(Stroke. 2000;31:766.)
© 2000 American Heart Association, Inc.
Original Contributions |
Is Pharmacological Neuroprotection Dependent on Reduced Glutamate Release?
From Clinica Neurologica (P.C., B.P., E.S., D.C., G.B.), Dipartimiento Neuroscienze, Università di Tor Vergata, Rome, Italy; I.R.C.C.S. Ospedale S. Lucia (D.C., G.B.), Rome, Italy; and the School of Pharmacy (A.H.H.), DeMontfort University, Leicester, UK.
Correspondence to Dr Paolo Calabresi, Clinica Neurologica, Dip. Neuroscienze, Universita’ di Tor Vergata, via di Tor Vergata, 00133 Rome, Italy. E-mail calabre{at}uniroma2.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Background and Purpose—The aim of this study was to determinate the possible role of the ionotropic glutamate receptor in the expression of irreversible electrophysiological changes induced by in vitro ischemia and to test whether the neuroprotective action of various neurotransmitter agonists and drugs of clinical interest is related to a presynaptic inhibitory action at glutamatergic synapses.
Methods—Intracellular and extracellular recordings have been performed in a rat corticostriatal slice preparation. Different pharmacological compounds have been tested on corticostriatal glutamatergic transmission in control conditions and in an in vitro model of ischemia (oxygen and glucose deprivation).
Results—In vitro ischemia lasting 10 minutes produced an
irreversible loss of the field potential recorded from striatal
slices after cortical stimulation. Preincubation of the slices with
3 µmol/L 6-cyano-7-nitroquinoxaline-2,3-dione (an
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
[AMPA] receptor antagonist) allowed a significant
recovery of the field potential amplitude (P<0.05,
n=6), whereas incubation with 30 µmol/L aminophosphonovaleric
acid (an N-methyl-D-aspartate receptor
antagonist) did not produce a significant recovery after 10
minutes of ischemia (P>0.05, n=7). Bath
application of 3 mmol/L glutamate for 5 minutes produced a
complete but reversible inhibition of the field potential amplitude.
When a similar application was coupled with a brief period of
ischemia (5 minutes), which produced, per se, only a transient
inhibition of the field potential, it caused an irreversible loss of
this parameter. We also tested the possible neuroprotective
effect of neurotransmitter agonists reducing the release of glutamate
from corticostriatal terminals. Agonists acting on purinergic
(adenosine), muscarinic (oxotremorine), and metabotropic
glutamate receptors (L-serine o-phosphate
[L-SOP]) significantly (P<0.001, n=8 for each
agonist) reduced glutamatergic synaptic potentials, with each showing
different potencies. The EC50 was 26.4 µmol/L for
adenosine, 0.08 µmol/L for oxotremorine, and 0.89
µmol/L for L-SOP. Concentrations of these agonists producing the
maximal inhibition of the synaptic potential were tested on the
ischemia-induced irreversible loss of field potential.
Adenosine (P<0.05, n=9) and oxotremorine
(P<0.05, n=8) showed significant neuroprotective
action, whereas L-SOP was ineffective (P>0.05, n=10).
Similarly, putative neuroprotective drugs significantly
(P<0.001, n=10 for each drug) reduced the amplitude of
corticostriatal potential, with different EC50 values
(phenytoin, 33.5 µmol/L; gabapentin, 96.8 µmol/L;
lamotrigine, 26.7 µmol/L; riluzole, 6 µmol/L; and
sipatrigine, 2 µmol/L). Concentration of these drugs producing
maximal inhibition of the amplitude of corticostriatal potentials
showed a differential neuroprotective action on the ischemic
electrical damage. Phenytoin (P<0.05, n=10),
lamotrigine (P<0.05, n=10), riluzole
(P<0.05, n=9), and sipatrigine
(P<0.001, n=10) produced a significant neuroprotection,
whereas gabapentin (P>0.05, n=11) was ineffective. The
neuroprotective action of transmitter agonists and clinical drugs was
not related to their ability in decreasing glutamate release, as
detected by changes in the paired-pulse facilitation protocol.
Conclusions—Ionotropic glutamate receptors, and particularly AMPA-like receptors, play a role in the irreversible loss of field potential amplitude induced by ischemia in the striatum. Drugs acting by reducing glutamatergic corticostriatal transmission may show a neuroprotective effect. However, their efficacy does not seem to be directly related to their capability to decrease glutamate release from corticostriatal terminals. We suggest that additional modulatory actions on voltage-dependent conductances and on ischemia-induced ion distribution at the postsynaptic site may also exert a crucial role.
