From the Clinica Neurologica (A.P., P.C., A.T., V.D.), Dipartimento di
Sanità, Università "Tor Vergata," and Ospedale S.
Lucia, IRCCS, Rome, Italy.
Correspondence to Antonio Pisani, MD, Clinica Neurologica, Dipartimento di Sanità, Università "Tor Vergata," Via di Tor Vergata 135, 00133 Rome, Italy. E-mail pisani{at}utovrm.it
Methods—Pyramidal cortical neurons were
intracellularly recorded from a slice preparation.
Electrophysiological recordings and
microfluorometric measurements of [Ca2+]i
were performed simultaneously in slices perfused with a
glucose-free physiological medium equilibrated with
a 95% N2/5% CO2 gas mixture.
Results—Eight to twelve minutes of O2/glucose
deprivation induced an initial membrane
hyperpolarization, followed by a delayed, large but
reversible membrane depolarization. The depolarization phase was
accompanied by a transient increase in
[Ca2+]i levels. When O2/glucose
deprivation exceeded 13 to 15 minutes, both membrane
depolarization and [Ca2+]i rise became
irreversible. The dihydropyridines nifedipine and
nimodipine significantly reduced either the membrane depolarization or
the [Ca2+]i elevation. In contrast,
tetrodotoxin had no effect on either of these parameters.
Likewise, antagonists of ionotropic and group I and II
metabotropic glutamate receptors failed to reduce the depolarization of
the cell membrane and the [Ca2+]i
accumulation. Finally, dantrolene, blocker of intracellular
Ca2+ release, did not reduce both electrical and
[Ca2+]i changes caused by
O2/glucose depletion.
Conclusions—This work supports a role of L-type Ca2+
channels both in the electrical and ionic changes occurring during the
early phases of O2/glucose deprivation.
Cortical neurons are among the most vulnerable cell subtypes in the
brain if exposed to energy deprivation.8 This
susceptibility is believed to derive from a high metabolic
demand and poor energy storage capability.12
Electrophysiological studies performed on rat and
human neocortical slices demonstrated that neocortical cells maintain
membrane potential for relatively long periods of oxygen or glucose
deprivation alone.6 7 However, when these
neurons were deprived of both oxygen and glucose, membrane potential
changes were much faster, suggesting that anaerobic
glycolysis has a crucial importance in the maintenance of ionic
gradients across the cell membrane in these cells.6 By
means of a combined approach of
electrophysiological recordings and
microfluorometric measurements of Ca2+ concentrations, we
have recently shown that in cortical neurons the time-course of the
anoxia-induced membrane potential changes closely resembles the data by
Jiang and Haddad.6 Additionally, we have reported that
[Ca2+]i elevations occur
simultaneously with electrical changes.7
Therefore, in an attempt to analyze further the source of
[Ca2+]i increase during
O2/glucose deprivation, we examined
simultaneously both the
electrophysiological alterations and the
[Ca2+]i changes, with the
Ca2+-sensitive dye fura 2, in a cortical slice preparation.
We then tested the efficacy of various pharmacological agents in
counteracting these changes and found that membrane depolarization
triggers an increase in [Ca2+]i, which is
mainly dependent on the opening of voltage-gated L-type
Ca2+ channels.
For simultaneous optical and electrical recordings,
the tip of the recording electrode was filled with a solution
of 2 mmol/L fura 2 (pentapotassium salt, Molecular Probes, The
Netherlands), biocytin (2 to 4%, Sigma), and 100 mmol/L KCl,
while the shank was backfilled with a 2 mol/L KCl solution. After cell
impalement, cells were loaded with both fura 2 and biocytin by
injecting, through the recording electrode, 0.1 to 0.5 nA
negative current for 10 to 15 minutes. This loading procedure
interfered neither with electrical nor with optical recordings.
