Traumatic Brain Injury Laboratory, Cara Phelan Centre for Trauma Research and the Department of Anaesthesia, St. Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1W8, Canada
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ABSTRACT |
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Tian, Guo-Feng and
Andrew J. Baker.
Glycolysis Prevents Anoxia-Induced Synaptic Transmission Damage
in Rat Hippocampal Slices.
J. Neurophysiol. 83: 1830-1839, 2000.
Prolonged anoxia can cause permanent
damage to synaptic transmission in the mammalian CNS. We tested the
hypothesis that lack of glucose is the major cause of irreversible
anoxic transmission damage, and that anoxic synaptic transmission
damage could be prevented by glycolysis in rat hippocampal slices. The
evoked population spike (PS) was extracellularly recorded in the CA1 pyramidal cell layer after stimulation of the Schaffer collaterals. When the slice was superfused with artificial cerebrospinal fluid (ACSF) containing 4 mM glucose, following 10 min anoxia, the evoked PS
did not recover at all after 60 min reoxygenation. When superfusion ACSF contained 10 mM glucose with or without 0.5 mM
-cyano-4-hydroxycinnate (4-CIN), after 60 min reoxygenation the
evoked PS completely recovered following 10 min anoxia. When
superfusion ACSF contained 20 mM glucose with or without 1 mM sodium
cyanide (NaCN), after 60 min reoxygenation the evoked PS completely
recovered even following 120 min anoxia. In contrast, when superfusion
ACSF contained 4 mM glucose, following 10 min 1 mM NaCN chemical anoxia
alone, without anoxic anoxia, the evoked PS displayed no recovery after 60 min reoxygenation. Moreover, when 16 mM mannitol and 16 sodium L-lactate were added into 4 mM glucose ACSF, following 10 min anoxia the evoked PS failed to recover at all after 60 min
reoxygenation. The results indicate that elevated glucose concentration
powerfully protected the synaptic transmission against anoxic damage,
and the powerful protection is due to anaerobic metabolism of glucose and not a result of the higher osmolality in higher glucose ACSF. We
conclude that lack of glucose is the major cause of anoxia-induced synaptic transmission damage, and that if sufficient glucose is supplied, glycolysis could prevent this damage in vitro.
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INTRODUCTION |
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Neurons in the mammalian CNS may be functionally
impaired following anoxic exposure (Fujiwara et al.
1987; Kass and Lipton 1982
, 1986
,
1989
; Krnjevi
and Leblond 1989
;
Lipton and Whittingham 1979
). Anoxia for 10-15 min in
vivo causes irreversible morphological (Pulsinelli et al.
1982
; Von Lubitz and Diemer 1983
) and functional (Keykham et al. 1978
; Smith et al. 1983
)
damage to brain tissue. Anoxia for 10 min in vitro also leads to
irreversible loss of synaptic transmission (Kass and Lipton
1982
, 1986
, 1989
; Lipton and Whittingham 1979
).
Because little glycogen is stored in brain tissue, a continuous supply
of glucose is crucial for neuronal function in the mammalian CNS
(Barinaga 1997; Nelson et al. 1968
).
Although many investigators have examined the changes in the intrinsic
membrane properties during anoxia (Belousov et al. 1995
;
Fried et al. 1995
; Hershkowitz et al.
1993
; Jiang and Haddad 1994
;
Krnjevi
and Leblond 1989
; Zhang and
Krnjevi
1993
) and protection against anoxic damage
(Clark and Rothman 1987
; Fried et al.
1995
; Schurr et al. 1995
), few papers have
addressed the issue of the energy supply during anoxia (Grigg
and Anderson 1989
; Lipton and Whittingham 1982
;
Schurr et al. 1987
; Zhang and Krnjevi
1993
) even though glucose deprivation causes transient or
permanent loss of neuronal functions (Fowler 1993
;
Shoji 1992
; Wada et al. 1997
).