Key Words: anticonvulsant • excitatory amino acids • ischemia • neuronal death
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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It has been postulated that the abnormal release of excitatory amino acids plays a crucial role in triggering the cellular events leading to neuronal death after brain ischemia. The potential role of glutamate in the ischemia-induced acute electrical changes at the neuronal level has not been fully clarified. Corticostriatal afferents represent one of the major excitatory glutamatergic projections in the brain. Moreover, striatal neurons express high sensitivity to ischemic insults and energy deprivation.1 2 Prolonged in vitro ischemia causes irreversible electrophysiological changes in the striatum, reflecting the permanent disruption of ionic homeostasis.1 2 The role of ionotropic glutamate receptors in these changes has been challenged by electrophysiological studies.1 For this reason, we have analyzed the effect of either
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA)-like or N-methyl-D-aspartate
(NMDA) glutamate receptor antagonists on the permanent loss
of the field potential amplitude induced by in vitro (combined oxygen
and glucose deprivation) ischemia in
corticostriatal slices. Dealing with this issue, we have also tested
whether application of exogenous glutamate is able to convert a
transient depression of the field potential after brief
ischemia into a permanent loss of this potential. Assuming that
glutamate release during ischemia is a critical factor in the
expression of the permanent electrical changes caused by this
pathological event, we should expect that agents interfering with
glutamate release might exert a neuroprotective effect in experimental
ischemia.2 Lamotrigine represents a
good example of this class of agents. This compound, which reduces the
release of glutamate in various brain areas,3 4 5 also
exerts a cerebroprotective effect after focal
ischemia.6 Nevertheless, a large body of evidence
demonstrates additional mechanisms underlying the action of
lamotrigine7 and other neuroprotective compounds, such as
phenytoin,7 8 gabapentin,9 10
riluzole,7 11 and sipatrigine, a new lamotrigine-like
agent.12 13 All of these agents, in fact, have also been
shown to modulate voltage-dependent sodium and calcium channels in the
postsynaptic neuron. To correlate the neuroprotective efficacy of these
pharmacological compounds with their presynaptic action at
glutamatergic synapses, we performed
electrophysiological experiments in
corticostriatal slices. The possible pharmacological modulation of this
pathological event in vitro might reflect the potential therapeutic
efficacy of drugs of clinical interest in reducing neuronal damage
after in vivo ischemia.14 15 Use of the in
vitro preparation may allow us to quantify not only the neuroprotective
efficacy of known doses of these drugs but also the presynaptic
inhibitory effect of these concentrations of agents at
corticostriatal glutamatergic synapses. In the present study, the
pharmacological analysis of the neuroprotective effect of
clinically related drugs was preceded by a detailed investigation of
the possible neuroprotective effect of various neurotransmitter
receptor agonists whose primary action is the reduction of
corticostriatal glutamatergic transmission via a presynaptic
mechanism. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Forty Wistar rats (150 to 250 g) were used. Animals were anesthetized with alotane and killed by severing the major blood vessels in the chest. The preparation and maintenance of slices have been
described previously.1 2 Briefly, corticostriatal coronal slices (200 to 300 µm) were prepared from tissue blocks of the brain with the use of a vibratome. A single slice was transferred to a recording chamber and submerged in a continuously flowing Krebs solution (36°C, 2 to 3 mL/min) gassed with 95% O2/5% CO2. To study the effects of in vitro ischemia, slices were deprived of both glucose and oxygen. Glucose was totally removed from the perfusate, and the NaCl concentration was increased to balance the osmolarity. Oxygen deprivation was obtained by bubbling the perfusing solution in a gas mixture containing 95% N2/5% CO2. Ischemic solutions entered the recording chamber no later than 20 seconds after turning a 3-way tap. Complete replacement of the medium in the chamber took 90 seconds. The composition of the control solution was (in mmol/L) NaCl 126, KCl 2.5, MgCl2 1.2, NaH2PO4 1.2, CaCl2 2.4, glucose 11, and NaHCO3 25.
For the extracellular experiments, the electrodes were filled with 2
mol/L NaCl (5 to 10 M
). The intracellular recording
electrodes were filled with 2 mol/L KCl (30 to 60 M) and placed in
the striatum close (1 to 3 mm) to the cortical areas. An Axoclamp
2A amplifier (Foster City) was used for recordings. Traces were
displayed on an oscilloscope and stored on a digital system. For
synaptic stimulation, bipolar electrodes were used (0.03- to
0.01-millisecond duration, 1 to 5 V). These stimulating electrodes were
located either in the cortical areas close to the recording
electrode or in the white matter between the cortex and the striatum to
activate corticostriatal fibers. The field potential amplitude
was defined as the average of the amplitude from the peak of the early
positivity to the peak negativity and the amplitude from the peak
negativity to peak late positivity. Quantitative data on modifications
induced by ischemia are expressed as a percentage of the
controls, with the latter representing the mean of
responses recorded during a stable period (15 to 20 minutes) before
the ischemic phase. Values given in the text and in the figures are
mean±SD of changes in the respective cell populations. Student
t test (for paired and unpaired observations) was used
to compare the means. Drugs were applied by dissolving them to the
desired final concentration in the saline and by switching the
perfusion from control saline to drug-containing saline. Drugs used
were as follows: adenosine (Sigma Chemical Co),
aminophosphonovaleric acid (APV, Tocris),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris), sipatrigine
(BW619C89, kind gift from Dr M.J. Leach, University of Greenwich,
London, UK), gabapentin (Parke-Davis), glutamate (Sigma), lamotrigine
(Glaxo Wellcome), L-serine o-phosphate
(L-SOP, Tocris), oxotremorine (RBI), phenytoin (Sigma), and riluzole
(RBI). Adenosine, oxotremorine, L-SOP, gabapentin, and
sipatrigine were solved in water; phenytoin, lamotrigine, and riluzole
were solved in dimethyl sulfoxide. The maximal final concentration of
the solvent was 1:300. This concentration of dimethyl sulfoxide did not
produce, per se, detectable
electrophysiological changes.
