Institut National de la Santé et de la Recherche Médicale U 29, INMED, 13273 Marseille Cedex 09, France
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ABSTRACT |
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Congar, Patrice, Jean-Luc Gaïarsa, Théodora Popovici, Yezekiel Ben-Ari, and Valérie Crépel. Permanent Reduction of Seizure Threshold in Post-Ischemic CA3 Pyramidal Neurons. J. Neurophysiol. 83: 2040-2046, 2000. The effects of ischemia were examined on CA3 pyramidal neurons recorded in hippocampal slices 2-4 mo after a global forebrain insult. With intracellular recordings, CA3 post-ischemic neurons had a more depolarized resting membrane potential but no change of the input resistance, spike threshold and amplitude, fast and slow afterhyperpolarization (AHP) or ADP, and firing properties in response to depolarizing pulses. With both field and whole-cell recordings, synaptic responses were similar in control and post-ischemic neurons. Although there were no spontaneous network-driven discharges, the post-ischemic synaptic network had a smaller threshold to generate evoked and spontaneous synchronized burst discharges. Thus lower concentrations of convulsive agents (kainate, high K+) triggered all-or-none network-driven synaptic events in post-ischemic neurons more readily than in control ones. Also, paired-pulse protocol generates, in post-ischemics but not controls, synchronized field burst discharges when interpulse intervals ranged from 60 to 100 ms. In conclusion, 2-4 mo after the insult, the post-ischemic CA3 pyramidal cells are permanently depolarized and have a reduced threshold to generate synchronized bursts. This may explain some neuropathological and behavioral consequences of ischemia as epileptic syndromes observed several months to several years after the ischemic insult.
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INTRODUCTION |
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The brain is critically dependent on its blood
flow for a continuous supply of oxygen and glucose (Siesjö
et al. 1978). Only a few minutes of severe global ischemia can
induce selective damage to particularly sensitive brain structures,
including the hippocampal formation. In this structure, an ischemic
episode induces in human and experimental animals a delayed selective
damage of CA1 pyramidal neurons (2 to 4 days after ischemic episode),
whereas CA3 and dentate gyrus neurons are largely resistant
(Petito et al. 1987
; Pulsinelli 1985
;
Zola-Morgan et al. 1992
). Most of the studies on
ischemia have focused on short-term effects (up to 1 wk) (for review
see Schmidt-Kastner and Freund 1991
), and despite
several immunohistological studies (Schmidt-Kastner and Freund
1991
), the long-term modifications and the physiology of the
post-ischemic adult hippocampal network are poorly known. Only
recently, long-term abnormal activities have been reported in
post-ischemic neocortical pyramidal neurons (Luhmann et al.
1995
; Mittmann et al. 1998
). The possibility of
persistent changes of synaptic network activity after an ischemic
stroke is of major interest as patients surviving this type of insult
often express delayed epileptic syndromes months or years after the
initial insult (Cocito et al. 1982
; Kilpatrick et
al. 1990
; McNamara 1979
). The CA3 region is a
good candidate to subserve these long-term alterations because this region is one of the most susceptible regions in the brain for the
generation of seizures (Green 1964
; Hablitz and
Johnston 1981
). In addition, the CA3 pyramidal cells have lost
their principal target cells (CA1 neurons) but still receive most of
their excitatory inputs including the granule cells of the fascia
dentata (Schmidt-Kastner and Freund 1991
), and it is
known that the neurons deprived of their projecting sites or after
axotomy are often hyperexcitable (Chung et al. 1993
;
Prince et al. 1997
).
Using post-ischemic hippocampal slices, we now report that several months after the ischemic insult, CA3 pyramidal cells display a more depolarized resting membrane potential and have a lower threshold to generate synchronized bursts than controls. Therefore in addition to the short-term effect of ischemia, i.e., the degeneration of CA1 pyramidal neurons, there are long-term changes in the activity of CA3 pyramidal cells that may modify the hippocampal network properties and may have several important pathophysiological implications.
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METHODS |
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Animal models
Experiments involving animals were approved by French ethical science committee (statement no. 04223). Rats had access to food and water ad libitum and were housed under a 12-h light-dark cycle at 22-24°C.