There are discrepant conclusions arising from the results of studies on
either mechanisms or the dose response of anoxic injury in brain slice
preparations. For example, after 10 min anoxia there was almost no
recovery of CA1 synaptic transmission in slices from young and adult
rats (Kass and Lipton 1989); in contrast, after up to 20 min anoxia, synaptic transmission almost completely recovered
(Grigg and Anderson 1989
). We suggest that the
concentration of glucose in the artificial cerebrospinal fluid (ACSF)
is a critical factor. The concentration of glucose in ACSF varied from
4 mM (Fried et al. 1995
; Kass and Lipton
1982
, 1986
, 1989
; Lipton
and Whittingham 1979
, 1982
), 7 mM (Clark
and Rothman 1987
), 10 mM (Belousov et al. 1995
;
Hershkowitz et al. 1993
; Jiang and Haddad 1994
; Krnjevi
and Leblond 1989
;
Schurr et al. 1987
; Zhang and Krnjevi
1993
), 11 mM (Fujiwara et al. 1987
; Grigg
and Anderson 1989
; Shoji 1992
), and up to 25 mM
(Jiang and Haddad 1994
). This has usually been neglected
in discussions of discrepant experimental results of in vitro studies
(i.e., Fujiwara et al. 1987
; Jiang and Haddad
1994
; Kass and Lipton 1989
). In this study, we
demonstrate that variations in glucose concentrations may account for
the differences in results (Grigg and Anderson 1989
;
Kass and Lipton 1989
) and that glucose powerfully
protects synaptic transmission against anoxia-induced damage in vitro.
Although some investigators (Grigg and Anderson 1989;
Schurr et al. 1987
) have noted that increased glucose
concentrations in ACSF improved recovery of neuronal function from
anoxic challenge, the duration of anoxia has never exceeded 60 min in
brain slices. Our results show that superfusion of hippocampal slices
with ACSF containing sufficient glucose sustains partial synaptic
transmission during anoxia and ensures its rapid recovery after hypoxia
is terminated. These results demonstrated that during anoxia, lack of
glucose impairs synaptic transmission. If sufficient glucose is
supplied during anoxia, synaptic transmission is partially supported
and completely protected by glycolysis.
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METHODS |
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Male Sprague-Dawley rats (150-200 g) were anesthetized
with 2.0-2.5% halothane in oxygen (Tian et al.
1999) and decapitated. Hippocampal slices from rat
brains were prepared as described previously (Zhang et al.
1998
). The brain was immediately removed and maintained in an
ice-cold ACSF for 3-5 min before slicing. The brain was mounted on an
aluminum block and transversely sliced (~400 µm) in ice-cold
(<3°C) ACSF using a vibratome (series 1000, Technical Products
International, St. Louis, MO). Slices then were kept in oxygenated ACSF
at room temperature (22-23°C) for at least 1 h before the
experiment and not more than 8 h after slice preparation. The
composition of the ACSF is (in mM) 126 NaCl, 3.0 KCl, 1.4 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaCO3, and 4 glucose (Kass and Lipton
1989
), pH 7.4 at 36.5 ± 0.5°C when aerated with 95%
O2-5% CO2.
For electrophysiological recording, the slice was placed in a
superfusion chamber, which was closed up like a box by adding removable
plates, and only a small slit remained, which gave access to the
electrodes. The slice was fully submerged in the superfusion chamber
and continuously superfused (7-8 ml/min) with ACSF equilibrated and
continuously bubbled with 95% O2-5%
CO2 (Zhang et al. 1998). Humidified, warmed 95% O2-5%
CO2 was blown over the chamber to ensure a warm
oxygenated local environment (Zhang et al. 1998
). All
recordings were made at slice temperature of 36-37°C. To achieve a
stable experimental temperature, the ACSF was warmed up before it
superfused the slice using a water bath controlled by a temperature controller, and the temperature of the ACSF in the superfusion chamber
was continuously monitored using a YSI 4600 series precision thermometer with a micro YSI 451 temperature sensor (YSI, Yellow Springs, OH). The slice was made "anoxic" by superfusing them with
ACSF preequilibrated and continuously bubbled with 95%
N2-5% CO2, and humidified,
warmed 95% N2-5% CO2 was
blown over the chamber to ensure a warm oxygen-free local environment.