In some experiments in which the activity of the cytosolic enzyme LDH was determined during reperfusion in the medium to assess cellular necrosis, 0.2 mL of medium was collected at the indicated times, and the amount of LDH, expressed as percentage of total activity of the tissue, was calculated. The activity of LDH was determined spectrophotometrically from the change in absorbance at 340 nm, with 0.18 mmol/L NADH and 0.72 mmol/L pyruvate in 50 mmol/L phosphate buffer, pH 7.4 at 25°C, used as substrate. All the experiments were performed according to the Animal Research Guidelines of the European Communities Council Directive (86/609/EEC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Effect of Different Periods of Ischemia in Corticostriatal Slices
As shown in Figure 1A
, a brief
period (5 minutes) of in vitro ischemia produced a reversible
inhibition of the field potential amplitude (n=10). Conversely, a
longer period of ischemia (10 minutes) caused the irreversible
disappearance of the corticostriatal field potential (n=12) (Figure 1B). Accordingly, intracellular recordings have
previously shown that striatal spiny neurons respond to in vitro
ischemia with a reversible membrane depolarization when the
period of energy deprivation lasts 
5 minutes. An
ischemic period lasting 
10 minutes leads to a permanent loss
of the resting membrane potential.1
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Role of Ionotropic Glutamate Receptors in Irreversible Loss of
Field Potential Induced by Ischemia
In the first part of the present study, we investigated the
potential role of ionotropic glutamate receptors in the expression of
the irreversible loss of the field potential amplitude after 10 minutes
of ischemia. As shown in Figure 2A, preincubation of the slices with
3 µmol/L CNQX (an AMPA receptor antagonist) produced
a significant depression of the field potential amplitude but allowed a
significant recovery of this potential after ischemia
(P<0.05, n=6). Higher concentrations (5 and 10
µmol/L) of this antagonist did not produce further
protection (n=7, data not shown). Conversely, incubation of the slices
with 30 µmol/L APV (an NMDA receptor antagonist)
produced minor changes of the field potential amplitude but induced
only a mild recovery that was not statistically significant
(P>0.05, n=7) after 10 minutes of ischemia.
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). Conversely, the partial
effect obtained by 30 µmol/L APV (
) was not significant. Note
that the control saline solution (•) did not modify the effect of
ischemia. B, Bath application of 3 mmol/L glutamate (GLU,
gray bar) induced a transient depression of the field potential
amplitude. Similarly, a brief period of ischemia (5 minutes,
black bar) also induced a transient depression of the potential. Note,
however, that when the 2 treatments were combined, they induced a
permanent loss of the field potential. The insert in panel B shows that
in some experiments similar data were obtained before any
pharmacological or experimental treatment of the slice to avoid the
possible induction of sensitization.
The potential role of glutamate receptors in the
electrophysiological abnormalities induced
by ischemia in the striatum was also investigated by using a
different experimental approach. As shown in Figure 2B
, bath
application of 3 mmol/L glutamate for 5 minutes produced a
complete but reversible inhibition of the field potential amplitude
(n=6). Similarly, as reported above, 5 minutes of ischemia
induced a reversible blockade of the field potential (n=6). However,
when the 2 treatments were associated, they caused an irreversible loss
of the field potential amplitude (n=6). In some experiments (n=5), as
shown in the insert of Figure 2B, the irreversible loss of the
field potential induced by the coadministration of glutamate plus
ischemia was obtained before any pharmacological or
experimental treatment of the slice to avoid the possible induction of
sensitization. These experiments clearly indicate that the activation
of glutamate receptors plays a role in the irreversible disruption of
an ionic gradient triggered by ischemia.