After the experiment, each biocytin-stained slice was fixed in
paraformaldehyde (in 0.1 mmol/L phosphate buffer
at pH 7.4) overnight and then processed according to standardized
protocols.14 Sections were processed further to make
permanent staining of biocytin-loaded cells, according to
Kawaguchi.15
An Axoclamp 2A amplifier (Axon Instruments) was used for
electrophysiology. Traces were displayed on an oscilloscope and stored
on a digital system. The recording chamber was mounted on the
stage of an upright microscope (Axioskop FS, Zeiss), equipped with a
60x water immersion objective (Olympus). Epi-illumination was provided
by a 75-W Xenon lamp. Excitation light passed through a shutter and was
filtered at 340 and 380 nm. Emission light was filtered by a long-pass
barrier filter (500 to 530 nm) and detected by a CCD camera (Photonic
Science, UK). Images were stored and analyzed off-line
(IonVision, ImproVision, UK). The background fluorescence was
measured in a part of the slice with no fura 2–filled neurons and
subtracted from the signal to obtain the basal fluorescence
level (F). Changes in [Ca2+]i are expressed
in terms of
Effects of O2/Glucose Deprivation on
Cortical Pyramidal Neurons
Role of L-Type Ca2+ Channels in In Vitro
Ischemia
In addition, we tested the action of nimodipine on this in vitro model
of ischemia. A maximal concentration of nimodipine (20
µmol/L) significantly reduced both the
[Ca2+]i elevation (Fig 5
Effects of Blockade of Synaptic Transmission, Glutamate Receptor
Activation and Intracellular Ca2+ Release
Both in vivo and in vitro experimental evidence supports the postulated
role of increase in cytosolic Ca2+ in
anoxic/ischemic brain damage.8 9 10 There is a
partial agreement that most of [Ca2+]i
increase derives from the extracellular space.22 23 24 25
Microfluorometric measurements of [Ca2+]i in
cerebellar and hippocampal slices showed that large
[Ca2+]i elevations induced in in vitro
ischemia were mostly due to Ca2+ influx from the
extracellular fluid.25 26 However, to date, the mode of
entry of Ca2+ still represents a matter of debate.
Several studies showed that the antagonism of both NMDA and non-NMDA
glutamate receptors attenuates Ca2+ overload in the course
of energy deprivation.23 27 28 However, the
efficacy of glutamatergic antagonists in models of focal
cerebral ischemia is still controversial.29 30 31 32 33 In
our experimental condition, the contribution of either the ionotropic
or metabotropic (group I and II) glutamate receptor to the membrane
potential and [Ca2+]i changes appears
negligible. It is noteworthy that, in most of the slice models of in
vitro ischemia, glutamate receptors antagonism did not exert
protective effects.34 35 In striatal and brain stem
neurons, intracellularly recorded from slice preparations, the
anoxia-induced membrane depolarization was not prevented by combined
antagonism of NMDA and non-NMDA glutamate receptors.4 5 In
a recent study, in fura 2–loaded cortical slices exposed to
ischemia, the combined antagonism of NMDA and AMPA receptors
slowed the rate of Ca2+ accumulation, but not the peak
concentration of [Ca2+]i.36 In
dissociated hippocampal neurons, Friedman and Haddad failed to prevent
the anoxia-induced [Ca2+]i rise either by
combined antagonism of NMDA and AMPA glutamate receptors or by
TTX.35 Moreover, comparing the effects of glutamate- and
anoxia-induced increase in [Ca2+]i, they
found that neurons exhibited a completely different time-course in
[Ca2+]i accumulation, suggesting that the
mechanisms underlying these two events are dissimilar. These results
were obtained from dissociated neurons, a preparation with no synaptic
inputs. It is possible that some of the reasons for the discrepancy
between the slice data and those obtained in cultures and "in
vivo," can be attributed to the different experimental model used.
Alternatively, it is possible that in our slice experiments we have
evaluated only the "acute" effects of ischemia, whereas
excitatory amino acids might play a major role in the "delayed
neuronal death." Our data prove that even in a slice preparation,
where synaptic inputs, neurotransmitter actions and glial functions are
partially preserved, the blockade of either synaptic transmission or of
glutamate receptors does not prevent the temporal profile of both
membrane potential and [Ca2+]i changes caused
by ischemia. The substantial attenuation of the modifications
of both membrane potential and [Ca2+]i in the
presence of nifedipine and nimodipine suggests that during
O2/glucose depletion, the raise in
[Ca2+]i is dependent, to a large extent, on
Ca2+ entry through L-type Ca2+ channels.