Field potentials were recorded extracellularly through glass pipettes
filled with 150 mM NaCl (tip resistance of 2-3 M
), and the
electrode was placed in the somatic layer of the CA1 region. Signals
were recorded using an Axopatch 200B amplifier (Axon Instrument, Foster
City, CA), and data were stored and analyzed with pCLAMP software
(version 6.0.4, Axon Instrument).
Electrical stimulation of Schaffer collaterals was performed using a bipolar tungsten electrode placed in the stratum radiatum of CA1. Stimulation pulses of constant current (0.2-0.5 mA, 0.1 ms) were generated by a Grass S88 stimulator (Grass Instrument, West Warwick, RI) and delivered through an isolation unit (PSIU6) every 30 s.
The extracellular postsynaptic orthodromic responses started with a
small downward stroke (arrows in Fig.
1A) just after the stimulus
artifact, the presynaptic volley (PV) (Fried et al.
1995). The PV was followed by an upward waveform [field
excitatory postsynaptic potential (fEPSP)]. During the fEPSP, there
was a sharp downward stroke, the postsynaptic population spike (PS;
asterisk in Fig. 1A) (Fried et al. 1995
). The
PS amplitude was evaluated by calculating the voltage difference
between the negative peak and the positive one preceding it.
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To examine the effect of glucose on synaptic transmission during and
after anoxia, slices were superfused with ACSF that contained different
concentrations of glucose. In one set of experiments, glucose
concentrations were 4, 10, and 20 mM. In another series, glucose was
used in combination with other chemicals [4 mM glucose plus 16 mM
mannitol, 4 mM glucose plus 16 mM sodium L-lactate, 4 mM
glucose plus 1 mM sodium cyanide (NaCN), 10 mM glucose plus 0.5 mM
-cyano-4-hydroxycinnate (4-CIN), 20 mM glucose plus 1 mM NaCN].
Different ACSFs were superfused for 15 min before anoxia, during
anoxia, and 60 min reoxygenation after different durations (10, 60, and
120 min) of anoxia, except ACSF containing 4 or 20 mM glucose plus 1 mM
NaCN, which were superfused only during anoxia. In this case ACSF
containing 4 or 20 mM glucose without NaCN was superfused for 15 min
before anoxia and during 60 min reoxygenation. Recovery of synaptic
transmission was assessed by expressing the 1-h postanoxic PS amplitude
as a percentage of control (preanoxic) amplitude.
All data are expressed as group means ± SD. Significance levels in all cases were determined by Student's t-test.
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RESULTS |
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Extracellular recordings of evoked responses
Figure 1A shows the synaptic responses recorded from
the CA1 pyramidal cell layer after electrical stimulation of the
Schaffer collaterals when the slice was superfused with ACSF containing 4, 10, and 20 mM glucose. The responses were similar to those reported
previously (Fried et al. 1995). The typical response consists of three parts: the PV, the fEPSP, and the PS. The PV is a
small downward stoke (arrow in Fig. 1A) just after the
stimulus artifacts (Fried et al. 1995
), representing
action potentials from the axonal terminals of the Schaffer
collaterals. The fEPSP is an upward waveform just after the PV. The PS
is a sharp downward stroke during the fEPSP (Fried et al.
1995
), consisting of the synchronized firings of postsynaptic
neurons, its magnitude largely determined by the number and
synchronization of their firings (asterisk in Fig. 1A). The
extracellular recordings were stable for up to 3 h when the slice
was superfused with ACSF containing 4, 10, and 20 mM glucose in normal
conditions without any anoxic challenge (Fig. 1B). The
maximal PS amplitudes averaged 7.1 ± 1.3 mV (mean ± SD,
range 5.0-13.0 mV, n = 86).
Effects of anoxia on evoked responses
When the slice was superfused with ACSF containing 4 mM glucose, the PV was abolished within 5 min after introduction of anoxia (n = 6; Fig. 2A). However, it persisted throughout 10 min (n = 6; Fig. 3A) and 60 min (n = 7; Fig. 3C) of anoxia at 10 mM glucose; it also persisted throughout 60 min (n = 6; Fig. 4A) and 120 min (n = 6; Fig. 4C) of anoxia at 20 mM glucose. Thus severe prolonged anoxia did not impair the ability of Schaffer collaterals to be excited and conduct action potentials when the slices were superfused with ACSF containing 10 or 20 mM glucose.
