Ion Transport and Membrane Potential in CNS Myelinated Axons
II. Effects of Metabolic Inhibition
LISA LEPPANEN AND PETER K. STYS
Loeb Research Institute, Ottawa Civic Hospital, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada
times may reflect intrinsic mechanisms designed to limit axonal
injury during anoxia / ischemia.
INTRODUCTION
Mammalian CNS axons are susceptible to anoxic injury
and sustain irreversible damage from an increase in [Ca 2/ ]i
(LoPachin and Stys 1995; Ransom et al. 1994; Stys et al.
1990; Waxman et al. 1991). Ca 2/ homeostasis becomes
disrupted as a result of energy depletion and the subsequent
loss of ion gradients. Under normal physiological conditions,
the low [Ca 2/ ]i is maintained by two membrane proteins,
the Na / /Ca 2/ exchanger and Ca 2/ -ATPase (Blaustein
1988), as well as buffering by intracellular stores and Ca 2/
binding proteins (Kostyuk and Verkhratsky 1994). The
Na / /Ca 2/ exchanger normally extrudes 1 Ca 2/ in exchange
for 3 Na / by using the free energy available through the Na /
gradient (Steffensen and Stys 1996). Factors influencing
the rate and direction of exchanger operation include the
transmembrane Na / gradient and membrane potential. The
exchanger will mediate Ca 2/ influx when the transmembrane
Na / gradient is reduced and/or the membrane depolarizes.
The exchanger is sensitive to membrane potential because
its ion transport is electrogenic, transferring one net charge
per cycle (Rasgado-Flores and Blaustein 1987). For these
reasons, given that the exchanger is the main pathway for
anoxic Ca 2/ overload in optic nerve axons (Stys et al. 1992b;
Waxman et al. 1992), the rate and extent of depolarization
during metabolic stress will influence the degree of exchanger-mediated Ca 2/ entry and thus ultimate cellular injury. We therefore examined the ions and channels involved
in mediating membrane depolarization during glycolytic inhibition and chemical anoxia in optic nerve. Preliminary
results have been published in abstract form (Leppanen and
Stys 1996).
METHODS
Detailed methods and recording techniques are described in the
companion paper (Leppanen and Stys 1997). The optic nerves
were dissected from Long-Evans male rats (150–175 g) anesthetized with 80% CO2-20% O2 and decapitated. The recorded axonal
compound resting membrane potential, Vg , was measured in vitro
with the grease gap technique (Stys et al. 1993). The middle
segment of one nerve was inserted into a slit silastic tube filled with
petroleum jelly. One end was perfused at 37.07C with oxygenated
artificial cerebrospinal fluid (aCSF) or test solutions. The opposite
end was depolarized using an isotonic K / solution (NaCl replaced
with equimolar KCl) containing 0.5 mM CaCl2 . Solutions were
0022-3077/97 $5.00 Copyright q 1997 The American Physiological Society
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Leppanen, Lisa and Peter K. Stys. Ion transport and membrane potential in CNS myelinated axons. II. Effects of metabolic inhibition. J. Neurophysiol. 78: 2095 – 2107, 1997. Compound resting membrane potential was recorded by the grease
gap technique ( 37 7 C ) during glycolytic inhibition and chemical
anoxia in myelinated axons of rat optic nerve. The average
potential recorded under control conditions ( no inhibitors ) was
0 47 { 3 ( SD ) mV and was stable for 2 – 3 h. Zero glucose
( replacement with sucrose ) depolarized the nerve in a monotonic fashion to 55 { 10% of control after 60 min. In contrast,
glycolytic inhibition with deoxyglucose ( 10 mM, glucose omitted ) or iodoacetate ( 1 mM ) evoked a characteristic voltage
trajectory consisting of four distinct phases. A distinct early
hyperpolarizing response ( phase 1 ) was followed by a rapid
depolarization ( phase 2 ) . Phase 2 was interrupted by a second
late hyperpolarizing response ( phase 3 ) , which led to an abrupt
reduction in the rate of potential change, causing nerves to
then depolarize gradually ( phase 4 ) to 75 { 9% and 55 {
6% of control after 60 min, in deoxyglucose and iodoacetate,
respectively. Pyruvate ( 10 mM ) completely prevented iodoacetate-induced depolarization. Effects of glycolytic inhibitors were delayed by 20 – 30 min, possibly due to continued,
temporary oxidative phosphorylation using alternate substrates
through the tricarboxylic acid cycle. Chemical anoxia ( CN 0 2
mM ) immediately depolarized nerves, and phase 1 was never
observed. However a small inflection in the voltage trajectory
was typical after É 10 min. This was followed by a slow depolarization to 34 { 4% of control resting potential after 60 min
of CN 0 . Addition of ouabain ( 1 mM ) to CN 0-treated nerves
caused an additional depolarization, indicating a minor glycolytic contribution to the Na / -K / -ATPase, which is fueled preferentially by ATP derived from oxidative phosphorylation.
Phases 1 and 3 during iodoacetate exposure were diminished
under nominally zero Ca 2/ conditions and abolished with the
addition of the Ca 2/ chelator ethylene glycol-bis ( b -aminoethyl
ether ) - N,N,N * ,N * -tetraacetic acid ( EGTA; 5 mM ) . Tetraethylammonium chloride ( 20 mM ) also reduced phase 1 and eliminated phase 3. The inflection observed with CN 0 was eliminated during exposure to zero-Ca 2/ / EGTA. A Ca 2/ -activated
K / conductance may be responsible for the observed hyperpolarizing inflections. Block of Na / channels with tetrodotoxin
( TTX; 1 m M ) or replacement of Na / with the impermeant
cation choline significantly reduced depolarization during glycolytic inhibition with iodoacetate or chemical anoxia. The
potential-sparing effects of TTX were less than those of choline-substituted perfusate, suggesting additional, TTX-insensitive Na / influx pathways in metabolically compromised axons.
The local anesthetics, procaine ( 1 mM ) and QX-314 ( 300
m M ) , had similar effects to TTX. Taken together, the rate and
extent of depolarization of metabolically compromised axons
is dependent on external Na / . The Ca 2/ -dependent hyperpolarizing phases and reduction in rate of depolarization at later
2096
TABLE
L. LEPPANEN AND P. K. STYS
1.