Moreover, the inability of dantrolene to prevent electrical and ionic
changes induced by O2/glucose deprivation
suggests that the release of Ca2+ from intracellular stores
does not seem to play a major role in the early events of energy
depletion. These findings appear to be in accordance with
fluorescence imaging studies obtained from dissociated
hippocampal CA1 neurons, in which, during anoxia, most of the
Ca2+ influx occurs through cobalt-sensitive
Ca2+ channels.35 In hippocampal slices, Lobner
and Lipton found significant attenuation of
45Ca2+ uptake during the second 2.5 minutes of
ischemia in the presence of
nifedipine.22 Interestingly, it has been shown
that cardiac arrest is associated with an elevation in the binding of a
L-type Ca2+ channel specific ligand in cortical
synaptosomes, suggesting that, during ischemia, the increase in
the number of L-type channels in the cell membrane may allow an
increased Ca2+ entry.37 Yet, in in vivo models
of both focal and complete cerebral ischemia, dihydropyridines
have been shown to ameliorate ischemic
[Ca2+]i accumulation.38 39 40
However, a major problem in interpreting data from in vivo studies is
identifying the site at which drugs act. In fact, these in vivo studies
cannot distinguish between a relaxant effect on blood vessels, thus
increasing blood flow, and an action at neuronal level.41
Our experimental model is represented by a slice
preparation that lacks blood vessels. Therefore, the present
results support a neuronal effect of L-type Ca2+ channel
blockers.
Our experiments show that a minor portion of membrane potential changes
(15±4.1%) and [Ca2+]i rise (16±3.9%)
observed during O2/glucose omission was not blocked by
dihydropyridine, suggesting the presence of an
additional mechanism involved in the
pathophysiological changes occurring in course of
ischemia. We are currently investigating the possible
mechanisms subtending these minor changes occurring in cortical cells
in conditions of energy depletion.
Clinical Implications
Our experimental model precludes the possibility to investigate the
involvement of blood vessels. Indeed, thrombolytic
agents are currently under evaluation in clinical trials; this approach
seems to be effective in the clinical outcome of acute ischemic
stroke.45 46 47 Understanding the pathophysiology of the
different stages of ischemic stroke is of crucial importance to
establish the exact criteria of the so-called therapeutic window and,
consequently, for drug development.
Received May 29, 1997;
revision received September 4, 1997;
accepted October 6, 1997.
Laboratory
of Cerebrovascular Biology and Stroke,
Department of Neurology,
University of Minnesota,
Minneapolis, Minnesota
This elegant study uses state-of-the-art approaches to address a
critical issue in the neurobiology of neuronal death secondary to
energy deprivation. The results are of interest, as they
provide new data in support of the importance of voltage-gated calcium
channels in the mechanism of cerebral ischemia. In addition,
the findings reemphasize that calcium channel blockers may be a viable
therapeutic modality for the treatment of stroke. Of interest is the
observation that in this preparation, glutamate receptor
antagonists did not block calcium entry. This finding leads
to the prediction that glutamate receptor antagonists are
not effective in preventing the deleterious effects of intracellular
calcium accumulation during energy failure. However, this conclusion is
not supported by the abundant literature demonstrating a protective
effect of glutamate receptor antagonists in focal cerebral
ischemia and in neuronal cultures subjected to oxygen-glucose
deprivation.3 4 While this discrepancy
underscores the importance of the experimental model used when studying
the consequences of cerebral ischemia, it also indicates that
our understanding of the role of calcium and glutamate receptors in
ischemic brain injury is far from complete. Future experiments
focusing on the ionic changes elicited by activation of glutamate
receptors would contribute to further elucidate the relation between
glutamate and calcium influx in this model.
Received May 29, 1997;
revision received September 4, 1997;
accepted October 6, 1997.
© 1998 American Heart Association, Inc.
Original Contributions
L-Type Ca2+ Channel Blockers Attenuate Electrical Changes and Ca2+ Rise Induced by Oxygen/Glucose Deprivation in Cortical Neurons
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Background and Purpose—Experimental
evidence supports a major role of increased intracellular calcium
[Ca2+]i levels in the induction of neuronal
damage during cerebral ischemia. However, the source of
Ca2+ rise has not been fully elucidated. To clarify further
the role and the origin of Ca2+ in cerebral
ischemia, we have studied the effects of various
pharmacological agents in an in vitro model of oxygen
(O2)/glucose deprivation.