Effect of Neurotransmitter Receptor Agonists on Irreversible Loss
of Field Potential Induced by Ischemia
In the second part of the present study, we tested the
hypothesis that a drug-induced decrease of the release of
glutamate from corticostriatal terminals is a major mechanism
underlying the pharmacological neuroprotection that occurs after a
period of in vitro ischemia lasting 10 minutes. To achieve this
goal, we have analyzed the possible neuroprotective effects of
various neurotransmitter agonists whose selective
inhibitory actions at corticostriatal terminals have been
previously demonstrated.17 18 19 Figure 3A shows the dose-response curve and the
EC50 values obtained for adenosine,
L-SOP, and oxotremorine (n=8 for each agonist). The pharmacological
effects of all these drugs on the excitatory synaptic potential (EPSP)
amplitude were fully reversed after 15 minutes of washout. Moreover,
these inhibitory effects were coupled neither with changes
of the resting membrane potential nor with alterations of the input
resistance of the recorded neurons (data not shown). These data
allowed us to chose for each drug a concentration producing the maximal
inhibition of the synaptic potential to be used in the experiments
dealing with ischemia. Drugs were applied 10 minutes before the
onset of ischemia, and their application was also maintained
during the period of ischemia. The drug-induced decrease of the
field potential was stable after 5 minutes of application, and the
amplitude of these depressions (expressed as percentage of the control
values) was similar to the decrease of the intracellularly recorded
EPSPs induced by each drug. Figure 3B shows that
adenosine (100 µmol/L) produced a recovery of the field
potential amplitude 30 minutes after the washout of the
ischemic solution whose amplitude was significantly higher than
that observed in the control medium (n=9). Oxotremorine (300 nmol/L), a
muscarinic agonist acting mainly at M2 receptors,19
exerted a similar neuroprotective action (n=8). Conversely, L-SOP
(30 µmol/L), an agonist acting at group III presynaptic
metabotropic glutamate receptors,18 did not show a
significant neuroprotective action when measured 30 minutes after
administration of the ischemic solution (n=10). The presynaptic
effect of adenosine, oxotremorine, and L-SOP was further
investigated by using a paired-pulse protocol. Paired-pulse
modification of neurotransmission has been studied extensively and is
attributed to a presynaptic change in release
probability.17 20 21 An increase in the ratio of the
second pulse response to the first pulse response (EPSP2/EPSP1)
indicates a decrease in the release probability. The decrease in
transmitter release probability is consistent with the
observations that manipulations depressing transmitter release usually
increase the magnitude of this ratio. Therefore, we measured the
magnitude of EPSP2/EPSP1 before and during the application of the
tested compounds. Synaptic responses to a pair of stimuli were
recorded with an interstimulus interval of 60 milliseconds. As
shown in Figure 3C, adenosine (n=8), oxotremorine (n=9),
and L-SOP (n=9) increased the magnitude of EPSP2/EPSP1 in the tested
neurons, confirming for all of them a presynaptic site of action.
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), L-SOP
(
) in relation to the drug-induced EPSP
inhibition. B, Histogram shows that 10 minutes of ischemia
caused a complete loss of the field potential, which did not recover
until 30 minutes of washout in control medium (n=12) and in the
presence of 30 µmol/L L-SOP (n=10). Conversely, a significant
recovery was observed either in the presence of 100 µmol/L
adenosine (n=9) or in the presence of 0.3 µmol/L
oxotremorine (n=8). C, Histogram demonstrates that these 3 receptor
agonists significantly increased the EPSP2/EPSP1 ratio, suggesting a
presynaptic mechanism of action (adenosine, n=8; L-SOP, n=9;
and oxotremorine, n=9). The insert at the top of panel C reproduces a
trace obtained from a single experiment showing the paired-pulse
protocol under control conditions. Significance was calculated by using
the Student t test. *P<0.05.
Effect of Putative Neuroprotective Drugs on Irreversible Loss of
Field Potential Induced by Ischemia
In the third part of the present study, by using a similar
electrophysiological approach, we
analyzed the neuroprotective efficacy of various
pharmacological agents expressed as the recovery of the field potential
amplitude measured 30 minutes after the washout of the ischemic
solution. As shown in Figure 4A, for each
drug, we obtained a dose-response curve and an
EC50 value showing the potency and the efficacy
of the drug to inhibit the EPSP amplitude (n=10 for each drug). Also in
this case, these experiments allowed us to chose a concentration for
each neuroprotective agent producing the maximal inhibition of the
potential. This maximal concentration was tested on the
ischemia-induced elecrophysiological
changes. As shown in Figure 4B, the most effective
neuroprotective drug was sipatrigine (30 µmol/L, n=10).
Pretreatment with this agent, in fact, allowed a recovery of the field
potential amplitude to 
50% of the control value. A significant
neuroprotection was also observed by pretreating the slices with 3
other putative neuroprotective drugs: lamotrigine (100 µmol/L,
n=10), phenytoin (100 µmol/L, n=10), and riluzole (30
µmol/L, n=9). Conversely, gabapentin (300 µmol/L, n=11) did
not significantly protect against the ischemic insult. The
analysis of the paired-pulse facilitation revealed that the
only drug that significantly increased the EPSP2/EPSP1 ratio measured
under control conditions was lamotrigine (n=10), whereas phenytoin
(n=11), gabapentin (n=10), riluzole (n=11), and sipatrigine (n=10)
reduced the EPSP amplitude without affecting this
electrophysiological parameter
(Figure 4C). Neither lamotrigine nor the other studied compounds
altered the resting membrane potential and the apparent input
resistance of the intracellularly recorded cells (n=11 for each
drug, data not shown).