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At 4 mM glucose, the fEPSP in all slices completely disappeared at ~2-4.5 min after introduction of anoxia (Table 1). At 10 mM glucose, however, the fEPSP in some slices did not disappear during 10 min (2 of 6) and 60 min (2 of 7) anoxia at all (Table 1); the fEPSP in some slices disappeared at ~3-8 min after introduction of anoxia, but the fEPSP in some slices recovered later during anoxia (Table 1). Similar to those at 10 mM glucose, at 20 mM glucose, and during 60 and 120 min anoxia, the fEPSP in some of slices did not disappear at all throughout the duration of anoxia (Fig. 5A, Table 1); the fEPSP in some slices disappeared ~4-10 min after introduction of anoxia, but the fEPSP in some slices recovered later during anoxia (Fig. 5B, Table 1). The retention of the fEPSP in some slices (Fig. 5A) and its reappearance in others (Fig. 5B) during anoxia at 10 or 20 mM glucose, particularly at 20 mM glucose during anoxia lasting 120 min, indicated that presynaptic terminals could still be excited and could retain the ability to release neurotransmitters, and that postsynaptic membrane receptors retained the ability to respond to released neurotransmitters.
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The PS was always depressed at ~0.5-1.5 min and completely abolished within 3 min after the introduction of anoxia regardless of glucose concentration (P > 0.05; Figs. 2-4).
At 4 mM glucose, the anoxic depolarization (Roberts and Sick
1988) always occurred within 2-5 min (3.3 ± 0.8 min,
n = 6) after introduction of anoxia (Fig.
2C). The occurrence of anoxic depolarization indicated that
the ion gradients across neuronal membrane were severely disturbed.
However, at 10 or 20 mM glucose, anoxic depolarization was not observed
during 10, 60, and even 120 min anoxia. Anoxic depolarization was not
observed during 10, 60, and even 120 min of anoxia, suggesting that the
extracellular potassium concentration was well maintained in ACSF
containing 10 or 20 mM glucose.
Recovery of synaptic transmission following anoxia
Table 2 summarizes the PS recovery after different durations of anoxia in the presence of different concentrations of glucose and combinations in ACSF.
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Effect of glucose concentration on recovery of synaptic transmission following anoxia
When the slice was superfused with ACSF containing 4 mM glucose, following 10 min anoxia, although the PV recovered (Fig. 2A), PS displayed no recovery after 60 min reoxygenation (Fig. 2, A and B, and Table 2). This indicated that synaptic transmission was completely damaged by 10 min anoxia at 4 mM glucose in ACSF. However, when anoxia was terminated at 30 s after anoxic depolarization occurred, the PS slowly recovered within 2-7 min (4.2 ± 1.8 min, n = 7) after reoxygenation (Fig. 6, A and B). Its recovery at the end of 60 min reoxygenation was 34 ± 15% (n = 7). At 4 mM glucose in ACSF, the PS did partially recover when anoxia was terminated at 30 s after the occurrence of anoxic depolarization, indicating anoxic duration after the occurrence of anoxic depolarization was strongly correlated with the degree of recovery of synaptic transmission following anoxic challenge.
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When the slice was superfused with ACSF containing 10 mM glucose, following 10 min anoxia, the PS rapidly recovered within 2.0-4.0 min after reoxygenation (Fig. 3B and Table 2), and its recovery at the end of 60 min reoxygenation was 105 ± 14% (Fig. 3, A and B, and Table 2). This suggests that glucose at 10 mM in superfusion ACSF prevents damage to synaptic transmission by 10 min anoxia. Even following 60 min anoxia, the PS still recovered within 1.5-6.0 min after reoxygenation (Fig. 3D and Table 2), and its recovery at the end of 60 min reoxygenation was 91 ± 27% (Fig. 3, C and D, and Table 2). Thus even following 60 min anoxia, glucose at 10 mM in ACSF still protects synaptic transmission from irreversible damage.
