1Department of Physiology and 2Department of Neurosurgery, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan
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ABSTRACT |
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Isagai, T., N. Fujimura, E. Tanaka, S. Yamamoto, and H. Higashi. Membrane dysfunction induced by in vitro ischemia in immature rat hippocampal CA1 neurons. We investigated differences between immature and mature hippocampal neurons in their response to deprivation of oxygen and glucose (in vitro ischemia), using intracellular recording techniques from CA1 pyramidal neurons in rat brain slices. The membrane was more depolarized in immature hippocampal CA1 neurons (postnatal day 7, P7) compared with the adult neurons (P140), and the apparent input resistance in immature neurons was higher than that in adult neurons. In immature neurons, the threshold for action potential generation was high, and the peak amplitude of the action potential was low in comparison with adult neurons. A time-dependent inward rectification, at potentials negative than the resting potential, was prominent in neurons of P14 and P21. After P21, the resting membrane potential, the apparent input resistance, and the threshold and the peak amplitude of the action potential did not significantly change with increasing age. In adult neurons, application of ischemia-simulating medium caused irreversible changes in membrane potential consisting of an initial hyperpolarization followed by a slow depolarization and a rapid depolarization. Once the rapid depolarization occurred, reintroduction of oxygen and glucose failed to restore the membrane potential, a state referred to as irreversible membrane dysfunction. In neurons of ages P7 or P14, the initial hyperpolarization was not apparent, whereas a slow depolarization followed by a rapid depolarization was observed. With development of the neurons, the latency for onset of the rapid depolarization became shorter and its maximal slope increased. Moreover, neurons of ages P14 or P21 showed a partial or complete recovery after reintroduction of oxygen and glucose, unlike mature neurons. In summary, the present study has demonstrated that the initial hyperpolarization and rapid depolarization induced by in vitro ischemia is age dependent. The rapid depolarization is not readily produced in the neurons in age less than P21 during ischemic exposure.
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INTRODUCTION |
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A considerable number of experimental studies have
demonstrated that the immature brain, including brain stem
(Ballanyi et al. 1992), hippocampus (Cherubini et
al. 1989
; Friedman and Haddad 1993
), and
cerebral cortex (Bickler et al. 1993
; Hansen
1977
; Luhmann and Kral 1997
; Luhmann et
al. 1993
), is markedly resistant to oxygen deprivation. There
have been some reports that the neural activity in immature rat
hippocampal neurons is also resistant to oxygen and glucose deprivation
(in vitro ischemia) (Kawai et al. 1989
; Nabetani
and Okada 1994a
,b
; Nabetani et al. 1995
, 1997
). This insensitivity of the immature brain to lack of oxygen or in vitro
ischemia probably results from developmental differences in brain
energy production and energy consumption (for review, see
Ben-Ari 1992
; Hansen 1985
; Luhmann
1996
).
We previously have reported that adult hippocampal CA1 neurons show an
initial hyperpolarization followed by a slow depolarization, which
leads to a rapid depolarization after ~6 min of exposure to oxygen
and glucose-deprived medium (Tanaka et al. 1997). When oxygen and glucose are reintroduced immediately after generating the
rapid depolarization, the membrane potential depolarizes further and
approaches 0 mV (the persistent depolarization) (Rader and Lanthorn 1989
). Thus the neuron shows no functional recovery
(Higashi 1990
; Higashi et al. 1990
;
Kudo et al. 1989
; Rader and Lanthorn 1989
; Tanaka et al. 1997
; also see Martin
et al. 1994
). Moreover, simultaneous recordings of changes in
intracellular Ca2+ concentration
([Ca2+]i) and membrane potential recorded in
Fura-2/AM-loaded slices revealed a rapid increase in
[Ca2+]i corresponding to the rapid
depolarization in all CA1 layers (Tanaka et al. 1997
;
also see Hansen and Zeuthen 1981
; Silver and
Erecinska 1990
; Uematsu et al. 1988
). Moreover,
pretreatment with a N-methyl-D-aspartic acid
(NMDA) receptor antagonist or a non-NMDA receptor antagonist inhibits
the persistent depolarization and restores the membrane potential to
preexposure levels when oxygen and glucose have been reintroduced
(Rader and Lanthorn 1989
; Tanaka et al.