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), lamotrigine (
), gabapentin (
), riluzole (
), and
sipatrigine (•) in relation to the drug-induced EPSP inhibition. B,
Histogram shows that 10 minutes of ischemia caused a complete
loss of the field potential, which did not recover until 30 minutes of
washout in control medium (n=12). Conversely, a significant recovery
was observed in the presence of 100 µmol/L phenytoin (n=10),
100 µmol/L lamotrigine (n=10), 30 µmol/L riluzole (n=9),
and 30 µmol/L sipatrigine (n=10). Gabapentin (300 µmol/L,
n=11) showed a small, but not significant, recovery of the field
potential amplitude. C, Histogram demonstrates that only lamotrigine
(100 µmol/L, n=10) significantly increased the EPSP2/EPSP1
ratio, whereas the other compounds did not significantly alter this
parameter (at least 10 experiments for each drug).
Significance was calculated by using the Student t test.
*P<0.05 and ***P<0.001; ns indicates no
significance.
LDH Release During Ischemia
To test an independent parameter to the slice
"stress condition" in addition to
electrophysiological variables, we
measured LDH release in the perfusate after different periods
of ischemia (5, 10, 20, and 30 minutes). Periods of
ischemia lasting <30 minutes did not produce significant
changes in LDH release compared with LDH release in control experiments
(data not shown). A small but significant increase in the release of
LDH from the slices was observed in the reperfusion phase after
prolonged ischemic conditions (30 minutes). This release was
time dependent, reaching a maximal value at 50 to 60 minutes of
reperfusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Ionotropic Glutamate Receptors Play a Role in Expression of Irreversible Electrical Changes in the Striatum After Ischemia
The striatum is the major afferent component of the basal ganglia and receives dense excitatory glutamatergic projections from the neocortex and thalamus.2 Glutamate receptor–mediated excitotoxicity has been proposed to cause striatal neuronal death after ischemia.22 23 24 It has been shown that glutamate receptor blockade reduces ischemic neuronal death within the striatum.25 A distinctive pathological feature of striatal ischemia is the specific neuronal vulnerability. In fact, ischemic insults cause a selective loss of striatal spiny neurons.2 26 27 This selective vulnerability has been related to the differential expression of ionotropic glutamate receptors on striatal neuronal subtypes.2 In the present study, we have shown that the blockade of AMPA-like glutamate receptors significantly protects the field potential amplitude from the irreversible damage induced by ischemia. We also found that the blockade of NMDA-like glutamate receptors slightly protects the field potential, but this protection did not reach a level of significance. This experimental finding seems to suggest that activation of AMPA-like but not NMDA glutamate receptors plays a role in the expression of the long-term disruption of the ionic homeostasis in striatal neurons. The critical role of glutamate receptors in the generation of the irreversible loss of the corticostriatal field potential has also been approached by using a different experimental paradigm. Under control conditions, bath application of glutamate produced a transient depression of field potential amplitude. This depression was probably related to the massive depolarization induced by this transmitter in the postsynaptic neurons and to the activation of presynaptic autoreceptors located on corticostriatal terminals.18 We observed that brief periods of ischemia (5 minutes), which did not induce, per se, a long-term blockade of this potential, were able to cause permanent changes when administered in conjunction with bath application of glutamate. This finding strongly supports the idea that energy deprivation and activation of postsynaptic glutamate receptors may concur in the expression of the permanent alteration of striatal electrical activity.
To correlate the permanent electrophysiological changes to the neuronal cell loss induced by ischemia, we have measured LDH release from the tissue. This parameter, in fact, is a biochemical marker of tissue necrosis. We found a significant increase of LDH release only in the reperfusion phase after 30 minutes of ischemia but not after shorter periods. This finding indicates that the irreversible loss of electrical signals is an early sign of neuronal damage, whereas changes in LDH release may be considered an indicator of neuronal injury after prolonged energy deprivation.
Neuroprotective Effect of Some Neurotransmitter Receptor Agonists
Is Not Solely Related to Presynaptic Inhibition of Glutamate Release
From Corticostriatal Terminals
In the present study, we have also analyzed the
dose-response curves and the EC50 values of 3
neurotransmitter receptor agonists known to decrease the
corticostriatal EPSP by presynaptic mechanisms: adenosine,
L-SOP, and oxotremorine. According to their presynaptic mechanisms of
action, all these agonists increased the paired-pulse facilitation (an
index of a presynaptic inhibition). For each agonist, we tested a
concentration producing the maximal inhibition of the EPSP on the
ischemia-induced permanent suppression of the field potential.
Surprisingly, we found that although adenosine was more
effective than oxotremorine in reducing EPSP amplitude, it protected
the field potential to the same extent as oxotremorine. Moreover, we
also observed that L-SOP produced a significant depression of the
synaptic potential but did not show a neuroprotective effect. These
data seem to suggest that the neuroprotective action of
neurotransmitter receptor agonists is not solely related to a
presynaptic inhibition of glutamate release from corticostriatal
terminals.