Forebrain ischemia
The ischemic episode was achieved by four-vessel occlusion as
described previously by Pulsinelli et al. (1979). On the
first day, male Wistar rats weighing 180-200 g (Charles River, France) were anesthetized with intraperitoneal injection of 5% chloral hydrate
(350 mg/kg). Both vertebral arteries were electrocauterized in the alar
foramina of the first cervical vertebra and atraumatic clamps set on
both carotid arteries. The animals were then allowed to recover from
anesthesia overnight. The next day, the carotid arteries were clamped
in the unanesthetized rats for 20-25 min. The carotid artery clamps
were then released and animals were allowed to survive until
experiments (2-4 mo later). Only rats that were unresponsive, had lost
their righting reflex, and developed fully dilated pupils during
carotid clamping were included in the study. Rats with only
electrocauterized vertebral arteries (sham-operated rats) and controls
rats of the same age were used as controls because we did not observe
significant differences between the two populations of rats. Within the
first 24 h after the ischemic insult, only 25-30% of the animals
displayed a status epilepticus and subsequently died. After this
critical postoperative period (around 48 h), we did not observe
apparent chronic seizure activity in the surviving rats. However, we
did not monitor [electroencephalograph (EEG) or video camera] these
post-ischemic rats and we cannot exclude that they could have presented
episodic seizures.
Hippocampal slices preparation
Experiments were performed in CA3 hippocampal neurons in slices
obtained from 350-500 g male Wistar rats, 2-4 mo after ischemia. The
animals were intracardially perfused, under chloral hydrate (350 mg/kg)
anesthesia, with an ice-cooled and oxygenated modified-artificial cerebrospinal fluid (mACSF, 0-3°C) containing (in mM): 2 KCl, 0.5 CaCl2, 7 MgCl2, 1.2 NaH2PO4, 26 NaHCO3, 11 glucose, and
250 sucrose equilibrated with 95% O2-5% CO2
(pH = 7.4). After decapitation, the brain was quickly removed from
the skull, hippocampi were dissected free, and 400-µM-thick
transverse slices were prepared by using a Leica VT 1000E tissue slicer
in the same 0-3°C ice-cooled mACSF equilibrated with 95%
O2-5% CO2 (pH = 7.4). The
slices were then incubated at room temperature in an oxygenated
artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3.5 KCl, 2 CaCl2, 1.3 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 11 glucose,
equilibrated with 95% O2-5% CO2
(pH = 7.4), as previously described (Cherubini et al.
1987). After a 2-h recovery period, hippocampal slices were
transferred one at a time to a submerged recording chamber and
continuously superfused (2.5-3 ml/min at 30-32°C) with ACSF.
Electrophysiological recordings
Electrophysiological recordings were obtained from CA3 pyramidal neurons by using the extracellular, intracellular, and whole-cell recording techniques. Extracellular recordings were performed by using a World Precision Instrument amplifier and glass microelectrode (2-3 MOhms) filled with ACSF. Intracellular recordings were performed in current-clamp mode by using an Axoclamp 2 amplifier (Axon Instruments) and KCl-filled glass microelectrode (3 M, 50-80 MOhms); bridge balance was checked repeatedly during the experiment and capacitive transients were reduced to a minimum by negative capacity compensation. Whole-cell recordings were performed in voltage-clamp mode by using an Axopatch 200A amplifier (Axon Instruments). Patch electrodes had a resistance of 4-6 MOhms when filled with a KGluconate (KGlu) internal solution that contained (in mM): 120 KGlu, 10 KCl, 10 NaCl, 1 CaCl2, 2 MgATP, 0.5 GTP, 10 EGTA, 10 HEPES, pH 7.3 (intracellular free Ca2+ = 100 nM). Biocytin (0.5%) was added to the patch pipette solution for morphological analysis and passively diffused into neurons. For synaptic recordings, slices were stimulated by a bipolar electrode placed into the hilus of dentate gyrus. Stimulation parameters were 20-50 µs duration, 10-80 V intensity, and 0.033 Hz frequency.
Histological procedures
The extent and the specificity of the lesion in the post-ischemic hippocampus was determined by cresyl violet staining. The avidin-biotinylated horseradish peroxidase complex (ABC) reaction was used to visualize the biocytin-filled cells. After recording, slices were fixed overnight at 4°C in a solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). After cryoprotection in 20% sucrose, slices were quickly frozen in dry ice and recut in 60-µm-thick transverse sections by using a cryostat. After several rinsings in a Tris buffer saline (TBS, 0.1 M Tris; 1% NaCl, pH = 7.3), the sections were processed overnight with the standard ABC solution (Vectastain Elite ABC Kit, Vector Laboratories). After several rinsings in Tris-HCl (0.1 M, pH = 7.3), the sections were reacted with diamino-benzidine (DAB, 50 mg/100 ml Tris-HCl), as a chromogen, and H2O2 (0.01%) for 15 min. The sections were then rinsed in Tris-HCl, stained with Giemsa, dehydrated in graded ethanol, cleared in xylene, and mounted in Permount. The labeled cells were then visualized and drawn with the aid of a Camera Lucida by using either a ×20 or a ×50 oil-immersion objective.