Composition of solutions
aCSF
NaCl
KCl
Choline Cl
DG
KCN
TEA-Cl
MgSO4
NaHCO3
Choline
Bicarbonate
NaH2PO4
KH2PO4
CaCl2
Dextrose
Sucrose
126
3
—
—
—
—
2
26
—
1.25
—
2
10
—
Zero-Na//Choline
—
1.75
Zero Glucose
DG
126
—
—
3
—
—
—
—
2
26
—
127
—
—
—
2
—
26
—
1.25
2
10
—
—
—
126
—
2
—
2
—
26
126
10
—
—
2
26
—
1.25
—
2
—
10
KCN
1.25
—
2
—
—
TEA
106
3
—
—
—
20
2
26
—
—
1.25
—
2
10
—
1
2
10
—
gassed with 95% O2-5% CO2 . The central nerve segment
was cooled to improve recording stability. Vg was recorded with
3 M KCl/agar bridge electrodes. Junction potentials (typically
°1–3 mV), which drifted linearly over time, were determined at
the beginning and end of each experiment, and Vg was corrected
by subtracting interpolated values of these potentials. The nerve
studied immediately after the dissection was denoted nerve A. The
second nerve (B) was placed in an oxygenated chamber (95%
O2-5% CO2 ) containing aCSF at room temperature for later recording. Although the grease gap technique provided stable, longterm recordings, it is unable to discern between different axonal
populations that may exhibit different responses to metabolic inhibition (Stys and LoPachin 1996). Our results therefore, represent
a mean population response, biased in favor of larger axons (see
companion paper Leppanen and Stys 1997). Because of the origin
of the recorded potential, we cannot exclude the possibility that
some drugs affected the short circuit factor and thus affected the
recorded potentials. Finally, given the heterogeneous structure of
myelinated axons, with the majority of the axolemma covered by
tight myelin wraps, diffusion barriers into this region may have
influenced the effects of some agents active at internodal loci.
Composition of aCSF and solutions of 2-deoxyglucose (DG;
Sigma), KCN (Fisher Scientific), tetraethylammonium chloride
(TEA; Sigma), zero-glucose, and zero-Na / /choline [choline Cl
(BDH)] are listed in Table 1. NaCN (BDH), iodoacetate-Na / salt
(IAA; Sigma), pyruvate (Sigma), ouabain (Sigma), ethylene
glycol-bis( b-aminoethyl ether)-N,N,N *,N *-tetraacetic acid (EGTA;
Sigma), procaine (Sigma), and QX-314 (a generous gift from
Astra Pharma) were dissolved in aCSF. Tetrodotoxin (TTX;
Sigma) was diluted from stock solution in distilled water.
Statistical differences were calculated using analysis of variance
with Dunnett’s test.
RESULTS
Glycolytic inhibition
After nerve insertion in normal aCSF, Vg typically stabilized within 90 min and ranged from 040 to 055 mV
[ mean 047 { 3 ( SD ) mV; Table 2 ] . We examined several
methods of inducing glycolytic inhibition in the rat optic
nerve ( RON ) , a preparation that is essentially 100% myelinated ( Foster et al. 1982 ) . In Fig. 1 A, the effect of
zero-glucose ( sucrose substitution ) on resting membrane
potential is shown. A delay of 28 { 6 min preceded the
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onset of a monotonic, gradual depolarization proceeding
at 1 { 0.4 mV /min ( Table 4 ) , to 55 { 10% of control
after 60 min ( Fig. 1 A, arrow, Table 2 ) . We compared
ratios of potentials over time, normalized to the baseline
reading obtained at time 0 ( defined as 90 min after nerve
insertion and stabilization ) . Using 080 mV as an estimate
of true optic axon membrane potential ( Stys et al. 1997 ) ,
these ratios were used to estimate absolute membrane potentials over time ( Tables 7 and 8 in DISCUSSION ) . In
contrast to simple omission of glucose, substitution of
glucose with DG [ a competitive antagonist of the glycolytic enzyme hexokinase ( Devlin 1992 ) ] , induced an initial hyperpolarizing response in five of seven nerves ( Fig.
1 B ) . This early hyperpolarization, termed phase 1, together with the delay before hyperpolarizing lasted for
38 { 8 min after application of DG before nerves began
to gradually depolarize ( phase 2 ) at 2 { 1 mV /min ( Table
4 ) . Again, in contrast to zero-glucose alone, the gradual
depolarization was interrupted by a second late hyperpolarizing response in four of seven nerves studied ( phase
3 ) , finally terminating in a slow depolarization ( phase 4 )
to 75 { 9% of control after 60 min of application ( Table
2. Effects of glycolytic block or chemical anoxia on
optic nerve membrane potential
TABLE
Condition
aCSF†
Zero glucose
Deoxyglucose, 10
mM
IAA, 1 mM
Pyruvate, 10 mM, add
IAA, 1 mM‡
NaCN, 2 mM
Time (min)
Ratio* (%)
Vg (mV)
n
0
60
100
55 { 10
047 { 3
025 { 5
284
6
60
30
60
120
60
30
60
75
79
55
37
{
{
{
{
9
7
6
3
104 { 2
51 { 6
34 { 4
035
038
026
018
{
{
{
{
4
4
3
2
7
24
24
19
049 { 5
024 { 3
016 { 2
2
15
15
All times corrected for dead space. Ratio and Vg values are means {
SD. * Ratio was calculated by dividing the potential at 30, 60, and 120 min
by the time 0 potential. † Data from companion paper (Leppanen and Stys
1997). ‡ Pyruvate applied 60 min before iodoacetate-Na/ salt (IAA).
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Values are in millimolar. aCSF, artificial cerebrospinal fluid; DG, 2-deoxyglucose; TEA, tetraethylammonium chloride.
