarization within the same population. Indication for this
may be found in the reports by Sugaya et al. (1975)
and
Czéh et al. (1993)
, who found a few anomalous
cells that retained nearly normal membrane characteristics in the midst
of the usual SD-like
Vo. In low
[Na+]o, the proportion of
units that did not participate in SD may have increased. The
theoretical possibility of liquid junction or diffusion potentials
generated by ion gradients in interstitial fluid needs to be
considered. Such potentials are, however, negligibly small compared
with the
Vo associated with SD
(Somjen 1973
). Finally, the puncture of the membrane by
the intracellular electrode may have exaggerated the hypoxiainduced
depolarization of neurons, but this effect need not be stronger in low
[Na+]o than in normal ACSF.
The hypoxic responses of extracellular and intracellular voltages
differed also in the combined presence of the three drugs, CPP, DNQX,
and TTX. Hypoxic Vo was absent in
half the cases when this drug combination was used, and its amplitude
was on average only 62 ± 18% of normal in the others. It seems
that in the presence of the triple drug combination the "ignition
point" for starting SD was not always reached. Yet when individual
neurons underwent SD, they depolarized, after a very prolonged delay,
to the same degree as those in the drug-free condition (Fig. 5).
Similarly, if SD occurred,
[K+]o eventually
increased to the same level in the presence of the three drugs as in
their absence (Fig. 4).
The unalterable final level of depolarization of neurons underscores the, already suspected, all-or-none character of SD. As long as any pathway for a persistent inward current is available, the potential to which the membrane tends is not governed by the number of channels that are still open but by the self-regenerating feedback. Lack of Na+ or partially blocked pathways for inward current may postpone SD and even prevent depolarization of some of the neurons but, once the process is set into motion, it will proceed to completion. The fact that the combined administration of DNQX and CPP had no more effect than either drug alone pointed to the same conclusion. This also indicated that as long as there was a remaining pathway for the influx of Na+, the ultimate level of the depolarization as well as of the ion concentration changes were governed by positive feedback.
It should be pointed out that the intracellular potential,
Vi, was referred to a "bath" ground
and it was not corrected for shifts of Vo. While
at rest, Vi correctly represents the
membrane potential, Vm; during SD, the
large Vo sums with the
intracellular signal. Therefore during SD,
Vm approaches 0 mV closer than is suggested by Vi shown in Figs. 5 and 6. In
comparing the effects of various treatments on the fast depolarization
(b-c segment of Figs. 5 and 6A), the
error is, however, small.
Alternative conductances that might mediate SD-like depolarization
We have previously reported that hypoxic SD was consistently
prevented by the combined application of glutamate antagonists plus
drugs inhibiting voltage-gated Ca2+ and
Na+ channels, namely DNQX + CPP + TTX + Ni2+ (Müller and Somjen
1998). In the experiments reported here, during the combined
application of DNQX, CPP, and TTX, only Ni2+ was
the missing ingredient. In the presence of the remaining three agents,
which should markedly decrease Na+ conductance,
SD failed in about half of the trials but it still occurred in the
others. It appears that, in the absence of the main voltage-gated
Na+ channels and ionotropic glutamate receptors,
other conductances can, if with difficulty, generate hypoxic SD.
Ni2+ is considered to be an antagonist of calcium
channels (Hille 1992
), although it has other actions as
well (Hille 1968
; Hille et al. 1975
). It
could therefore be suspected that after blockade of glutamate- and
voltage-controlled Na+ channels,
Ca2+ currents may have mediated SD. Yet when
[Na+]o was reduced to 25 mM, SD was effectively suppressed, indicating that, by themselves,
Ca2+ ions are not capable of carrying the
necessary current. The minimal contribution of the influx of
Ca2+ to the depolarization is also evident from
the fact that SD can occur in the absence of external
Ca2+ (Basarsky et al. 1998
).