Key Words: calcium channels • electrophysiology • fura 2 • glucose • ischemia • oxygen
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Ischemic stroke
remains a common cause of severe neurological disability. In the recent
past, an intensive effort has been directed at clarifying the
pathophysiological mechanisms underlying neuronal
degeneration during ischemic injury. Several studies have shown
that the acute changes in neuronal electrical properties induced by
either anoxia, aglycemia, or a combination of both, are extremely
variable, depending on the brain area
analyzed.1 For example, nigral dopaminergic and CA1
and CA3 hippocampal neurons hyperpolarize in response to
anoxia,2 3 whereas brain stem and striatal neurons
depolarize.4 5 During anoxia, cortical neurons exhibit an
early hyperpolarization followed by a delayed
membrane depolarization.6 7 The differential responses
observed during energy deprivation are thought to underlie
the diverse vulnerability of neuronal subtypes to energy
metabolism failure.1 Compelling evidence led to
the hypothesis that calcium [Ca2+]i overload
is involved in neuronal injury occurring in cerebral hypoxia or
ischemia.8 9 10 Mechanisms responsible for the
elevation of [Ca2+]i during ischemia
include opening of transmitter- or voltage-gated channels, mobilization
from intracellular pools, inhibition of ion pumps, imbalance of
sodium/Ca2+ exchanger.11 However, despite these
advances in the understanding of the ischemia-related events, the origin of Ca2+
influx has not been defined precisely.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Seventy-two male Wistar rats (age, postnatal day 18 to 24) were
used for the experiments. Preparation and maintenance of the
slices have been described5 7 13 previously and followed
the institutional guidelines. Briefly, animals were killed under ether
anesthesia by cervical dislocation, the brain was removed,
and coronal slices (200-µm thick), containing cortex and striatum,
were cut with a vibratome. Slices were maintained at 34°C in an
oxygenated solution (see composition below) for about 1
hour. A single slice was then transferred to a recording
chamber and fully submerged in a continuously flowing Krebs solution
(33°C, 3 ml/min) gassed with 95% O2/5% CO2.
The composition of the solution was (in millimoles per liter): 126
NaCl, 2.5 KCl, 1.2 MgCl2, 1.2
NaH2PO4, 2.4 CaCl2, 11 glucose, 25
NaHCO3. The ischemic medium was a glucose-free
solution equilibrated with a 95% N2/5% CO2
gas mixture. Glucose was replaced with saccharose to balance
osmolarity. Complete replacement of the medium in the chamber took 60
to 90 seconds. Only one single experiment was performed on each
slice. 
F/F, where F is the normalized change in
fluorescence and F is the background-subtracted basal
fluorescence. The F/F value can be interpreted as changes in
[Ca2+]i.16 17 Changes in
mitochondrial NADH levels during energy deprivation are
known to interfere with fura 2 signals.18 Therefore, some
undyed slices were exposed to O2/glucose
deprivation, and the resulting raw fluorescence was
detected, averaged, and subtracted from the control signal obtained in
dyed slices. In most of the experiments, the intensity of excitation
light was small; thus the fluorescence signals resulting from
photo-bleaching of the dye were negligible. Values in the text and in
the table are expressed as mean±SEM. Student's t test was
used to compare the means. The experiments performed to check the
possible effect of the pharmacological agents (nimodipine, D-APV, CNQX,
MCPG, BAY K 8644, and dantrolene) on both the membrane potential
changes and the elevation of the [Ca2+]i of
the recorded cells, were first statistically analyzed using
an ANOVA test. The result from this test has indicated a large and
significant difference between groups (P<.001) so that we
proceeded with the Student's t test to compare the means of
each group referred, one by one, to the control.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Electrophysiological and Morphological Features
of Cortical Cells
Data were obtained from
electrophysiologically and visually
identified cortical pyramidal neurons.
Pyramidal cortical neurons showed a resting membrane
potential of -73±6 mV. As previously described,19 20
during depolarizing current pulses, two types of pyramidal
cells were observed: "regular-spiking" and "bursting" neurons.