METABOLIC INHIBITION AND AXONAL MEMBRANE POTENTIAL
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FIG . 1. Effect of glycolytic inhibition on recorded (Vg ) and calculated absolute (Vm ) compound resting membrane potential
in rat optic nerve. Vm was normalized to 080 mV for this and all subsequent figures. A: zero glucose/sucrose application
evoked a gradual depolarization after a delay of É30 min. Ratio was calculated by dividing the potential at 60 min ( ➞ ) by
the time 0 potential. Deoxyglucose substituted zero-glucose (B) or iodoacetate (C) both elicited a characteristic response
consisting of 4 distinct phases: an early hyperpolarizing phase, P-1, which was followed by a more rapid depolarization
(P-2). P-2 was interrupted by a second late hyperpolarization, P-3, and the voltage trajectory then entered a final, more
gradual depolarization at a lower rate (P-4). Final levels of depolarization were similar at the end of 2 h for iodoacetate
and zero-glucose. Extent of depolarization was less for deoxyglucose than for iodoacetate or zero-glucose. D: pyruvate (10
mM) completely prevented iodoacetate-induced depolarization.
2 ) . Because energy production still could occur from residual glucose in the extracellular space or from glycogen
stores in astrocytes ( Cataldo and Broadwell 1986 ) , IAA
( 1 mM ) , a relatively specific irreversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase ( Sabri and Ochs
1971 ) , was used to directly inhibit glycolytic metabolism.
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As with DG, four phases were observed ( Fig. 1C ) : an
early distinct hyperpolarizing response ( phase 1, in 14 of
24 nerves ) was followed by a rapid depolarization ( phase
2 ) at a rate of 5 { 2 mV /min ( Table 4 ) . Phase 2 began
20 { 3 min after IAA application ( Table 4 ) , which was
significantly faster than in zero-glucose / sucrose, 28 { 6
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L. LEPPANEN AND P. K. STYS
min ( P õ 0.001 ) , or in DG / zero-glucose, 38 { 8 min
( P õ 0.01 ) . As with DG, a second late hyperpolarizing
response ( phase 3 ) interrupted phase 2. Finally, a gradual
depolarization after phase 3 ( at a reduced rate in comparison to phase 2 ) reached 55 { 6% of control potential
after 60 min ( Table 2 ) . Addition of pyruvate ( 10 mM ) , a
substrate used by the tricarboxylic acid cycle, completely
prevented the IAA-induced changes ( Fig. 1 D, Table 2 ) .
Chemical anoxia
Role of Ca 2/ during glycolytic inhibition and chemical
anoxia
The hyperpolarizing inflections observed with IAA and,
to a far lesser extent, CN 0 were unexpected and may reflect
interesting mechanisms of axonal response to energy failure.
Ca 2/ -activated K / conductances (KCa2/ ) have been implicated in other tissues (Leblond and Krnjevic 1989), therefore we investigated the role of Ca 2/ on potential trajectories
during IAA and CN 0 treatment. In Fig. 3A, simultaneous
application of IAA and nominally zero Ca 2/ caused an accelerated onset of depolarization, beginning within 5 { 2 min,
compared with 20 { 3 min in IAA alone (Table 4). The
depolarizing response, at a rate of 2 { 0.3 mV/min (Table
4), was interrupted by a less prominent phase 3 (Fig. 3A,
➞ ). Resting potential depolarized to 65 { 2% and
44 { 2% of control at 30 and 60 min (Table 3), respectively,
significantly more than with IAA alone at these times
(79 { 7% and 55 { 6%, P õ 0.01 and P õ 0.05, respectively). Pretreating the nerves with nominally zero Ca 2/ for
60 min before IAA application caused phase 3 to become a
brief response without a distinct hyperpolarization (Fig. 3B,
arrow, Table 3) after the addition of IAA. A 60-min application of zero-Ca 2/ /EGTA (5 mM) elicited a small but rapid
depolarization to 96 { 1% of control (Fig. 3C, Table 6).
As with nominally zero Ca 2/ and IAA, the nerve depolarized
quickly, beginning within 3 { 1 min (Table 4), but distinct
phases were not observed in the voltage trajectory. Exposure
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Role of Na / during glycolytic inhibition and chemical
anoxia
The role of transmembrane Na / flux during metabolic
inhibition was studied using a specific Na / channel blocker
and ion substitution experiments. In Fig. 5A, TTX (1 mM)
alone elicited a hyperpolarizing response (Table 6), and
with IAA addition, the extent of depolarization (to 95 { 6%
of control after 60 min, Table 5), was reduced greatly in
comparison with IAA alone. This gradual depolarization
continued for ¢2 h after IAA addition and did not attain a
steady state level during the times examined. Notably, no
hyperpolarizing inflections were ever seen (i.e., phases 1 or
3) in IAA-poisoned nerves pretreated with TTX. Further
support for Na / -mediated depolarization was provided by
experiments where Na / was replaced with the impermeant
cation choline. Zero-Na / /choline alone caused a prompt
hyperpolarization followed by depolarization to a stable plateau (Fig. 5B; see also Fig. 3A in companion paper). Addition of IAA produced a small but prompt hyperpolarization
(Fig. 5B, ➞ ) with little depolarization during 60 min of
application (Table 5).
For IAA and TTX, the trajectory of Vg was similar for
nerves A ( studied immediately after dissection ) and
nerves B ( maintained in oxygenated aCSF at room temper-
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Differences were observed between glycolytic block
and chemical anoxia induced with 2 mM NaCN, an inhibitor of mitochondrial cytochrome oxidase aa3 ( Albaum et
al. 1946; Kauppinen and Nicholls 1986a; Tadic 1992 ) .
CN 0 resulted in a rapid depolarization ( phase 2 ) at 4 {
2 mV /min ( Table 4 ) with minimal delay after application;
in contrast to IAA and DG, the transient initial hyperpolarization ( phase 1 ) was never seen ( Fig. 2 A ) . A small inflection ( Fig. 2 A, ➞ ) , which was far less pronounced
than with IAA, followed the rapid depolarizing response,
leading to an abrupt slowing of the rate of depolarization
( phase 4 ) . Nerves depolarized to 34 { 4% of control after
60 min ( Table 2 ) . To test the hypothesis that Na / ,K / ATPase was still partially active during CN 0 poisoning,
ouabain ( 1 mM ) was added to the perfusate after 90 min of
CN 0 . An additional depolarization confirmed that residual
pump activity was present during chemical anoxia ( Fig.