1997
; Yamamoto et al. 1997b
). Thus the
activation of non-NMDA and NMDA receptors and the accumulation of
[Ca2+]i have important roles in the membrane
dysfunction induced by in vitro ischemia. Nevertheless, the potential
responses induced by in vitro ischemia in immature CA1 hippocampal
neurons are still unclear.
The rapid depolarization induced by in vitro ischemia (Tanaka et
al. 1997) corresponds to the terminal depolarization (phase II
depolarization) produced by in situ ischemia or asphyxia (Hansen 1985
). The differences in the mechanisms underlying the
generation of the rapid depolarization after in vitro ischemia between
immature and adult rats are of interest because the rapid
depolarization is crucial for the irreversible membrane change that
leads to neuronal death (Tanaka et al. 1997
). Thus the
present study addresses processes involved in the membrane dysfunction
induced by in vitro ischemia in hippocampal CA1 neurons in slice
preparations from rats of different ages. We have examined whether or
not in vitro ischemia produces the rapid depolarization in immature
hippocampal CA1 neurons and whether the rapid depolarization triggers
membrane dysfunction. Preliminary accounts of some of this data have
been presented previously (Fujimura et al. 1997a
).
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METHODS |
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The preparation and recording techniques employed were similar
to those described in the previous paper (Tanaka et al.
1997). Briefly, the forebrain of immature [postnatal day (P)7,
P14, P21, P28, P35, and P42] and adult (P140) male Wistar rats was
removed quickly under ether anesthesia and placed in chilled (4-6°C)
Krebs solution aerated with 95% O2-5% CO2.
The composition of the solution was (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose.
The hippocampus was dissected and then sliced with a Vibratome (Oxford)
at a thickness of ~400 µm. The slice preparation was submerged
completely in superfusing solution (preheated to 36.5 ± 0.5°C).
Intracellular recordings from CA1 pyramidal neurons were made with
glass micropipettes filled with K acetate (2 M). The electrode
resistance ranged between 40 and 80 M
.
Slices were made "ischemic" by superfusing them with medium equilibrated with 95% N2-5% CO2 and deprived of glucose, which was replaced with NaCl isoosmotically (ischemia-simulating medium). When switching the superfusing media, there was a delay of 15-20 s before the new medium reached the chamber due to the volume of the connecting tubing. Thus there was an ~30-s delay in saturating the bath with test solution after initial switching to test solution.
The response to deprivation of oxygen and glucose mainly consists of an
initial hyperpolarization, a slow depolarization, a rapid
depolarization and a persistent depolarization (Tanaka et al.
1997). The onset of the rapid depolarization produced by ischemia-simulating medium was estimated by extrapolating the slope of
the slow depolarization to the rapid depolarization. The latency of the
rapid depolarization was measured from onset of superfusion to onset of
the rapid depolarization (Tanaka et al. 1997
). Recovery
after reintroduction of oxygen and glucose was defined as follows: no
recovery, 30-60 min after reintroduction the membrane potential lay
between 0 and
19 mV; complete recovery, the membrane potential was
more negative than
60 mV; partial recovery, membrane potential
repolarized to values between
20 and
59 mV (Yamamoto et al.
1997b
). In most neurons with complete recovery, action
potentials and fast excitatory postsynaptic potentials elicited by
direct and focal stimulation, respectively, were similar to those
observed during the preexposure period, i.e., in normal medium. All
quantitative results were expressed as means ± SD. The number of
neurons examined is given in parentheses. The one-way ANOVA with
Scheffé post hoc comparisons was used to compare data, with
P < 0.05 considered significant, unless specified otherwise.