Does Modulation of Additional Postsynaptic Events Play a Role in
Neuroprotective Effects of Various Drugs of Clinical Interest?
The finding that the neuroprotective effect of neurotransmitter
receptor agonists does not correlate with the reduction of glutamate
release is not in contrast with the hypothesis that the neuronal death
caused by ischemia involves an excitotoxic
mechanism.14 15 In fact, it is possible that although some
of these agents do not directly affect the glutamate release within the
striatum, they may modulate some events secondarily induced by
glutamate receptor stimulation at the postsynaptic level, such as the
activation of voltage-dependent inward conductances. It is also
possible that some neuroprotective agents exert their pharmacological
effect during ischemia by modulating a calcium-independent
glutamate release. This pathological form of neurotransmitter release
seems to be important during ischemia, and it may occur via
different mechanisms.24 The pharmacological modulation of
this calcium-independent glutamate release would not be detected by
measuring the changes in paired-pulse facilitation, a calcium-dependent
form of short-term synaptic plasticity.
To further explore the potential role of postsynaptic mechanisms of action in the pharmacological neuroprotection of the striatum after ischemia, we characterized the dose-response curve and the EC50 values of various putative neuroprotective drugs concerning their inhibitory action on the corticostriatal EPSP amplitude. The concentration of each agent producing the maximal inhibition of these potentials was tested on the permanent loss of field potential induced by ischemia. We observed that although all the putative neuroprotective drugs used in the present study depressed the EPSP amplitude, only lamotrigine increased the paired-pulse facilitation (suggesting a possible presynaptic action), whereas the neuroprotection exerted by phenytoin, sipatrigine, gabapentin, and riluzole was not coupled with significant changes of this parameter. Taken together, these data seem to suggest also that the modulation of postsynaptic membrane properties may play a crucial role in the mechanisms underlying neuroprotection in central neurons. Accordingly, inhibition of postsynaptic voltage-dependent calcium and/or sodium currents has been reported for phenytoin,7 8 lamotrigine,3 4 5 gabapentin,9 10 riluzole,7 11 and sipatrigine.12 13 It is important to note that none of the neuroprotective agents used, at least not at the concentration used in the present study, affects resting membrane potential and apparent input resistance of the recorded spiny neurons. This observation clearly indicates that a possible postsynaptic site of action does not involve resting membrane conductances but rather implicates the modulation of currents operating at depolarized levels. Alternatively, it is also possible that these neuroprotective agents interact either with conductances that are specifically activated during ischemia, such as ATP-dependent potassium currents, or metabolic processes and conductances operated by intracellular calcium accumulation.28 29 We have recently shown that some neuroprotective agents used in the present study do not alter the amplitude of reversible membrane depolarizations recorded from striatal spiny neurons during brief (3- to 5-minute) periods of in vitro ischemia.1 The efficacy of these drugs in the present study might suggest that prolonged ischemia (10 minutes) triggers a more complex chain of events that can be modulated by these neuroprotective agents.
Limits of the Study and Future Perspectives
We are aware that the main limitation of the present study is
that the mechanisms underlying the neuroprotection have been
investigated by using an in vitro model of ischemia. In fact,
it is possible that the drugs that were used show a different
pharmacological efficacy when used in vivo. However, the use of the in
vitro model has allowed us to correlate the neuroprotective effect of
known concentrations of drugs in a certain brain area with their
efficacy in inhibiting glutamatergic transmission. This goal would
never have been achieved by using an in vivo model of
ischemia.
It is possible that concentrations of drugs higher than those tested in the present study could exert more pronounced neuroprotective effects in vitro. However, it should be noted that these higher concentrations might not have a clinical relevance, because they would be achieved in vivo only after the administration of doses of drugs exerting toxic effects.
It would be interestingly to measure, during in vivo experiments, glutamate concentrations with microdialysis during ischemia in control conditions and after treatment with the putative neuroprotective drugs. These experiments would allow researchers to correlate the decrease of glutamate release exerted by each pharmacological agent with its neuroprotective action. This interesting issue should be developed in future studies.
The pursuit of effective neuroprotective therapy has often been frustrating because the promise of efficacy in preclinical animal studies has not been realized in clinical trials. Nevertheless, it is worth noting that some of the drugs whose neuroprotective activity has been analyzed in the present study are currently under investigation in clinical trials.30
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Acknowledgments |
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This study was supported by a MURST grant (Cofinanziamento), a BIOMED grant (BMH4-97-2215), and a Telethon grant(E.729) to Dr Calabresi and a CNR grant (Biotecnologie 95/95) and a MURST 98 grant (Cofin) to Dr Bernardi. We wish to thank M. Tolu for the technical assistance.
Received November 3, 1999; revision received November 3, 1999; accepted December 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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1. Calabresi P, Marfia GA, Centonze D, Pisani A, Bernardi G. Sodium influx plays a major role in the membrane depolarization induced by oxygen and glucose deprivation in rat striatal spiny neurons. Stroke.. 1999;30:171–179.