2 B ) . In contrast, no change in voltage trajectory was observed by adding ouabain to IAA-poisoned nerves
(Fig. 2C).
to zero-Ca 2/ /EGTA plus IAA resulted in a slower rate of
depolarization (1 { 0.1 mV/min) compared with IAA alone
(5 { 2 mV/min, P õ 0.05, Table 4). Although zero-Ca 2/ /
EGTA slowed the rate of depolarization because of a more
rapid onset in Ca 2/ -depleted conditions, nerves depolarized
to a greater degree at 30 and 60 min, 61 { 3% and 43 {
4%, respectively, versus IAA alone, 79 { 7% and 55 {
6%, (P õ 0.0001 and P õ 0.001, respectively) (Table 3).
Similarly, the abrupt alteration in the depolarization rate
after phase 3 was reduced progressively as the Ca 2/ removal
became more severe (compare Figs. 1C and 3, A–C), until
the trajectory became monotonic under zero-Ca 2/ /EGTA
conditions.
TEA (20 mM), a broad-spectrum K / channel antagonist
(including some KCa2/ ) (Hille 1992) depolarized nerves to
a surprisingly small extent when applied alone (Fig. 3D,
Table 6). TEA exposure during IAA application reduced
phase 1 and eliminated phase 3, with depolarization beginning within 17 { 2 min (similar to IAA alone, 20 { 3 min;
Table 4) to 61 { 4% of control after 60 min (similar to
IAA alone, 55 { 6%; Table 3). Although phase 3 was
abolished, a distinct change in the rate of depolarization
from phase 2 to phase 4 was still present.
During chemical anoxia and a nominally zero Ca 2/ perfusate that was applied 60 min before CN 0 , nerves depolarized
at a rate of 8 { 2 mV/min (Table 4) to 30 { 7% of control
at 60 min (Table 3) with an inflection apparent (Fig. 4A).
Similar to the voltage trajectory observed with IAA and
zero-Ca 2/ /EGTA, addition of CN 0 after 60 min of zeroCa 2/ /EGTA treatment evoked a depolarization at a rate of
9 { 3 mV/min, without an inflection, to 27 { 4% at 60 min
(Fig. 4B, Table 3). For both nominally zero Ca 2/ and zeroCa 2/ /EGTA treatments, nerves depolarized at a significantly
faster rate than with CN 0 alone (4 { 2 mV/min, P õ 0.05).
METABOLIC INHIBITION AND AXONAL MEMBRANE POTENTIAL
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FIG . 2. Effect of chemical anoxia and ouabain on resting membrane potential in rat optic nerve. A: NaCN (2 mM) caused
an immediate depolarizing response commonly interrupted by an inflection in the voltage trajectory ( ➞ ), followed by a
slower depolarization. Addition of ouabain (1 mM) produced an additional, although minor depolarization, in chemically
anoxic nerves (B), but not in glycolytically inhibited nerves ( C). This suggests that glycolysis is able to fuel the Na / ,K / ATPase to a minor extent during chemical anoxia. See Fig. 1 legend for definition of Vg and Vm .
ature for several additional hours ) , which was not the case
for CN 0 . For TTX-treated nerves A, addition of CN 0
resulted in a fast, albeit limited ( compared with CN 0 without TTX, P õ 0.0001 ) , depolarization to 76 { 13% of
control after 60 min of treatment ( Table 5 ) . In contrast,
nerves B failed to depolarize after CN 0 was added to the
TTX perfusate (Vg Å 99 { 7% of control after 60 min of
CN 0 ) , which was significantly different from nerve A
( P õ 0.05; Fig. 5C, Table 5 ) . The nerve A and B differ-
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ence also was observed for CN 0 and zero-Na / / choline
( P õ 0.001 ) . Similar to the CN 0 and TTX nerves A, CN 0
exposure after 60 min of zero-Na / / choline produced an
immediate, but slow depolarization ( Fig. 5 D ) to 81 { 5%
of control after 60 min of CN 0 ( Table 5 ) . In contrast to
nerves A, nerves B responded by hyperpolarizing ( Fig.
5 D, arrow ) in CN 0 , then modestly depolarized to 98 {
4% of control ( Table 5 ) . Results were similar regardless
of whether CN 0 was applied as the Na / or K / salt ( KCN
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2/
FIG . 3. Effect of Ca -depleted perfusate on membrane potential during glycolytic inhibition with iodoacetate (IAA, 1
mM). A: exposure to iodoacetate and nominally zero Ca 2/ simultaneously, accelerated the onset of depolarization, eliminated
phase 1, and reduced the size of phase 3 ( ➞; compare with Fig. 1C). B: pretreatment with zero Ca 2/ caused phase 3 to
become even less pronounced ( ➞ ). C: zero-Ca 2/ /ethylene glycol-bis( b-aminoethyl ether)-N,N,N *,N *-tetraacetic acid
(EGTA) solution alone elicited a small depolarization ( ➞ ). Addition of the Ca 2/ chelator eliminated all hyperpolarizing
phases. D: tetraethylammonium chloride (TEA; 20 mM) alone depolarized nerves slightly (r ) due to its broad-spectrum
K / -channel–blocking properties (Hille 1992). TEA reduced the early hyperpolarizing response, P-1, and abolished the
second late hyperpolarizing response, P-3. A Ca 2/ -dependent K / conductance(s) is a possible mechanism for the early and
late hyperpolarizing responses. See Fig. 1 legend for definition of Vg and Vm .
solution, Table 1 ), indicating that the small amounts of
Na / from NaCN had little effect on the nominally zeroNa / / choline responses ( data not shown ) .
We studied Vg during chemical anoxia with the use-dependent Na / channel blockers, procaine and QX-314. In Fig.
6A, procaine (1 mM) applied for 60 min had a hyperpolariz-
/ 9k1d$$oc25 J011-7
ing effect on Vg to 106 { 2% of control (Table 6). Procaine
greatly reduced the CN 0-induced depolarization (Vg Å 84 {
11% of control after 60 min, Table 5). QX-314 [300 mM, a
concentration that does not block action potential generation
(Stys et al. 1992a)], a quaternary analogue of lidocaine,
hyperpolarized Vg to 105 { 2%; this effect developed much
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METABOLIC INHIBITION AND AXONAL MEMBRANE POTENTIAL
Role of Ca2/ during glycolytic inhibition
and chemical anoxia
TABLE
3.