In both of these types of pyramidal neurons, spikes were
followed by a pronounced afterhyperpolarization. As
described above (see Materials and Methods section) electrodes were
filled with both fura 2 and biocytin. This loading procedure allowed an
immediate visualization of the recorded cell (Fig 1A
), due to fluorescence emission
of the Ca2+-sensitive dye fura 2. In addition, subsequent
processing of the loaded slices permitted morphological
analysis. Pyramidal (IV layer) cells had a large
soma (28 to 33 µm) and an extensive dendritic tree densely
studded with spines (Fig 1B and 1C).
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Figure 1. Morphological identification of cortical
pyramidal neurons. A, The figure represents a
computerized reconstruction of images (average of 256 frames) of a fura
2–loaded pyramidal cell. White arrow indicates the
recording electrode. B, Biocytin-staining of the same cell in
A. C, Permanent staining of the same neuron in A and B. Note the large
size of the soma, the prominent apical dendrite, and the dendritic tree
studded with spines. Scale bars=25 µm (A), 12 mm (B),
11 mm (C).
Perfusion with an ischemic solution for short periods
induced small membrane potential changes in cortical neurons. In fact,
2 to 6 minutes of O2/glucose deprivation caused
either no change or a small, not significant
hyperpolarization of the recorded neurons
(-5±3 mV, P>.01, n=118). Likewise, these changes in
membrane potential were not coupled with significantly relevant
[Ca2+]i changes (98±0.7 expressed as
percentage of control, in terms of F/F, see Materials and Methods
section; P>.01, n=118). Longer periods of omission of
O2 and glucose (8 to 12 min) caused an early membrane
hyperpolarization followed by a delayed, large
depolarization of the cell membrane (48±12 mV, P<.005,
n=96). This membrane depolarization was associated with a transient,
delayed increase in [Ca2+]i (54±9%,
P<.001). Fig 2 shows that an
8-minute perfusion with a solution lacking both O2 and
glucose, produced a late, significant increase in
[Ca2+]i (Fig 2A), which was fully reversible
on washout. Simultaneously, a delayed, large membrane
depolarization occurred (Fig 2B), followed, after the washout, by a
pronounced afterhyperpolarization. When
O2/glucose depletion exceeded 13 to 15 minutes, both the
electrical changes and the increase in
[Ca2+]i were irreversible (n=18, not
shown).
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Figure 2. O2/glucose
deprivation-induced membrane depolarization and increase in
[Ca2+]i in a cortical neuron. A, Eight
minutes of energy depletion caused a delayed, reversible increase
[Ca2+]i as indicated by changes in normalized
fura 2 fluorescence intensity ( F/F). B, Concurrently,
O2/glucose deprivation induced an initial
membrane hyperpolarization of the recorded cell
followed by a large depolarization. Note the pronounced
afterhyperpolarization after the end of the
exposure to O2/glucose depletion. In both A and B the
horizontal bar represents the period of exposure to
O2/glucose deprivation. C, Pseudocolor ratio
images of Ca2+ signals expressing changes in F/F at
times shown in A. Color bar codes for F/F. Resting membrane
potential was -77 mV.
To address the possible involvement of L-type voltage-dependent
Ca2+ channels in the early events that follow the onset of
O2/glucose deprivation, we tested the effects
of nifedipine, a dihydropyridine-derivative, on the
electrical and [Ca2+]i changes caused in this
experimental condition. The incubation with nifedipine (10
to 15 min), before starting perfusion with the
O2/glucose-deprived medium, did not affect the intrinsic
membrane properties of the recorded cells (resting membrane
potential, input resistance, action potential discharge, not shown).
Fig 3 shows that in the presence of a
saturating concentration of nifedipine (20 µmol/L),
both the [Ca2+]i rise and the membrane
depolarization caused by 10 minutes of O2/glucose
deprivation (Fig 3A), were significantly reduced (Fig 3B, 17±3% of control, P<.001 and 21.7±1.7%,
P<.003, respectively, n=18). After washout of the drug, the
membrane response and [Ca2+]i rise induced by
O2/glucose depletion returned to the control values (Fig 3C). Both the changes in membrane potential and
[Ca2+]i were reduced by
nifedipine in a dose-dependent manner (Fig 4). The EC50 value for the
effect on membrane potential changes was 1.826 µmol/L, whereas
the EC50 for the inhibition of
[Ca2+]i increase was 3.288 µmol/L.