Materials
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonovaleric acid (D-APV) were a gift from Novartis (Switzerland). Biocytin, kainate, and all other drugs were purchased from Sigma (St. Louis, MO). CNQX, D-APV, and kainate were diluted in oxygenated ACSF and bath-applied.
Data analysis
Membrane responses were displayed on a Nicolet digital oscilloscope and digitized and stored with a personal computer by using a Labmaster interface card (DIPSI, Asnieres, France). Data were analyzed off-line by using Acquis1 software (G. Sadoc, France). To study passive membrane properties and spike parameters, the pyramidal cells were recorded intracellularly. The input resistance was measured by using small (20 pA) 300-ms-square negative-current pulses and calculated from the slope of the voltage-current (V-I) curve. The reference points used to measure functional spike parameters from digitized traces are depicted in Fig. 1D. The spike parameters included the following: 1) spike threshold (a); 2) spike amplitude (the voltage difference between a and b); 3) fast afterhyperpolarization (AHP) amplitude (the voltage difference between a and c); 4) afterdepolarizing potential (ADP) amplitude, if present (the voltage difference between c and d). Data are presented as means ± standard error (SE). Statistical significance (P < 0.05) was assessed by using the Student's t-test analysis (unpaired data).
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RESULTS |
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In the present study, the intrinsic and network properties of the
post-ischemic CA3 neurons were examined 2-4 mo after a transient global ischemic insult when most of CA1 pyramidal neurons have degenerated (Fig. 1A). Some of those post-ischemic pyramidal
cells were labeled with biocytin to be reconstructed (n = 7). Reconstruction of these neurons showed that the CA3 post-ischemic
cells conserved pyramidal features with basal and apical dendrites as
the control neurons. Both long-shaft (with a long and thin apical
dendrite, Fig. 1B) and short-shaft (with a short and thick
apical dendrite, not shown) pyramidal neurons were observed in
post-ischemic hippocampi as in control hippocampi (Fitch et al.
1989).
CA3 pyramidal neurons are more depolarized several months after ischemia
With intracellular recordings, 2-4 mo after the ischemic insult,
the resting membrane potential of CA3 pyramidal neurons shifted from
72.6 ± 1.8 mV (n = 17) to
67.6 ± 1.4 mV
(n = 16, P = 0.0174, Fig.
2A). Therefore post-ischemic
pyramidal cells displayed a resting membrane potential closer to the
spike threshold than control neurons (see Fig. 2B and Table
1). In contrast, the input resistance of
post-ischemic neurons was similar to that of control cells (see Table
1). A brief (3-5 ms) and a long (200-1000 ms) depolarizing pulse
evoked similar responses in both control and post-ischemic cells,
respectively, a solitary spike and a burst of action potentials (Fig.
1, D and E). Several other parameters were also
not modified after ischemia including: spike threshold and amplitude,
fast AHP and ADP amplitudes (see Table 1), and peak amplitude of the
slow AHP (see Fig. 1F). Therefore post-ischemic CA3
pyramidal cells have a resting membrane potential closer to the spike
threshold and may fire more easily than control neurons.
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CA3 pyramidal neurons are more excitable several months after ischemia
Because post-ischemic CA3 pyramidal neurons have a more
depolarized resting-membrane potential, we hypothesized that the CA3 pyramidal network may generate more readily synchronized burst discharges in post-ischemic slices than in control ones. To test this
assumption, we first studied the evoked synaptic responses in normal
conditions in the post-ischemic CA3 pyramidal cells by using whole-cell
(VH = 60 mV) and extracellular
recordings. The synaptic response was evoked by hilar stimulation and
compared with the synaptic response recorded in hippocampal slices from control rats. In those conditions, the post-ischemic-evoked synaptic response presented a classical waveform without synchronized burst discharges as in control slices (n = 23, see Fig.
1G, and Fig. 3A).
In addition, there was not a significant change in the ratio of the
inhibitory postsynaptic current (IPSC) (including the GABA-A and GABA-B
receptor-mediated responses) versus the excitatory postsynaptic current
(EPSC) in the post-ischemic cells (n = 11) compared
with the control neurons (n = 11, P = 0.79, see Fig. 1H). Therefore in normal conditions, there
was no apparent change in the excitability of the post-ischemic CA3
pyramidal network.