Condition
Zero Ca2/ / 1 mM IAA
(applied simultaneously)
Zero Ca2/, add 1 mM IAA*
Zero Ca2/ / 5 mM EGTA, add
1 mM IAA*
TEA (20 mM), add 1 mM IAA*
Zero Ca2/, add 2 mM NaCN†
Zero Ca2/ / 5 mM EGTA, add
2 mM NaCN†
Time
(min)
30
60
30
60
30
60
60
30
60
30
60
Ratio
(%)
65
44
60
42
61
43
61
38
30
34
27
{
{
{
{
{
{
{
{
{
{
{
2
2
2
1
3
4
4
7
7
7
4
Vg (mV)
031
021
029
020
030
021
030
017
013
016
013
{
{
{
{
{
{
{
{
{
{
{
0.2
2
1
1
2
1
2
1
2
3
1
n
3
3
4
4
4
4
4
3
3
3
3
more slowly than that of procaine (Fig. 6B, Table 6). The
nerve A and B difference observed during CN 0 /TTX and
CN 0 /zero-Na / /choline treatment also occurred for CN 0
and QX-314. Nerves A depolarized to 80 { 9% of control
whereas Vg for nerves B remained unchanged at 101 { 6%
(P õ 0.05, Table 5).
DISCUSSION
Glucose and oxygen are instrumental in sustaining adequate cellular energy metabolism because ATP is derived
almost exclusively from a continuous supply of glucose in
the brain (Erecinska and Silver 1989). Although a number
of studies have been carried out on the effects of anoxia/
glycolytic inhibition on peripheral myelinated axons
(Brismar 1981; Lindstrom and Brismar 1991; Lundberg and
Oscarsson 1954; Maruhashi and Wright 1967; Schoepfle and
Bloom 1959), there is far less data on metabolically compromised central axons. Several studies have examined the effects of anoxia on action potential conduction in CNS fibers
(Davis and Ransom 1987; Fern et al. 1995; Lee et al. 1993;
Stys et al. 1990), to our knowledge there is no detailed
account of the ionic determinants of resting membrane potential in central mammalian axons during anoxia or glycolytic block. We therefore investigated the effect of metabolic
compromise on membrane potential in the RON, a representative model of mammalian CNS axons.
A large proportion of the ATP synthesized from glycolysis
and oxidative phosphorylation is used by Na / ,K / -ATPase
to maintain membrane potential (Vm ) in neural cells (Ritchie
1967). Unexpectedly, various methods of blocking glycolysis that, in theory, possessed similar modes of action, produced different depolarization trajectories. IAA likely
blocked glycolysis profoundly, causing a more synchronous
disruption of energy-dependent mechanisms, eliciting a response consisting of four distinct phases. Conversely, the
energy depletion from zero glucose exposure occurred more
gradually (probably due to slow removal of glucose from
the extracellular space), resulting in a less severe insult and
consequently a more gradual collapse of ion gradients and
/ 9k1d$$oc25 J011-7
Vm (Tables 7 and 8). Axons also may have derived energy
from the degradation of astrocytic glycogen (Cataldo and
Broadwell 1986; Dringen and Hamprecht 1992; Swanson
and Choi 1993), possibly via lactate transfer (Fern and Ransom 1996; Tsacopoulos and Magistretti 1996). Adding DG,
an inhibitor of hexokinase (Devlin 1992), may have produced a somewhat harsher disruption of glycolysis, with
emergence of the four characteristic phases typical of IAA
(Fig. 1). Interference with glial glycogen breakdown by DG
(Dringen and Hamprecht 1993) also may have contributed
to the observed differences. Pyruvate completely prevented
nerve depolarization induced by IAA, making it unlikely
that nonspecific effects of this inhibitor were responsible for
Vm changes in agreement with previous reports (Ochs and
Smith 1971; Sabri and Ochs 1971). The results also indicated that RON axons are fueled preferentially by aerobic
metabolism, which generates the majority of required ATP
at rest, as in other parts of the CNS (Erecinska and Dagani
1990); glycolysis contributed insufficient and only minor
amounts of ATP for the maintenance of Vm (Fig. 2).
A feature common to all three methods of glycolytic inhibition was a significant delay (20–30 min) before any effect
on Vm was noticeable. Although it could be argued that slow
washout of glucose was responsible for zero-glucose ( {
DG) treatments, a delayed penetration of IAA into the cells
is unlikely as this inhibitor produced almost immediate effects under zero-Ca 2/ /EGTA conditions (Fig. 3C). It is
possible that zero Ca 2/ solutions altered the axon-myelin
relationship as well as cell membranes (Schlaepfer and
Bunge 1973), allowing more rapid entry of IAA. Alternatively, operation of the tricarboxylic acid cycle/oxidative
phosphorylation may have continued temporarily in IAA
poisoned nerves, using alternate substrates such as amino
acids (Stryer 1988). Evidence for persistent oxidative phosphorylation activity during glycolytic inhibition was shown
in cultured rat astrocytes and neurons (Pauwels et al. 1985)
and guinea pig synaptosomes (Kauppinen and Nicholls
1986b). These studies suggested that the cells generated
4. Time to onset and rate of rapid depolarization
induced by glycolytic inhibition and chemical anoxia
TABLE
Condition
Zero glucose
Deoxyglucose (10 mM)
IAA (1 mM)
IAA (1 mM) / zero Ca2/
(applied simultaneously)
Zero Ca2/, add 1 mM IAA†
Zero Ca2/ / 5 mM EGTA, add
1 mM IAA†
TEA (20 mM), add 1 mM IAA†
NaCN (2 mM)
Zero Ca2/, add 2 mM NaCN‡
Zero Ca2/ / 5 mM EGTA, add
2 mM NaCN‡
Time to
Onset
(min)
Rate of Initial
Rapid Phase*
(mV/min)
n
28 { 6
38 { 8
20 { 3
1 { 0.4
2{1
5{2
6
7
24
5{2
3{1
2 { 0.3
2 { 0.1
3
4
3{1
17 { 2
—
—
1 { 0.1
—
4{2
8{2
4
4
15
3
—
9{3
3
All times corrected for dead space. Middle two column values are
means { SD. * Rate shown as dVg/dt. † Zero Ca2/, zero Ca2//EGTA or
TEA were applied 60 min before IAA. ‡ Zero Ca2/ or zero Ca2//EGTA
were applied 60 min before NaCN.