Nonetheless, when in the presence of the triple combination of
inhibitors SD did occur, the SD-related drop of
[Na+]o was depressed by
only 18 ± 5%. This indicates ample influx of
Na+ into cells, bypassing glutamate- and
voltage-controlled Na+ channels. We therefore
conclude that, in the presence of DNQX, CPP, and TTX, either the influx
of Ca2+ triggers the process that, eventually,
leads to the influx of Na+, or else, that the
residual conductance that is available for Na+ is
sensitive to blockade by Ni2+.
The identity of the Na+ conductances that mediate
SD after blockade of glutamate- and voltage-controlled channels remains
to be discovered. The commonly observed persistent
Na+ current is excluded, because it is highly
sensitive to TTX (Crill 1996). The existence of
TTX-insensitive Na+ currents in neurons has been
reported by Hoehn et al. (1993)
and Cummins et
al. (1999)
but deemed to be a laboratory artifact by
Chao and Alzheimer (1995)
. It is not certain whether
theirs will be the last word in this dispute. An inward
K+ current through open K+
channels could not contribute to the depolarization because, in spite
of the very large increase of
[K+]o during hypoxic SD,
the transmembrane K+ gradient does not reverse
(Müller and Somjen 2000
) and during the rapid
phase of simulated SD-like depolarization, the computed K+ equilibrium potential remains negative
relative to the prevailing membrane potential (Kager et al.
2000
). Other possible candidates include the nonspecific cation
current described by Alzheimer (1994)
,
Ca2+-activated group I metabotropic glutamate
receptor-controlled nonselective cation currents characterized by
Congar et al. (1997)
, or an acetylcholine and
Ca2+-dependent current reported by Fraser
and MacVicar (1996)
and Kawasaki et al. (1999)
.
Changes in extracellular ion levels
Ion currents across membranes of neurons and glial cells are the major source of the massive ionic changes that occur during hypoxic SD. The unusual magnitude of these responses is made possible by the narrow extracellular space, which is restricted even more during hypoxia. Diffusional ion flow can only slightly counteract these changes by blunting the increase in [K+]o and the drop in [Na+]o. Therefore the analysis and interpretation of our data concentrates on ionic currents.
Once SD occurred, ionic homeostasis was not markedly improved by the presence of the channel blocking drugs (Figs. 2 and 3). Glutamate-receptor inhibition curtailed the maximal decrease in [Na+]o by only 19%, but the Na+ plateau level was not at all affected by any of the treatments (Fig. 2). The origin of the Na+ plateau level is not clear. It could be thought that the initial surge of Na+ represents the rapidly inactivating "classical" Na+ current, INa,T, while the plateau is maintained by the persistent Na+ current, INa,P. Against this idea stands the fact that the plateau in the [Na+]o trace was not changed by TTX. The plateau may simply reflect the fact that most of the Na+ that has left interstitial space during the initial inward surge cannot be restored while hypoxia prevails, due to the impaired extrusion of Na+ from cells by the Na+-K+ pump.
The considerable and consistent acceleration of the initial rise
of [K+]o under the
influence of glutamate receptor antagonists (Table 2) was surprising.
The reason is not clear, but a factor could be impaired clearance of
excess K+ from interstitial space by glial cells.
Glial cells do have glutamate receptors (Barres et al.
1990); whether these could be involved in "K-siphoning"
is not known. In the presence of blocking drugs, [K+]o rose before the
onset of SD to levels that greatly exceeded the so-called
K+ "ceiling" of 10-11 mM defined by
Heinemann and Lux (1977)
(Fig. 2B). When SD
failed to occur under the influence of DNQX, CPP, and TTX during
prolonged hypoxia, [K+]o
reached a plateau level of 16 mM (Fig. 4C) and neurons
progressively depolarized to about
17 mV. In the absence of a large
Vo, this value of
Vi is probably close to the real
Vm. In computer simulation, the ignition
point of SD appeared to be a joint function of neuron membrane voltage
and [K+]o (Kager
et al. 2000
; Somjen et al. 2000
). With some of
the available channels blocked, this ignition point has apparently
shifted to higher levels.