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We then hypothesized that the post-ischemic pyramidal cells may have a
lower threshold to generate synchronized burst discharges in the
presence of convulsive agents. To test this assumption, different
concentrations of K+ and kainate were bath
applied (Ben-Ari and Gho 1988; Korn et al.
1987
). When the external concentration of
K+ was raised to 5 mM, the hilar stimulation
evoked an all-or-none interictal burst of population spikes (50-100 ms
duration) in the post-ischemic CA3 pyramidal cells (n = 6) in contrast to the control CA3 pyramidal cells (n = 6, Fig. 3, A and B, and Fig. 4). In the presence of higher
extracellular concentration of K+ (7.5 mM), which
generates network-driven synaptic events in CA3 area (Ben-Ari
and Gho 1988
; Korn et al. 1987
), the
synchronized all-or-none burst discharge was much more robust in the
post-ischemic CA3 pyramidal cells (n = 5) than the
control cells (n = 5, Fig. 3, A and
B). We also observed that, in presence of 5 and 7.5 mM [K+]0, spontaneous
interictal burst discharges developed more frequently in post-ischemic
CA3 pyramidal cells (n = 5) than in control cells (n = 5; Fig. 3B). Similarly, a low dose of
kainate (50 nM, 4 min duration) triggered a burst of population spikes
in post-ischemic pyramidal cells (n = 5) but not in
control neurons (n = 5, Fig. 3C). With
higher concentrations of kainate (100 or 200 nM, 4 min duration), the
number of population spikes increased more significantly in
post-ischemic neurons (n = 5) than in control cells
(n = 6, Fig. 3C). Spontaneous interictal
bursts of population spikes also developed more frequently in the
post-ischemic CA3 pyramidal neurons (n = 5) than
in control cells (n = 6) in the presence of 100 and 200 nM kainate (Fig. 3C). The burst discharges evoked by bath application of high concentration of K+ (5 and
7.5 mM) or in the presence of kainate were mediated by glutamatergic
receptors, because they were abolished in the presence of 10 µM CNQX
and 50 µM D-APV (not shown). Therefore several months after the ischemic insult, CA3 pyramidal neurons had a lower threshold to generate glutamatergic network-driven bursts than control cells.
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Paired-pulse stimulation evoked a burst discharge in post-ischemic neurons
A paired-pulse protocol has been frequently used to test the
excitability of CA1 pyramidal cells in epileptic rats in normal conditions (Bernard and Wheal 1995; Cornish and
Wheal 1989
). In an attempt to test the capability of
post-ischemic CA3 pyramidal cells to generate synchronized bursts in
absence of any convulsive agent, we used the same type of paradigm. A
conditioning (first stimulation) and a test (second stimulation) pulse
of equal intensity of stimulation were applied to the hilar area with
an interpulse interval ranging from 10 to 100 ms. The strength of the
intensity of the first pulse was adjusted to evoke a single population
spike in the conditioning response. In control CA3 pyramidal cells, only one or two population spikes were evoked by the test pulse at all
interpulse intervals (n = 10, Fig.
5, A and B). In
contrast, in post-ischemic CA3 pyramidal cells (n = 8),
the test pulse evoked a burst discharge when the interpulse interval
ranged from 60 to 100 ms (Fig. 5, A and B).
Interestingly, in two post-ischemic slices out of eight, a burst
discharge was evoked by both the conditioning and the test pulse during
the paired-pulse protocol; this bursting response could be still evoked
by a single pulse after the protocol, suggesting a permanent alteration
of the network properties (not shown). Using this protocol, we also
studied the change of the slope of the synaptic transmission (recorded
in the stratum radiatum) and the amplitude of the first population spike evoked by the test pulse (recorded in the pyramidal cell layer).
In both control (n = 10) and post-ischemic neurons
(n = 8), the paired-pulse protocol induced a
statistically identical facilitation of the slope and the population
spike (see Fig. 5, C and D).
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Therefore several months after ischemia, it is possible to evoke a burst discharge in CA3 pyramidal cells by using a paired-pulse protocol without convulsive agent. This observation confirms that the post-ischemic network becomes hyperexcitable after an ischemic insult.
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DISCUSSION |
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The principal result of the present report is that 2-4 mo after an ischemic insult, CA3 pyramidal cells have a more depolarized resting membrane potential and a greater susceptibility to generate all-or-none interictal burst discharges. Therefore in addition to the short-term effect of ischemia, i.e., the degeneration of CA1 pyramidal neurons, there are long-term changes in the activity of CA3 pyramidal cells that may modify the hippocampal network properties.