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Figure 3. Effects of nifedipine on
membrane depolarization and [Ca2+]i increase
caused by O2/glucose depletion. A, In control condition,
perfusion with a O2/glucose-deprived solution (10 minutes)
caused a late, but significant, rise in
[Ca2+]i expressed in terms of normalized
fluorescence signal (upper graph). Simultaneously,
an early hyperpolarization occurred, followed by a
large, transient depolarization of the cell membrane (lower graph). B,
In the presence of a saturating dose of nifedipine (20
µmol/L), the Ca2+ signal observed during exposure to
O2/glucose deprivation was significantly
smaller (upper graph). Likewise, in the presence of
nifedipine (20 µmol/L), the amplitude of the
membrane depolarization of the recorded cell was reduced (lower
graph). C, After a 20-minute washout of nifedipine, a full
recovery both of Ca2+ signal and membrane depolarization
was observed (upper and lower graph, respectively). Horizontal bar
indicates the period of exposure to O2/glucose
deprivation (10 minutes). Resting membrane potential was
-74 mV.
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Figure 4. Dose-response curve of the effects of
nifedipine on both membrane potential and
[Ca2+]i during exposure to
O2/glucose depletion. Each point represents the
average of at least four single experiments (± SEM). Normalized data
points were fitted according to the formula y = m3
(m0m1/(m0m1 +
m2m1)), where m2 defines the
EC50 values: the EC50 value for the effect on
membrane potential changes ( Vm) was 1.826 µmol/L, whereas the
EC50 for the inhibition of
[Ca2+]i increase (F/F) was 3.288
µmol/L., 18±2% of control,
P<.003, n=7), and the concurrent depolarization of the cell
membrane (Fig 5, 22±3.1%, P<.001). Finally, we tested the
effect of BAY K 8644 (3 µmol/L), an agonist of L-type
Ca2+ channels. BAY K 8644, per se, produced significant
changes neither in the passive properties of the recorded cells,
nor in the resting [Ca2+]i. Interestingly,
3 µmol/L BAY K 8644 did not accelerate the effects of
O2/glucose deprivation, but it significantly
increased the peak amplitude and delayed the recovery time of both the
membrane depolarization and of [Ca2+]i rise
(Fig 5; 124±4%, P<.001, and 140±5.3%,
P<.005, respectively, n=4).
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Figure 5. Pharmacological modulation of the changes in
[Ca2+]i and membrane depolarization induced
by O2/glucose deprivation. Values are expressed
in terms of percentage of the response to O2/glucose
deprivation obtained with no pharmacological agents. Each
bar represents the average of at least four experiments
(±SEM). Nimodipine (20 µmol/L) significantly reduced both the
rise in [Ca2+]i and membrane depolarization,
whereas either TTX (1 µmol/L) or a combination of 50
µmol/L D-APV, plus 10 µmol/L CNQX, plus 500 µmol/L
MCPG. Also dantrolene, 30 µmol/L, was unable to affect
electrical and ionic alterations caused by O2/glucose
deprivation. Note that application of BAY K 8644, 3
µmol/L, enhanced significantly the amplitude of depolarization and
the increase in [Ca2+]i.
The blockade of synaptic transmission by 1 µmol/L TTX did
not modify the alterations caused by O2/glucose depletion
on cortical neurons. Fig 5
shows that neither the electrical changes
(96±5%, P<.001) nor the elevation in
[Ca2+]i (97±5.2%, P<.005) were
affected by TTX. To analyze the possible involvement of
glutamate receptors in the induction of membrane potential changes and
[Ca2+]i increase caused by in vitro
ischemia, we preincubated the slices in a solution containing
either ionotropic or metabotropic (group I and II) glutamate receptor
antagonists. Fig 5 shows that in the presence of 50
µmol/L D-APV, an antagonist of NMDA receptors, plus
10 µmol/L CNQX, an antagonist of AMPA receptors,
plus 500 µmol/L MCPG, an antagonist of both group I
and II metabotropic glutamate receptors, the membrane potential changes
and [Ca2+]i rise induced by
O2/glucose deprivation were unaffected
(98.9±2.1, P<.001, and 98.6±1.9, P<.003,
respectively, n=7). We finally addressed the possible role of
intracellular Ca2+ stores by using dantrolene, a drug known
to inhibit the intracellular release of Ca2+ by interfering
with the Ca2+-induced Ca2+
release.21 Dantrolene (10–30 µmol/L) failed to
significantly affect both membrane potential changes and
[Ca2+]i rise induced by
O2/glucose depletion (Fig 5, 96.9±2.1, P<.001
and 97.9±1.9, P<.005, n=6).