When the slice was superfused with ACSF containing 20 mM glucose, following 60 min anoxia, the PS rapidly recovered within 1.0-3.5 min after reoxygenation (Fig. 4B and Table 2), and its recovery at the end of 60 min reoxygenation was 119 ± 25% (Fig. 4, A and B, and Table 2). Even following 120 min anoxia, the PS rapidly recovered within 1.0-2.5 min after reoxygenation as well (Fig. 4D and Table 2), and its recovery at the end of 60 min reoxygenation was 112 ± 12% (Fig. 4, C and D, and Table 2). This indicated that the 20 mM glucose provided virtually complete protection against anoxia-induced damage to synaptic transmission by prolonged anoxia even up to 120 min.
Effect of osmolality on recovery of synaptic transmission following anoxia
The protection of synaptic transmission during anoxia by higher
glucose concentration could be due to higher osmolality in ACSF
(Huang et al. 1996) rather than the glucose itself. To
evaluate this possibility, 16 mM mannitol was added into ACSF
containing 4 mM glucose to yield an ACSF with an osmolality equal to
that of ACSF containing 20 mM glucose. When slices were superfused with
this ACSF, following 10 min anoxia the PS still did not recover at all
after 60 min reoxygenation (Fig. 6, C and D, and
Table 2). These results indicated that the protection against
anoxia-induced disruption of synaptic transmission was due to glucose
itself, not to higher osmolality.
Effect of superfusion rate on recovery of synaptic transmission following anoxia
The superfusion rate varied from 1-2 ml/min (Khazipov et
al. 1993; Shurr et al. 1987
), 4-5 ml/min
(Takata and Okada 1995
), 7-9 ml/min (Fujiwara et
al. 1987
), 25-35 ml/min (Kass and Lipton 1982
;
Lipton and Whittingham 1982
), up to 60-70 ml/min
(Fried et al. 1995
; Kass and Lipton 1986
)
at different laboratories. Because the anoxic depolarization is very
transient in the slices (Fig. 2C), the potassium might be
washed out at the superfusion rate of 7-8 ml/min. To evaluate this
possibility, the slices were superfused at a slower flow rate. At ~2
ml/min superfusion rate, the results were similar to those at the
superfusion rate of 7-8 ml/min. When slices were superfused with ACSF
containing 20 mM glucose at ~2 ml/min (1.8-2.2 ml/min), following
120 min anoxia, the PS still rapidly recovered within 1.0-3.0 min
after reoxygenation (Fig. 7B),
and its recovery at the end of 60 min reoxygenation was 114 ± 19% (n = 6; Fig. 7, A and B).
However, the PS was abolished at a slower rate after introduction of
anoxia when slices were superfused at the slower flow rate (3.0-5.0
min vs. 2.0-3.0 min). This indicated that when the slice was
superfused with ACSF containing 20 mM glucose, the superfusion rate did
not affect recovery of synaptic transmission following 120 min anoxia.
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Effect of metabolism on recovery of synaptic transmission following anoxia
Our recordings were made at least 150 µm below the upper surface
of the slice. In this region during anoxia, no oxygen is available to
neurons (Fujii et al. 1982; Fujiwara et al.
1987
). To confirm the absence of oxygen during anoxic
challenge, the following experiments were performed. First, if during
anoxia the tissue was able to acquire some oxygen, lactate could be
used to produce ATP via aerobic metabolism, and then provide some
protective effect on synaptic transmission against anoxic damage.
Therefore 16 mM sodium L-lactate was added into ACSF
containing 4 mM glucose, to confirm whether aerobic metabolism of
glucose during anoxia was responsible for the protection against
anoxia-induced damage. With this ACSF, following 10 min anoxia, the PS
failed to recover after 60 min reoxygenation (Fig. 7, C and
D, and Table 2), suggesting that the production of ATP via
aerobic metabolism of glucose during anoxia (with consequent protection
against anoxia) was not responsible for the protection against
anoxia-induced damage. Second, 1 mM NaCN was added to ACSF containing
20 mM glucose during anoxia to cause anoxic anoxia combined with
chemical anoxia (Wind et al. 1997
). Before and after
this combined anoxia, the slice was superfused with ACSF containing 20 mM glucose without NaCN. The results were similar to those during
anoxia when slices were superfused with ACSF containing 20 mM glucose
without 1.0 mM NaCN. The fEPSP either persisted or recovered during
anoxia, and the PV persisted throughout the anoxic period of 120 min.