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RESULTS |
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This study was based on intracellular recordings from 60 CA1 pyramidal neurons of immature (P7, n = 10; P14, n = 13; P21, n = 10; P28, n = 4; P35, n = 7; P42, n = 6) and adult (P140, n = 10) rats with stable resting membrane potentials.
Membrane properties of immature and adult neurons
Table 1 summarizes resting and active membrane properties of hippocampal CA1 neurons of different ages. The membrane was more depolarized in immature (P7 and P14) CA1 neurons compared with adult CA1 neurons (P < 0.02 and P < 0.001, respectively). The apparent input resistance was significantly higher in immature CA1 neurons (P7) (P < 0.05). Figure 1 shows typical action potentials elicited by brief depolarizing current pulses through the recording electrode at different ages. The action potential was abolished reversibly by tetrodotoxin (TTX 0.3 µM) in immature (P7, n = 6 and P14, n = 6) and adult CA1 neurons (P140, n = 6; not shown). The threshold for action potential generation in immature (P7 and P14) CA1 neurons was depolarized significantly compared with that in adult CA1 neurons (P < 0.001 and P < 0.05, respectively; Table 1). The peak amplitude of the action potential was significantly lower in immature CA1 neurons (P7; P < 0.001) compared with adult neurons. The duration of the action potential was not significantly different between immature and adult CA1 neurons.
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Figure 2A illustrates typical potential responses to injection of hyperpolarizing and depolarizing current pulses in CA1 neurons at different ages. The resting membrane was more hyperpolarized in adult neurons (P140) compared with immature neurons (P7-P21). The apparent input resistance decreased as the age increased. In addition, a time-dependent inward rectification elicited at potentials negative to the resting potential was prominent in neurons of P14 and P21. The V-I relationship of these neurons also showed the presence of the time-dependent inward rectification in neurons of P14 and P21 but not in neurons of P7 and P140 (Fig. 2B).
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Responses to perfusion with ischemia-simulating medium in immature and adult CA1 neurons
As described previously (Tanaka et al. 1997),
deprivation of oxygen and glucose produced a sequence of potential
changes consisting of an initial hyperpolarization, a slow
depolarization followed by a rapid depolarization in adult neurons (at
age more than P56). All responses were accompanied by decreases in the
apparent input resistance. Reintroduction of oxygen and glucose after
the rapid depolarization failed to restore the membrane potential to
control levels. The membrane continued to depolarize progressively to 0 mV (a persistent depolarization), and this was accompanied by a further
decline in the apparent input resistance (Fig.
3D). These changes in membrane
potential and input resistance were never restored to control levels
even when the slice was perfused with normal medium for >60 min
(Onitsuka et al. 1998
; Tanaka et al.
1999
). At ages older than P21, the membrane potential changes induced by in vitro ischemia were essentially the same as those described in adult neurons. In neurons of P21, the potential changes were similar, but reintroduction of oxygen and glucose restored the
membrane potential partially or completely; 1 of 10 neurons showed
complete recovery, 4 neurons showed partial recovery (Fig. 3C), and the remaining 5 neurons showed no recovery.
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At ages P7 and P14, the initial hyperpolarization was, however, absent and the slow depolarization occurred after superfusion of ischemia-simulating medium (Fig. 3, A and B). In all neurons at age P14, the slow depolarization was followed by the rapid depolarization (Fig. 3B). Reintroduction of oxygen and glucose restored the membrane potential partially or completely; 1 of 13 neurons showed complete recovery, 3 neurons showed partial recovery, and the remaining 9 neurons showed no recovery. At age P7, the rapid depolarization occurred after 43.3 min superfusion with ischemia-simulating medium in only one of seven neurons (Fig. 3A). When oxygen and glucose were reintroduced, the membrane potential did not recover but further depolarized to 0 mV. In the remaining six neurons, intracellular recording was suddenly lost from impaled neurons after 40-55 min superfusion without obvious tissue movement.