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All times corrected for dead space. Ratio and Vg values are means { SD.
* Zero Ca2/ , zero Ca2//ethylene glycol-bis(b-aminoethyl ether)-N,N,N*,N*tetraacetic acid (EGTA) or TEA were applied 60 min before IAA. † Zero
Ca2/ or zero Ca2//EGTA were applied 60 min before NaCN.
2101
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L. LEPPANEN AND P. K. STYS
energy using substrates other than glycolytically derived pyruvate. Further support for maintenance of Vm mainly by
aerobic metabolism (with minimal glycolytic contribution)
was provided by the observation that nerves depolarized at
a similar rate (phase 2) with IAA and CN 0 , despite a delayed onset with the former.
Role of Ca 2/ during metabolic stress
2/
FIG . 4. Effect of zero Ca
and zero Ca 2/ /EGTA on membrane potential during chemical anoxia. A: nominally zero
Ca 2/ significantly accelerated the rate of CN 0-induced depolarization (Table 4), but inflections, characteristic of induction
of anoxia alone, were preserved. B: in contrast, addition of EGTA also caused accelerated depolarization with CN 0 but also
abolished inflections in the voltage trajectory, suggesting the possible modulation of Ca 2/ -dependent conductances during
chemical anoxia that were less pronounced than with glycolytic inhibition (compare with Figs. 1C and 3). See Fig. 1 legend
for definition of Vg and Vm .
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The early distinct hyperpolarizing response (phase 1) observed in the majority of nerves during glycolytic inhibition
with IAA and DG raised the possibility of a K / conductance
activation. These results are similar to guinea pig (Hansen
et al. 1982) and rat (Belousov et al. 1995; Fujiwara et al.
1987; Leblond and Krnjevic 1989) hippocampal neurons in
which hyperpolarization occurred during anoxia secondary
to K / -conductance increase. It was suggested that a rise in
[Ca 2/ ]i was responsible for the hyperpolarizing response
stimulating a KCa2/ channel (Leblond and Krnjevic 1989).
Our data are also consistent with such a mechanism because
phase 1 was Ca 2/ dependent. Phase 3, which interrupted the
rapid nerve depolarization with a hyperpolarizing inflection
during glycolytic inhibition, was also dependent on Ca 2/ ,
raising the possibility that phase 3 is a continuation of phase
1 sharing similar mechanisms. Moreover, reduction or elimination of these responses by TEA further supports our hypothesis of a mechanism involving activation of a KCa2/
channel. These hyperpolarizing responses were reduced progressively with more severe Ca 2/ -depleting treatments (Fig.
3), suggesting that a relatively inaccessible source of Ca 2/
may have been the trigger, perhaps originating from internal
Ca 2/ stores (Belousov et al. 1995) known to be present
in myelinated axons (Takei et al. 1992). The absence of
hyperpolarizing responses during CN 0 treatment, despite
rapid and massive depolarization, may indicate preferential
maintenance of internal Ca 2/ stores by glycolytically derived
ATP (Xu et al. 1995). Ca 2/ -depleted conditions accelerated
the rate of depolarization during chemical anoxia, consistent
with a more rapid loss of RON excitability (Stys, unpublished data). This may be due to removal of charge screening
by the divalent cation, resulting in a leakier membrane (Hille
et al. 1975; Woodhull 1973). However, zero-Ca 2/ conditions markedly slowed the depolarization rates in glycolytically inhibited nerves, suggesting that Ca 2/ depletion has
more complex effects than simple charge screening removal
at the membrane. We believe that the hyperpolarizing responses are not likely due to activation of the K / conductance opened by low levels of ATP (KATP ) (Jonas et al.
1991) because phases 1 and 3 (early and late hyperpolarizing
responses, respectively) were abolished during zero Ca 2/
conditions and, in addition, the KATP channel antagonist,
glibenclamide, failed to blunt the hyperpolarizing phases
(data not shown).
Phase 1 occurred without delay during zero-Na / /choline
and IAA exposure as well as during zero-Na / /choline and
CN 0 (nerve B) application. Moreover, it was absent during
the concomitant application of IAA and the Na / channel antagonist, TTX. The findings suggest a mechanism dependent on
Na / channels but not on Na / influx (the response was still
present with zero-Na / /choline application). It is possible that
during IAA alone, the Na / /Ca 2/ exchanger removed Ca 2/
entering through Na / channels (DiPolo et al. 1982; Stys and
LoPachin 1997) delaying the rise in [Ca 2/ ]i . During zero-Na /
perfusion, the exchanger was unable to buffer Ca 2/ , thereby
permitting a faster increase in [Ca 2/ ]i and activation of the
Ca 2/ -dependent hyperpolarizing mechanism. We propose a
METABOLIC INHIBITION AND AXONAL MEMBRANE POTENTIAL
2103
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/
/
FIG . 5. Role of Na during glycolytic inhibition and chemical anoxia. A: block of tetrodotoxin (TTX)-sensitive Na
channels elicited a small hyperpolarizing effect. Under these conditions, glycolytic inhibition with iodoacetate (1 mM)
resulted in a greatly reduced depolarization. B: replacement of Na / with the impermeant cation choline caused a transient
hyperpolarizing response. Addition of iodoacetate caused a prompt hyperpolarization ( ➞ ) with complete preservation of
membrane potential for ¢60 min. C: in contrast, TTX and NaCN application elicited significantly different responses between
nerves A (recorded immediately) and nerves B (recorded after several additional hours of in vitro incubation, see text). For
nerves A, NaCN application evoked a prompt depolarization that was much less extensive than without TTX. In contrast,
nerves B failed to depolarize. These results suggest an additional Na / influx pathway into RON axons, distinct from TTXsensitive Na / channels. This pathway appears to be downregulated after several hours of in vitro incubation (see text). D:
similarly, NaCN and zero-Na / /choline solution caused a blunted but consistent depolarization for nerves A. Nerves B (Vm
not normalized to 080 mV baseline) instead hyperpolarized slightly ( ➞ ) after addition of CN 0 with little further depolarization.