After reoxygenation, the PS returned within 1.5-3.5 min, and its
recovery at the end of 60 min reoxygenation was 118 ± 9% (Fig.
8, A and B, and
Table 2). To confirm whether the cyanide in the
bicarbonate/CO2 ACSF endangered the slice
preparation, the slice was challenged with only 1.0 mM NaCN chemical
anoxia alone without anoxic anoxia for 10 min at 4 mM glucose. When the
slice was superfused with ACSF containing 4 mM glucose, following 10 min 1.0 mM NaCN chemical anoxic challenge, the PS failed to recover
after 60 min reoxygenation (n = 6; Fig. 8, C and
D). Therefore the cyanide endangered the slice preparation,
and 10 min 1.0 mM NaCN chemical anoxia alone without anoxic anoxia
could completely damage the synaptic transmission in 4 mM glucose
bicarbonate/CO2 ACSF. These results also
confirmed that the protection afforded by glucose was not due to ATP
produced via aerobic metabolism during anoxic challenge.
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The lactate produced by glucose during anoxia may play a very important
role in the recovery of synaptic transmission after reoxygenation
(Schurr et al. 1997a,b
). When the slice was superfused with 10 mM glucose ACSF containing 0.5 mM 4-CIN (a lactate transporter inhibitor that inhibits the lactate transportation from glia to neurons), the results were similar to those when slices were superfused with ACSF containing 10 mM glucose without 0.5 mM 4-CIN. The PV persisted throughout the anoxic period, and the fEPSP either persisted or reappeared during anoxia. The PS returned after reoxygenation, and
their recoveries at the end of 60 min reoxygenation were 107 ± 6% and 100 ± 13% following 10 and 60 min anoxia, respectively (Fig. 9 and Table 2). These results
indicate that the protection of glucose is not due to lactate produced
by glia, but is due to glucose metabolism by the neuron itself.
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DISCUSSION |
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During anoxia, neuronal energy supply decreases because neurons
cannot produce energy via efficient oxidative metabolism; to
compensate, there is a rapid increase in anaerobic metabolism (Duffy and Pulsinelli 1979; Lutz and Nilsson
1997
). During anoxia, brain tissue requires more glucose via
glycolysis to compensate for the inefficiency of anaerobic metabolism,
because 1 molecule of glucose produces only 2 molecules of ATP via
anaerobic metabolism, whereas 1 molecule of glucose produces 38 molecules of ATP via aerobic metabolism (Lutz and Nilsson
1997
; Schurr et al. 1997a
; Wass and
Lanier 1996
). Thus we hypothesized that glucose in elevated supply would provide a sufficient source of energy via glycolysis to
preserve important neuronal function during anoxia. There is precedence
for this phenomenon in turtle brain tissue (Lutz 1992
; Lutz and Nilsson 1997
).
General inhibition to synaptic transmission by anoxia
Our results showed that the PS amplitude began to decrease
~0.5-1.5 min after the introduction of anoxia. This decrease might be caused by a decrease in ATP in the synaptic region of the
hippocampal slice, i.e., in the molecular layer (Lipton and
Whittingham 1982), and the increase in extracellular
K+ due to inhibition of the
Na+/K+ ATP pump
(Fujiwara et al. 1987
; Lipton and Whittingham
1979
). The PS was completely abolished ~1.5-3.0 min after
the exposure to anoxia. This effect might be due to a neuronal
depolarization because extracellular K+ increased
(Fujiwara et al. 1987
; Lipton and Whittingham
1979
).