Table 2 summarizes the latency of onset and the maximal slope of the rapid depolarization in different age groups. The onset of the rapid depolarization was earlier and its maximal slope increased as the age was increased. The onset was significantly later in neurons at age P14 (P < 0.002) and P21 (P < 0.05) compared with adult (P140) neurons. The maximal slope was significantly reduced in neurons at age P14 and P21 (P < 0.002 and P < 0.02, respectively) compared with adult neurons. These results suggest that the rapid depolarization is not readily produced in the immature neurons.
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DISCUSSION |
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Resting and active membrane properties of immature and adult neurons
The present study demonstrates that the membrane was more
depolarized in immature hippocampal CA1 pyramidal neurons compared with
adult neurons, and the apparent input resistance in immature neurons
was higher than that of adult neurons. In the immature neurons, the
peak amplitude of the action potential was low in comparison with that
of adult neurons. These results are comparable with previous reports of
developmental changes in the electrophysiological properties of
hippocampal CA1 neurons (Cherubini et al. 1989; Mueller et al. 1981
; Schwartzkroin 1982
;
Schwartzkroin and Altschuler 1977
; Zhang et al.
1991
), neocortical pyramidal neurons (McCormick and
Prince 1987
), and trigeminal neurons (Guido et al.
1998
). It is possible that a positive shift of the resting
potential in the immature neurons is due to a lower activity of Na,
K-ATPase because the rate of energy metabolism in the immature brain is 5-20% of the adult (Duffy et al. 1975
; Hansen
and Nordstrom 1979
; Thurston and McDougal 1969
).
Moreover, there is a previous report that in the first postnatal week
the Na, K-ATPase density on the membrane of hippocampal CA1 neurons is
low and insufficient to allow substantial activity of Na, K-ATPase
(Fukuda and Prince 1992
). The high apparent input
resistance in the immature neurons may be the result of the presence of
fewer active voltage-dependent ion channels compared with adult CA1
neurons (Costa et al. 1994
; Spigelman et al.
1992
) or relatively small cell soma and less branched dendrites
(Pokorny and Yamamoto 1981
). A time-dependent inward
rectification elicited at potentials negative to the resting potential
was prominent in neurons of P14 and P21. This result is also comparable
with previous reports in hippocampal CA1 neurons (Krnjevi
et al. 1989
; Schwartzkroin 1982
), neocortical
pyramidal neurons (McCormick and Prince 1987
), and
trigeminal neurons (Guido et al. 1998
). The present
study demonstrates that the threshold for generating the TTX-sensitive
action potential was high in immature neurons. It is likely that
Na+ channels in immature neurons are activated at more
positive potentials than those in adult neurons because the activation
curve for the Na+ current and the potential corresponding
to half-activation in immature neurons is shifted in the depolarizing
direction (Costa 1996
). After the age P21, values of the
resting membrane potential, the apparent input resistance, and the
threshold and the peak amplitude of the action potential were not
different between immature and adult neurons. These results suggest
that the resting and the active membrane properties reached maturity
between P14 and P21.
Responses to in vitro ischemia in immature and adult neurons
Deprivation of oxygen and glucose produced a sequence of potential
changes consisting of an initial hyperpolarization, a slow depolarization, a rapid depolarization, and a persistent depolarization in adult neurons. When oxygen and glucose were reintroduced immediately after generating the rapid depolarization, the neuron did not repolarize and the membrane potential finally became 0 mV after ~5
min. At ages P7 and P14, the initial hyperpolarization was, however,
absent, and the slow depolarization was followed by the rapid
depolarization. Previously, we have reported that the initial hyperpolarization is mediated mainly by activation of ATP-sensitive K+ channels (Fujimura et al. 1997b;
Fujiwara et al. 1987
; Yamamoto et al.