2. Calabresi P, Centonze D, Pisani A, Sancesario G, Gubellini P, Marfia GA, Bernardi G. Striatal spiny neurons and cholinergic interneurons express differential ionotropic glutamatergic responses and vulnerability: implications for ischemia and Huntington’s disease. Ann Neurol. 1998;43:586–597.[Medline] [Order article via Infotrieve]
3. Leach MJ, Marden CM, Miller AA. Pharmacological studies on lamotrigine, a novel potential antiepileptic drug, II: neurochemical studies on the mechanism of action. Epilepsia. 1986;29:490–497.
4.
Calabresi P, Siniscalchi A, Pisani A, Stefani A,
Mercuri NB, Bernardi G. A field potential analysis on the
effects of lamotrigine, GP 47779, and felbamate in neocortical slices.
Neurology. 1996;47:557–562.
5. Wang SJ, Huang CC, Hsu KS, Tsai JJ, Gean PW. Presynaptic inhibition of excitatory neurotransmission by lamotrigine in the rat amygdalar neurons. Synapse. 1996;24:248–255.[Medline] [Order article via Infotrieve]
6.
Smith SE, Meldrum BS, Chir MB. Cerebroprotective
effect of lamotrigine after focal ischemia in rats.
Stroke. 1996;26:117–121.
7. Stefani A, Spadoni F, Bernardi G. Differential inhibition by riluzole, lamotrigine, and phenytoin of sodium and calcium currents in cortical neurons: implication for neuroprotective strategies. Exp Neurol. 1997;147:115–122.[Medline] [Order article via Infotrieve]
8.
McLean MJ, MacDonald RL. Multiple actions of
phenytoin on mouse spinal cord neurons in cell culture. J
Pharmacol Exp Ther. 1983;227:779–789.
9. McLean MJ. Clinical pharmacokinetics of gabapentin. Neurology. 1994;44(suppl 5):S17–S22.
10. Stefani A, Spadoni F, Bernardi G. Gabapentin reduces voltage-gated calcium currents in central neurons. Neuropharmacology. 1998;37:83–91.[Medline] [Order article via Infotrieve]
11. Centonze D, Calabresi P, Pisani A, Marinelli S, Marfia GA, Bernardi G. Electrophysiology of the neuroprotective agent riluzole on striatal spiny neurons. Neuropharmacology.. 1988;37:1063–1070.
12. Xie XM, Garthwaite J. State-dependent inhibition of Na+ currents by the neuroprotective agent 619C89 in rat hippocampal neurons and in mammalian cell line expressing rat brain type IIA Na+ channels. Neuroscience.. 1996;73:951–962.[Medline] [Order article via Infotrieve]
13. Stefani A, Hainsworth AH, Spadoni F, Bernardi G. On the inhibition of voltage activated calcium currents in rat cortical neurones by the neuroprotective agent 619C89. Br J Pharmacol. 1998;125:1058–1064.[Medline] [Order article via Infotrieve]
14. Greene JG, Greenamyre JT. Bioenergetics and glutamate excitotoxicity. Prog Neurobiol. 1996;48:613–634.[Medline] [Order article via Infotrieve]
15. Obrenovitch TP, Ureniajak J. Altered glutamatergic transmission in neurological disorders: from high extracellular glutamate to excessive synaptic efficacy. Prog Neurobiol. 1994;51:39–87.
16. Pedersen JZ, Bernardi G, Centonze D, Pisani A, Rossi L, Rotilio G, Calabresi P. Hypoglycemia, hypoxia, and ischemia in a corticostriatal slice preparation: electrophysiologic changes and ascorbyl radical formation. J Cereb Blood Flow Metab. 1998;18:868–875.[Medline] [Order article via Infotrieve]
17.
Calabresi P, Centonze D, Pisani A, Bernardi G.
Endogenous adenosine mediates the presynaptic
inhibition induced by aglycemia at corticostriatal synapses.
J Neurosci. 1997;17:4509–4516.
18. Pisani A, Calabresi P, Centonze D, Bernardi G. Activation of group III metabotropic glutamate receptors depresses glutamatergic transmission at corticostriatal synapse. Neuropharmacology. 1997;36:845–851.[Medline] [Order article via Infotrieve]
19. Calabresi P, Centonze D, Gubellini P, Pisani A, Bernardi G. Blockade of M2-like muscarinic receptors enhances long-term potentiation at corticostriatal synapses. Eur J Neurosci. 1998;10:3020–3023.[Medline] [Order article via Infotrieve]
20.
Manabe T, Wyllie DJI, Nicoll RA. Modulation of
synaptic transmission and long-term potentiation: effects on paired
pulse facilitation and EPSC variance in the CA1 region of the
hippocampus. J Neurophysiol. 1993;70:1451–1459.
21. Schulz PE, Cook EP, Johnston D. Changes in paired-pulse facilitation suggest presynaptic involvement in long-term potentiation. J Neurosci. 1994;14:5325–5337.[Abstract]
22.