Damage to synaptic transmission by anoxia
When the hippocampal slice was superfused with ACSF containing 4 mM glucose, there was no recovery of the PS following exposure to
anoxia for only 10 min. This finding indicates that synaptic transmission in CA1 is completely damaged by 10 min exposure of anoxia
under these conditions, a result consistent with those of Kass
and Lipton (1986, 1989
). This irreversible
damage to synaptic transmission might be due to both a decreased ATP
level and an increased cytosolic calcium level (Kass and Lipton
1982
, 1986
, 1989
). However, at 4 mM glucose in ACSF, when anoxia was terminated at 30 s after
anoxic depolarization occurred, the PS could partially recover after
reoxygenation. When the slice was superfused with 4 mM glucose ACSF,
the anoxic depolarization always occurred after anoxia. The anoxic
duration after anoxic depolarization was 5-8 min (Fig. 1C)
when the slice was challenged by 10 min anoxia at 4 mM glucose in ACSF,
so the ion gradients across the neuronal membrane might be severely
disturbed. Our results further confirm that the anoxic duration after
anoxic depolarization is strongly correlated with the irreversible
transmission damage (Roberts and Sick 1988
; Sick
et al. 1987
).
Glucose protection against damage to synaptic transmission by anoxia
Although previous reports have stated that elevated glucose
improves functional recovery from anoxic challenge in vitro
(Grigg and Anderson 1989; Schurr et al.
1987
), it was unpredicted and remarkable in the present study
to find that glucose offers such an extremely powerful protection
against damage to synaptic transmission by anoxia in vitro.
During 60 min and even 120 min of anoxia, when the slice was superfused
with ACSF containing 20 mM glucose, the evoked PV and fEPSP could be
sustained throughout the duration of anoxia (Fig. 4A). This
finding indicated that during up to 120 min of exposure to anoxia, the
presynaptic terminal could still be excited and could retain the
capability to release neurotransmitters, and the postsynaptic receptors
could still be excited by those neurotransmitters. These novel findings
in the anoxic hippocampal slice demonstrated that mammalian neuronal
synaptic functions could be sustained under severe anoxic conditions if
there were enough glucose in the brain tissue. As pointed out
previously, these conditions are somewhat like those of turtle brain
tissue (Lutz 1992; Lutz and Nilsson
1997
), which can survive a prolonged anoxic challenge. During
anoxia there is a marked hyperglycemia in the turtle, with plasma
glucose rising to as much as 17.7 mM (Penney 1974
)
accompanied by an increase in blood flow to the brain by as much as
260% (Davies 1990
). During prolonged anoxia (up to
6 h), extracellular potassium concentration
[K+]o remained near
baseline in intact turtle brain (Sick et al. 1982
). In
isolated turtle cerebellum, with 20 mM glucose in ACSF, ATP levels
could be maintained and significant increase in
[K+]o could be prevented
during prolonged anoxia (up to 6 h) (Perez-Pinzon et al.
1992
). Although evoked field potentials were significantly depressed at the end of 4 h anoxia, evoked field potentials could restore to the initial values (Perez-Pinzon et al.
1992
).
However, in most of our hippocampal slices, during anoxia the fEPSP was
restored after its disappearance at the beginning of the anoxia (Fig.
5B). We interpreted this observation as follows. At the
beginning of anoxia, the brain tissue cannot produce enough high-energy
phosphates such as ATP to sustain the fEPSP. The glucose anaerobic
metabolism pathway, which might be inhibited in normoxia, becomes more
active as anoxia progresses. It has been suggested that this Pasteur
effect, i.e., increased glycolysis as compensation for inhibition of
oxidative metabolism, may exist during anoxia in turtle brains
(Lutz 1992; Perez-Pinzon et al.
1992
). Therefore the fEPSP progressively recovered after its
initial disappearance (Fig. 5B). When the brain tissue
produced enough high-energy phosphates to support synaptic functions,
the fEPSP was slowly increased or restored (Fig. 5, A and
B).