See Fig. 1 legend for definition of Vg and Vm .
dual role for the Na / /Ca 2/ exchanger: at the beginning of
metabolic stress, it operates to extrude Ca 2/ , but in the later
stages of anoxia/ischemia, when the Na / gradient has collapsed, it imports damaging quantities of Ca 2/ (Stys et al.
/ 9k1d$$oc25 J011-7
1990, 1992b). The inflection recorded during CN 0 exposure
alone may be analogous to the hyperpolarization in CN 0 and
zero-Na / /choline (Fig. 5D, nerve B), truncated by the massive
and rapid depolarization.
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L. LEPPANEN AND P. K. STYS
Role of Na/ during glycolytic inhibition and
chemical anoxia
TABLE
5.
Condition
60
60
60
Ratio (%)
Vg (mV)
n
Condition
Time
(min)
95 { 6
97 { 7
046 { 5
047 { 3
6
4
76 { 13
99 { 7
038 { 7
049 { 4
3
3
Pyruvate (10 mM)
TEA (20 mM)
Zero Ca2/ / 5 mM EGTA
TTX (1 mM)*
Procaine (1 mM)
QX-314 (300 mM)
60
60
60
20
60
60
81 { 5
98 { 4
039 { 2
047 { 1
6
4
84 { 11
038 { 5
4
Ratio
(%)
102
98
96
104
106
105
{
{
{
{
{
{
2
2
1
1
2
2
Vg (mV)
n
048
048
045
049
047
049
2
4
8
23
4
6
{
{
{
{
{
{
5
1
4
3
1
2
60
60
60
80 { 9
101 { 6
037 { 7
048 { 2
3
3
All times corrected for dead space. Ratio and Vg values are means {
SD. TTX, tetrodotoxin. * TTX applied 20 min before 60 min of either IAA
or NaCN. † IAA or NaCN were applied after 60 min of zero-Na//choline.
‡ Procaine or QX-314 were applied 60 min before NaCN.
The abrupt reduction in depolarization rate coinciding
with phase 3 (Fig. 1C), persisted in TEA despite the abolition of distinct hyperpolarizations by this blocker. This suggests that the mechanisms underlying the rate change are
distinct from those mediating the hyperpolarizing phenomena, although both appear to be relatively Ca 2/ -dependent.
Although the hyperpolarizing shifts were likely due to K /
conductance activations, given the rate limiting effect on
depolarization of Na / conductance (see below), it is likely
All times corrected for dead space, normalized to time 0 (defined after
90 min of stabilization post-insertion into recording chamber). Ratio and
Vg values are means { SD. * Data from companion paper.
that shifts in rate of decay of Vm were due to reductions in
Na / conductance, rather than increases in K / conductance.
Other studies have shown that anoxia decreases Na / permeability and shifts the steady state inactivation curve to increasingly negative potentials (Brismar 1981, 1983; Cummins et al. 1991; Haddad and Jiang 1993). The mechanism
for this presumed conductance change is unknown but may
involve Ca 2/ -dependent phosphorylation of Na / channels
by protein kinase (Li et al. 1993). The reduction of Na /
permeability may represent a stereotyped response to metabolic stress, which would at the same time reduce the drain
on already strained energy reserves and diminish the degree
of depolarization and deleterious Na / /Ca 2/ exchange-mediated Ca 2/ influx.
Role of Na / during metabolic stress
The immediate depolarizing response elicited by CN 0
demonstrated a major dependence of axonal polarization on
FIG . 6. Local anesthetics reduce anoxic RON depolarization. A : procaine ( 1 mM ) alone had a rapid hyperpolarizing
effect on membrane potential and blunted the depolarizing effect of chemical anoxia. B : QX-314 ( 300 mM ) alone also
elicited a small hyperpolarization, which developed much more slowly that with procaine, likely reflecting the limited
rate at which this permanently charged compound could cross the axolemma to gain access to the cytosolic side of the
Na / channel. Differences were observed for nerves A and B ( see Fig. 5 legend and text for details ) after CN 0 was
applied, causing limited depolarization in nerves A that was further reduced for nerves B. See Fig. 1 legend for definition
of Vg and Vm .
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TTX (1 mM), add 1 mM IAA*
Zero Na//Choline, add 1 mM IAA†
TTX (1 mM), add 2 mM NaCN*
Nerve A
Nerve B
Zero Na//Choline, add 2 mM
NaCN†
Nerve A
Nerve B
Procaine (1 mM), add 2 mM
NaCN‡
QX-314 (300 mM), add 2 mM
NaCN‡
Nerve A
Nerve B
Time
(min)
TABLE 6. Effect of various experimental conditions on
recorded compound resting membrane potential
METABOLIC INHIBITION AND AXONAL MEMBRANE POTENTIAL
TABLE
7.
Calculated absolute compound resting membrane
potential
Condition
Zero glucose
Deoxyglucose (10 mM)
IAA (1 mM)
Pyruvate (10 mM), add 1 mM IAA*
Pyruvate (10 mM)
IAA (1 mM), add 1 mM ouabain†
NaCN (2 mM)
NaCN (2 mM), add 1 mM ouabain†
Zero Ca2/, add 1 mM IAA (applied
simultaneously)
Zero Ca2/, add 1 mM IAA‡
Zero Ca2/ / 5 mM EGTA, add 1 mM IAA‡
TEA (20 mM), add 1 mM IAA‡
TEA (20 mM)
Zero Ca2/, add 2 mM NaCN‡
Zero Ca2/ / 5 mM EGTA, add 2 mM
NaCN‡
Zero Ca2/ / 5 mM EGTA
Time
(min)
60
60
30
60
120
60
60
20
30
60
20
30
60
30
60
30
60
60
60
30
60
30
60
60
Vm (mV)
044
060
063
044
029
083
081
028
041
027
020
052
035
048
034
049
034
049
078
031
024
027
021
077
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
8
7
5
5
3
2
2
4
5
3
15
2
2
2
1
2
3
3
1
5
6
5
3
1
n
6
7
24
24
19
2
2
2
15
15
3
3
3
4
4
4
4
4
4
3
3
3
3
8
All times corrected for dead space. Resting membrane potential (Vm)
values are means { SD. * Pyruvate applied 60 min before IAA. † Ouabain
applied after 90 min of either IAA or NaCN. ‡ Zero Ca2/, zero Ca2//EGTA
or TEA were applied 60 min before IAA. § Zero Ca2/ or zero Ca2//EGTA
were applied 60 min before NaCN.