Globus MYT, Busto R, Martinez E, Valdés I,
Dietrich WD, Ginsberg MD. Comparative effect of transient global
ischemia on extracellular levels of glutamate, glycine,
and 
-aminobutyric acid in vulnerable and nonvulnerable brain
regions in the rat. J Neurochem. 1991;57:470–478.[Medline]
[Order article via Infotrieve]
23. Obrenovitch TP, Urenjak J, Richards DA, Ueda Y, Curzon G, Symon L. Extracellular neuroactive amino acids in the rat striatum during ischaemia: comparison between penumbral conditions and ischaemia with sustained anoxic depolarization. J Neurochem. 1993;61:178–186.[Medline] [Order article via Infotrieve]
24.
Seki Y, Feustel PJ, Keller RW Jr, Tranmer BI,
Kimelberg HK. Inhibition of ischemia-induced glutamate release
in rat striatum by dihydrokinate and an anion channel blocker.
Stroke. 1999;30:433–440.
25. Le Peillet E, Arvin B, Moncada C, Meldrum BS. The non-NMDA antagonists, NBQX and GYKI 52466, protect against cortical and striatal cell loss following transient global ischaemia in the rat. Brain Res. 1992;571:115–120.[Medline] [Order article via Infotrieve]
26. Francis A, Pulsinelli W. The response of GABAergic and cholinergic neurons to transient cerebral ischemia. Brain Res. 1992;243:271–278.
27. Chesselet MF, Gonzales C, Lin CS, Polsky K, Jin BK. Ischemic damage in the striatum of adult gerbils: relative sparing of somatostatinergic and cholinergic interneurons contrasts with loss of efferent neurons. Exp Neurol. 1990;110:209–218.[Medline] [Order article via Infotrieve]
28. Haddad GG, Jiang C. O2 deprivation in the central nervous system: on mechanisms of neuronal response, differential sensitivity and injury. Prog Neurobiol. 1993;40:277–318.[Medline] [Order article via Infotrieve]
29. Hara H, Sukamoto T, Kogure K. Mechanisms and pathogenesis of ischemia-induced neuronal damage. Prog Neurobiol. 1993;40:645–670.[Medline] [Order article via Infotrieve]
30. Hickenbottom SL, Grotta J. Neuroprotective therapy. Semin Neurol. 1998;18:485–492.[Medline] [Order article via Infotrieve]
Editorial Comment
Cardiovascular Sciences, DuPont Pharmaceuticals Co, Wilmington, Delaware
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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One important criterion I keep in mind when reviewing papers for Stroke is whether the readers of the journal, both clinicians and experimentalists, are going to find the data relevant and useful to stroke research, in its broadest sense. In this respect, the difficulties in promoting glutamate receptor antagonists for clinical use for stroke have already resulted in serious doubts about whether glutamate receptor antagonists are important strategies for ischemic neuroprotection, as 2 decades of frustrations to materialize NMDA, AMPA, or other manipulation of the glutamatergic receptors/channels have passed. So, what can an in vitro, slice-based electrophysiological study still teach us in the art to keep us interested and even reenergized to continue to explore glutamatergic strategies in ischemic brain injury? In this report, the authors used a preparation that includes excitatory glutamatergic synapses to examine the effect of "ischemia" (glucose and oxygen deprivation) and an array of pharmacological agents on electrophysiological consequences (commonly believed to represent "en route" cell demise) of these neurons. An important component of the study is the use of pharmacological agents that have been in clinical development or are currently in clinical development for stroke. This is important, because it will allow, in due time, feedback from the clinical trials back to the "bench theory" for proof/disproof of the hypothesis. Several important points are made in the study by Calabresi et al, including the association of glutamate injury with ischemia; the role of the AMPA-like receptors in irreversible loss of cell potential during ischemia; and that neuroprotection (as assessed by preservation of electrophysiological parameters) may not correlate to glutamate release inhibition from presynaptic elements. Therefore, other features of drug interactions with the ionotropic glutamate channels need to be explored.
The article offers an interesting model system for pharmacological evaluation of compounds that needs to be further explored in conjunction with in vivo models. If the data generated in this in vitro system are confirmed in "main line" in vivo stroke models (eg, focal ischemia), including electrophysiological monitoring, coupled with direct monitoring of glutamate release and ultimately direct assessment of neuronal viability and neurobehavioral outcome, a powerful scheme to evaluate pharmacological agents could emerge. While the expertise of the authors does not span to the in vivo dimension, it is hoped that this in vitro "classical" pharmacological-electrophysiological study generates interest to extend the studies in view of seeking in vivo confirmation based on the in vitro results. Such database may be most valuable as it awaits results from clinical trials (negative or positive) which are the ultimate litmus test for the validity of the approach presented in the article by Calabresi et al. I hope that somewhere within the readers’ community of stroke this will happen.
Received November 3, 1999; revision received November 3, 1999; accepted December 6, 1999.
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