Administration of the drugs or drug combinations under normoxic conditions did not induce any detectable changes in the baseline concentrations of extracellular Na+ and K+ (see e.g., Fig. 3). Since drug penetration into interfaced slices is rather slow, minor drug-induced ion changes may have been masked by equilibration with the bath.
The cascade of events generating hypoxic SD: a hypothetical flow chart
The diagram of Fig. 7 attempts
to organize the most salient published findings into a coherent flow
chart of the events that generate hypoxic SD. Its intention is to
clarify both the parallel and the sequential organization of the
numerous processes that interact in the triggering and generation of
hypoxic SD. Soon after oxygen withdrawal and much before the onset of
SD, K+ channels are activated (Erdemli et
al. 1998; Fujimura et al. 1997
; Hansen et
al. 1982
; Leblond and Krnjevi
1989
),
causing hyperpolarization of neurons (Fig. 5). Extrusion of
K+ from neurons into the restricted interstitial
space (Mazel et al. 1998
; McBain et al.
1990
; Pérez-Pinzón et al. 1995
)
raises [K+]o (Fig. 2),
gradually turning the initial hyperpolarization into a slow
depolarization (Fig. 5). At a critical point, the initially gradual
depolarization starts to accelerate and becomes self-regenerating under
the influence of the following factors. 1) Persistent
Na+ current is activated by depolarization and it
is reinforced by rising
[K+]o and decreasing
pO2 (Crill 1996
; French et
al. 1990
; Hammarström and Gage
1998
; Somjen 2000
; Somjen and Müller,
unpublished observations). 2) Glutamate receptors,
especially NMDA receptors, are activated. Glutamate is released by both
Ca2+-dependent (vesicular) and
Ca2+-independent (nonvesicular) processes from
presynaptic terminals and from glial cells (Attwell et al.
1993
; Basarsky et al. 1999
; Drejer et al.
1985
; Fujikawa et al. 1996
; Kimelberg et
al. 1990
; Szerb 1991
). 3) Slowly
inactivating voltage-gated Ca2+ channels are also
activated. The influx of Ca2+ itself carries some
current and, more importantly, elevated
[Ca2+]i, then activates
additional conductances. The effect of ion fluxes is further amplified
and interaction and intercellular crosstalk is intensified due to
shrinkage of interstitial volume and increase in electrical tissue
resistance caused by cell swelling (Bure
et al.
1974
; Hansen and Olsen 1980
; Jing et al.
1994
; Müller 2000
; Müller and
Somjen 1999
). The importance of cell swelling is demonstrated
by the fact that hypertonic cell shrinkage prevents hypoxic SD and
improves the recovery of function following hypoxia (Huang et
al. 1996
). Glial cells are known to have stretch-activated channels (e.g., Kimelberg and Kettenmann 1990
;
Kimelberg et al. 1990
) but their presence in neurons has
not been demonstrated. Neuron swelling does, however, influence such
functions as the release of stored calcium (Borgdorf et al.
2000
) and synaptic currents (Huang et al. 1997
).
The positive feedback generated by this multitude of interacting
variables stops when electrochemical driving forces decrease to near
zero. Recovery does not start until and unless reoxygenation restores
cell metabolism.
|
The simultaneous activation of numerous processes acting in parallel
explains why blocking just some of the feedback loops will postpone but
not prevent SD, while inhibiting all of them does suppress it. Influx
of Na+ through parallel pathways is the main
player because, when
[Na+]o was reduced to 24 mM, the Vo amplitude became so
small that it no longer met the diagnostic criteria for an SD-related
extracellular potential shift.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-18670.
Present address of M. Müller: Zentrum Physiologie und Pathophysiologie, Abteilung Neuro- und Sinnesphysiologie, Georg-August-Universität Göttingen, D-37073 Gottingen, Germany.
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FOOTNOTES |
---|
Address for reprint requests: G. G. Somjen, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710 (E-mail: g.somjen{at}cellbio.duke.edu).
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 1 March 2000; accepted in final form 26 May 2000.
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REFERENCES |
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