/ 9k1d$$oc25 J011-7
8. Calculated absolute compound resting membrane
potential during metabolic inhibition and altered Na/
conductance
TABLE
Time
(min)
Condition
TTX (1 mM), add 1 mM IAA*
Zero Na//Choline, add 1 mM IAA†
TTX (1 mM), add 2 mM NaCN*
Nerve A
Nerve B
Zero Na//Choline, add 2 mM NaCN†
60
60
60
Procaine (1 mM), add 2 mM NaCN‡
QX-314 (300 mM), add 2 mM NaCN‡
Nerve A
Nerve B
TTX (1 mM)§
Procaine (1 mM)
QX-314 (300 mM)
60
60
60
20
60
60
Vm (mV)
n
076 { 4
077 { 6
6
4
061
080
065
079
067
{
{
{
{
{
10
5
4
3
9
3
3
6
4
4
064
081
083
085
084
{
{
{
{
{
7
5
1
2
2
3
3
23
4
6
All times corrected for dead space. Vm values are means { SD. * TTX
applied 20 min before 60 min of either IAA or NaCN. † IAA or NaCN
were applied after 60 min of zero-Na//choline. ‡ Procaine or QX-314 were
applied 60 min before NaCN. § Data from companion paper.
effects to TTX. Both agents caused a small but reproducible
hyperpolarization, indicating block of a persistent Na / conductance at rest (Stys et al. 1993). These compounds act at
the cytoplasmic side of the neuronal Na / channel; the onset
of the procaine effect was rapid likely due to its ability to
permeate the membrane in its uncharged form. In contrast,
the time of onset of the permanently charged QX-314 was
much longer, reflecting the much slower movement across
the axolemma by this molecule. As with TTX, both compounds greatly reduced nerve depolarization during chemical
anoxia. Unlike TTX, however, QX-314 does not abolish
electrogenesis at the concentration used (Stys et al. 1992a),
and its membrane potential sparing effect is likely due to
this drug’s preferential action at open, noninactivating Na /
channels (Khodorov 1991; Wang et al. 1987). QX-314 appeared to reduce the extent of depolarization for nerves A
even more effectively than TTX alone (compare Figs. 5C
and 6B), similar to complete Na / replacement with impermeant choline (Fig. 5D). Given the evidence suggesting
additional Na / influx pathways in RON axons (see above),
perhaps QX-314 has activity at these as yet unidentified
influx routes. One possibility is the inward rectifier, known
to be present on RON axons (Eng et al. 1990) and to possess
finite Na / permeability (Karst et al. 1993; Solomon and
Nerbonne 1993). This idea is supported further by observations that blocking this channel with Cs / was highly protective against RON anoxia (Stys and Hubatsch 1996), likely
due to a reduction of depolarization and Na / influx.
In conclusion, we determined that the resting potential of
the RON depends on energy generated primarily from aerobic metabolism with a minor contribution from glycolytic
ATP. Inhibition of glycolysis or oxidative phosphorylation
depolarized the recorded potential but with quite different
trajectories. The effects of glycolytic inhibition alone were
delayed, suggesting a limited ability of axons to generate
aerobic ATP from alternate substrates. The inevitable mas-
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aerobic metabolism, consistent with the rapid loss of excitability previously observed within minutes of anoxia (Stys
1996; Stys et al. 1990). Nerve depolarization by glycolytic
inhibition or chemical anoxia was reduced by blocking TTXsensitive Na / channels. One explanation may be that Na /
influx is rate limiting and will in turn affect the rate of K /
loss for reasons of electroneutrality: reducing Na / influx
therefore will reduce loss of internal K / and accumulation of
extracellular K / (Ransom et al. 1992), thereby maintaining
membrane polarization. Relative preservation of membrane
potential during metabolic inhibition where Na / was replaced with the impermeant cation choline further supports
this possibility.
Nerves A (studied immediately after dissection) and
nerves B (stored in oxygenated aCSF at room temperature
for several additional hours) exposed to Na / channel blockers (TTX, QX-314) or Na / -depleted perfusate, responded
very differently to the addition of CN 0 : nerves A depolarized
(although not nearly to the same extent as with CN 0 alone),
whereas nerves B did not (Fig. 5, C and D). Curiously,
glycolytic inhibition did not distinguish between the two
nerve populations. The persistent (albeit blunted) depolarization observed during anoxia in nerves studied promptly,
despite Na / channel block with TTX, may suggest an additional Na / influx path that appeared to be downregulated
with prolonged in vitro incubation, possibly due to washout
of modulatory factors (for example see Zhang and Krnjevic
1993).
The local anesthetics procaine and QX-314 had similar
2105
2106
L. LEPPANEN AND P. K. STYS
sive depolarization was largely, although not exclusively,
dependent on Na / influx through TTX-sensitive Na / channels [likely the noninactivating Na / conductance previously
demonstrated in the RON (Stys et al. 1993)], with evidence
for additional TTX-insensitive Na / influx pathways. The
membrane potential-sparing effect of local anesthetics likely
contributes to their neuroprotective actions. Finally, CNS
axons may possess autoprotective mechanisms (Fern et al.
1996), including possible activation of a Ca 2/ -dependent
K / conductance(s) to delay depolarization onset, and perhaps a controlled reduction of Na / permeability to slow the
decay of Vm , which also appears to be Ca 2/ dependent. We
therefore suggest that during the initial stages of metabolic
insult, the presumed rise in free [Ca 2/ ]i may be transiently
beneficial, reflecting the axon’s intrinsic mechanisms designed to limit injury during anoxia/ischemia.
Received 6 January 1997; accepted in final form 17 June 1997.
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