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Four main findings emerge from our current study: (1) cortical
pyramidal neurons respond to combined
O2/glucose deprivation with an early
hyperpolarization followed by a large, delayed
depolarization of the cell membrane potential; (2) a
simultaneous increase in [Ca2+]i
occurs only in the latter phase of these electrical changes; (3) both
the membrane potential changes and the elevation in
[Ca2+]i could be strongly attenuated by
L-type Ca2+ channel blockers, and enhanced by BAY K 8644,
an agonist of L-type channels. (4) Conversely, glutamate receptor
antagonists, TTX or dantrolene, failed to prevent the
O2/glucose deprivation-induced electrical and
ionic alterations. Taken together, these data suggest that during the
early phases of ischemia a major part of the acute changes of
both membrane potential and [Ca2+]i in
cortical neurons is likely due to a net Ca2+ influx through
voltage-gated L-type Ca2+ channels.
Over the past years the involvement of intracellular
Ca2+ in the ischemic cell death has become
evident.8 9 This observation led to the hypothesis that the
inhibition of Ca2+ influx might prove useful in the
treatment of acute stroke. Various antagonists of
voltage-dependent Ca2+ channels were investigated in a
variety of stroke models with different degrees of
efficacy.42 The overall results of several double-blind,
randomized trials with nimodipine did not give the expected results.
However, a recent meta-analysis of the nimodipine trials
demonstrated that previous analyses had methodological flaws,
the major of which was that patients were randomized up to 48 hours
after stroke. In the meta-analysis it emerged that patients who
had nimodipine within 12 hours after acute stroke had a beneficial
outcome, supporting the idea that an early therapeutic intervention
with L-type channel blockers might be useful.43 Another
group of drugs recently developed, aimed at counteracting
Ca2+ overload, was represented by glutamate
receptor antagonists, in particular both competitive and
noncompetitive NMDA antagonists. Unfortunately, most
recently, clinical trials performed with a competitive NMDA
antagonist were stopped, because it increased brain-related
death rate.44
Selected Abbreviations and Acronyms
AMPA
=
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate
CNQX
=
6-cyano-7-nitroquinoxaline-2,3-dione
D-APV
=
D-2-amino-5-phosphonovalerate
EC50
=
median effective concentration
MCPG
=
(S)-
-methyl-4-carboxyphenylglycine
TTX
=
tetrodotoxin
Acknowledgments
This work was partially supported by a M.U.R.S.T. grant
(PNR/Neurobiologia-Tema 1) to G.B. We wish to thank Mr. M Tolu for his
excellent technical assistance.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
A large body of evidence indicates that intracellular calcium
accumulation is a critical step in the chain of events leading to
ischemic neuronal death.1 2 However, the
mechanisms of the increase in intracellular calcium and the calcium
accumulation and anoxia-induced changes in membrane potentials have not
been clearly defined. In the accompanying article, Pisani et al have
used calcium-sensitive dyes and intracellular recordings in
brain slices subjected to oxygen-glucose deprivation to
provide additional insights into the mechanisms of calcium
accumulation. By monitoring both intracellular calcium and membrane
potential in morphlogically identified neurons, they were able to study
the effect of pharmacological agents that block selected calcium entry
pathways during oxygen-glucose deprivation. The results
suggest that the majority of calcium enters neurons through
voltage-gated L-type channels toward the late stage of membrane
depolarization. Interestingly, the contribution of glutamate receptors
and intracellular calcium pool was found to be negligible. The data
reaffirm the importance of voltage-gated L-type calcium channels in the
mechanisms of calcium influx in neurons after energy failure.
Selected Abbreviations and Acronyms
AMPA
=
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate
CNQX
=
6-cyano-7-nitroquinoxaline-2,3-dione
D-APV
=
D-2-amino-5-phosphonovalerate
EC50
=
median effective concentration
MCPG
=
(S)-
-methyl-4-carboxyphenylglycine
TTX
=
tetrodotoxin
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
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