1997a
). The binding densities of the sulfonylurea receptor antagonist, glibenclamide in rat brain are lower within 3 wk of birth
compared with adults (Xia et al. 1993
). It is,
therefore, possible that in neurons of ages P7 and P14 the lack of the
initial hyperpolarization is the result of an absence of sulfonylurea receptors.
We already have reported that the rapid depolarization is Na, K-ATPase
dependent and is due to a nonselective increase in permeability to all
participating ions in the adult neurons (Tanaka et al.
1997). In addition, the latency of the rapid depolarization is
prolonged and the maximal slope is decreased at low temperatures (27-33°C). The temperature coefficient (Q10) of the
latency and the maximal slope is 2.5 and 2.9, respectively
(Onitsuka et al. 1998
). Okada (1988)
reported that in
adult hippocampal slice preparations of the rat, the level of ATP and
creatine phosphate (CrP) is reduced to 30 and 10% of the preexposure
level respectively, after 5 min of in vitro ischemia, and the reduction
of the high energy phosphates is suppressed markedly at low
temperatures (28 and 21°C); Q10 of the high energy
phosphates is ~2. Energy metabolism in immature animals is much lower
than that in adult animals, as described previously (Duffy et
al. 1972
; Kawai et al. 1989
; Lowry et al. 1964
; Thurston and McDougal 1969
), and anaerobic
glycolysis is sufficient for the relatively small energy requirements
of the immature brain (Duffy et al. 1975
; Kawai
et al. 1989
; Samson et al. 1960
). The present
study showed that in immature neurons, the latency of the rapid
depolarization was prolonged and the maximal slope was reduced. It is
therefore possible that the prolonged latency of the rapid
depolarization in immature neurons is due to a relatively slow
depletion of ATP (Lowry et al. 1964
).
When oxygen and glucose were reintroduced to the slices immediately
after the rapid depolarization, the neurons at age P14 and P21 showed
partial or complete recovery. We previously have reported that in adult
neurons, at temperatures <33°C, the persistent depolarization is
fully or partially reversible after reintroduction of oxygen and
glucose (Onitsuka et al. 1998). Taken together, these
results suggest that a low-energy metabolism in immature neurons may
have a central role in recovery after in vitro ischemia. In adult rat
neurons, the membrane potential is well restored when the slices are
pretreated with inorganic Ca2+ antagonists, low
Ca2+ medium, or antagonists for Ca2+-induced
Ca2+ release from intracellular stores (Yamamoto et
al. 1997b
). Moreover, an increase in intracellular
Ca2+ concentration ([Ca2+]i)
caused by in vitro ischemia in immature neurons shows relatively slow
onset and the slope of raised [Ca2+]i is much
reduced in comparison with those of adult neurons (Nabetani et
al. 1997
). It is therefore possible that the recovery of the membrane potential in immature neurons is partly the result of a
decrease in the accumulated [Ca2+]i during in
vitro ischemia. However, most of the neurons at age P7 did not show the
rapid depolarization, but the intracellular recording of the impaled
neurons was suddenly and abruptly lost. It is likely, although not
proven, that swelling of the cell following a prolonged application of
ischemia-simulating medium is the cause of the displacement of the
neuron relative to the recording electrode.
In summary, the present study has demonstrated that the initial hyperpolarization and the rapid depolarization produced by ischemic exposure are age dependent. The resistance of immature neurons against ischemic exposure is probably due to a low-energy metabolic rate.
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ACKNOWLEDGMENTS |
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We thank Dr. D. C. Spanswick for comments and suggestions on the manuscript.
This work was supported in part by a Grant-in-Aid for Scientific Research of Japan and an Ishibashi Foundation Grant.
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FOOTNOTES |
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Address for reprint requests: E. Tanaka, Dept. of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
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 8 September 1998; accepted in final form 1 December 1998.
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REFERENCES |
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