Our results demonstrated that higher concentrations of glucose
prevented damage to synaptic transmission by anoxia, resulting in
faster and complete recovery of PS amplitudes when the slices were
reoxygenated. Indeed, 20 mM glucose provides virtually complete protection against damage to synaptic transmission by anoxia lasting as
long as 120 min. This in vitro powerful protection against anoxia-induced synaptic transmission damage is impressive. Synaptic transmission in our slices was impaired less compared with the results
of an earlier report (Schurr et al. 1987). In that
study, only 39 and 93% of hippocampal slices recovered synaptic
function following 10 min anoxia in the presence of 10 and 20 mM
glucose, respectively. In our study, following 10 min anoxia, 100% of
slices recovered synaptic function and the PS amplitude recovered to a
mean of 105% of preanoxic value at 10 mM glucose. The effect of the
superfusion rate on these results was evaluated. The results at the
superfusion rate of ~2 ml/min were similar to those at the
superfusion rate of 7-8 ml/min (Fig. 7, A and
B), indicating that the superfusion rate does not affect
recovery of synaptic transmission following anoxia. The better
"performance" of our tissues might be due to the following factors.
First, the hippocampal slices in this study may have less traumatic
injury (Lipton et al. 1995
), because our slices
were obtained by a vibratome slicer and cutting speed was very slow
(<5 mm/min), whereas theirs were obtained by McIIwain chopper. Second,
the hippocampal slices were obtained from younger rats in this study
(weighing 150-200 g vs. 200-350 g). Finally, there were differences
in the method of interpreting recovery of synaptic function. This study
compared the PS amplitude at the end of 60 min reoxygenation of each
slice to its own preanoxic level.
Mechanism of glucose protection against anoxic synaptic transmission damage
PROTECTIVE EFFECT OF HIGH OSMOLALITY ON ANOXIA SYNAPTIC
TRANSMISSION DAMAGE.
Although it has been shown that when 100 mM mannitol or fructose was
added into 10 mM glucose ACSF, such a hyperosmolality environment
improved functional recovery from anoxia (Huang et al.
1996), in our study, 4 mM glucose plus 16 mM mannitol or 16 mM
sodium L-lactate in ACSF did not show any protection
against 10 min anoxic damage to synaptic transmission. In contrast, 10 mM glucose and 20 mM glucose in ACSF provided complete protection against 10 and 120 min anoxic damage to synaptic transmission, respectively. These results therefore confirmed that the protection offered by high concentrations of glucose against damage to synaptic transmission by anoxia was not due to an increase in osmolality.
OTHER SOURCES OF OXYGEN IN THE PREPARATION.
Our recordings were performed at depths of at least 150 µm from the
upper surface of the hippocampal slice so that in the recording area
there was no oxygen that could be used by neurons (Fujii et al.
1982; Fujiwara et al. 1987
). Furthermore, if
neurons could use the extra glucose to produce energy via oxidative
metabolism, then they could certainly use lactate to produce energy via
oxidative metabolism to protect synaptic transmission from anoxic
damage. However, there was no recovery of the PS when the hippocampal slice was perfused with an ACSF containing 4 mM glucose and 16 mM
Na+ L-lactate.
ROLE OF LACTATE PRODUCED BY GLIA IN SYNAPTIC TRANSMISSION RECOVERY.
The lactate produced by glia might be an obligatory aerobic energy
substrate for functional recovery after anoxia (Schurr et al.
1997a,b
). In this study, when 0.5 mM 4-CIN was added into 10 mM
glucose ACSF, the lactate transportation was inhibited by 4-CIN
(Izumi et al. 1997
). However, the recoveries of synaptic transmission following 10 and 60 min anoxia were similar to those at 10 mM glucose without 4-CIN in ACSF. Therefore the lactate produced by
glia might not play an important role in the synaptic transmission
recovery following anoxia in this study.
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ACKNOWLEDGMENTS |
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The authors thank Drs. J. Duffin, S. Iscoe, and Y. T. Wang for critical comments on manuscript, D. Wigglesworth for help in figure preparation, and Dr. M. Zhao and K. McClenaghan for excellent assistance.
This work was supported by the St. Michael's Hospital Foundation.
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FOOTNOTES |
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Address for reprint requests: G.-F. Tian, Traumatic Brain Injury Laboratory, Cara Phelan Centre for Trauma Research, Rm. 7080, 7th Fl., Bond Wing, St. Michael's Hospital, 30 Bond St., Toronto, Ontario M5B 1W8, Canada.
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 30 June 1999; accepted in final form 8 December 1999.
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REFERENCES |
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