Specific intrinsic properties of the post-ischemic CA3 pyramidal neuron
Study of the intrinsic membrane properties of the
post-ischemic CA3 pyramidal cells revealed that there was a clear shift of the resting membrane potential of CA3 pyramidal neurons toward positive values. In contrast, those cells did not display significant changes of the input resistance, the spike threshold, the spike amplitude, the accommodation, the fast AHP, the ADP, and the slow AHP
in comparison with control cells. The resting membrane potential of
pyramidal neurons is a key factor in the regulation of the cell
excitability and mainly depends on the
Na+/K+ ATPase
(Haglund et al. 1985; Haglund and Schwartzkroin
1990
) and potassium channel activities (for review see
Storm 1990
). Down-regulation of the
Na+/K+ ATPase and potassium
channel activities has been described in different models of lesions.
For example, a decrease of the
Na+/K+ ATPase has been
reported in CA1 area several weeks after lesion of the CA3 pyramidal
cells (Anderson et al. 1994
) and postaxotomic mammalian
motoneurons display down-regulated potassium currents (Gustafsson 1979
; Laiwand et al. 1988
).
Therefore similar phenomena may take place in the partially
deefferented post-ischemic CA3 pyramidal neurons and underlie the
depolarization of the resting membrane potential. The reduction of the
membrane potential (without a change of input resistance) after an
ischemic insult is not a unique feature of CA3 pyramidal cells.
Previous studies have shown similar results in neocortical neurons
submitted to a forebrain ischemic insult (Luhmann et al.
1995
). In contrast, neocortical pyramidal cells recorded in the
infarct border zone induced by a focal ischemia do not display
modifications of their resting membrane potential (Mittmann et
al. 1998
). Therefore the modification of the membrane potential
in the post-ischemic-resistant neurons will depend on the type of
ischemic insult.
Synaptic network properties of the post-ischemic CA3 pyramidal cells
Examination of the synaptic transmission in the post-ischemic CA3
pyramidal cells showed that there is no seizure-like activity in normal
conditions. However, we observed that those neurons are more
susceptible than the control ones to display evoked and spontaneous
synchronized burst discharges when low doses of convulsive agents (as
K+ and kainate) were bath applied. We suggest
that the positive shift of the resting membrane potential toward the
spike threshold may facilitate the generation of the synchronized burst
discharges in post-ischemic CA3 pyramidal neurons. However, we cannot
exclude alternative hypothesis. Indeed, raising the extracellular
concentration of K+ or bath application of
kainate have pleiotropic effects in addition to inducing a
depolarization (Ben-Ari and Gho 1988; Rutecki et al. 1985
). This includes a reduction of GABAergic inhibition
(Fisher and Alger 1984
; Korn et al. 1987
;
Rovira et al. 1990
; but see also Cossart et al.
1998
; Frerking et al. 1998
) and a
down-regulation of potassium current as
IQ and IC
(Gho et al. 1986
). Therefore the
generation of synchronized burst discharge may be due to a greater
vulnerability of these conductances to the convulsive agents in
post-ischemic neurons. We also showed that synchronized burst
discharges could be induced in normal ACSF conditions when a
paired-pulse protocol was used. We observed that these burst discharges
developed when the interpulse interval ranged from 60 to 100 ms. At
this interpulse interval the GABA-A receptor-mediated inhibition is
reduced by 25-50% (Davies et al. 1990
). We suggest that the same paired-pulse depression of GABAergic inhibition facilitates more efficiently the firing of the post-ischemic cells than
the control neurons, because the post-ischemic cells have a more
depolarized resting membrane potential. However, we cannot exclude that
the paired-pulse depression of GABAergic inhibition is more robust in
the post-ischemic neurons compared with the control neurons. Further
experiments will be necessary to clarify this point. All together these
different observations clearly demonstrate that the post-ischemic CA3
pyramidal cells are more excitable than the control ones and that
synchronized burst discharges can be induced in normal ACSF if two
synaptic events are successively evoked within the appropriate time
window. Interestingly, these results are reminiscent of previous
observations performed in neocortical post-ischemic neurons. However,
in contrast to the present study, these neurons display epileptiform
activities evoked by a single shock of stimulation even in normal
conditions, due to a drastic reduction of GABAergic inhibition in favor
of excitatory responses (Luhmann et al. 1995
;
Mittmann et al. 1998
). The role of the ischemic insult
and/or the degeneration of the CA1 pyramidal cells in the change of
excitability of the CA3 pyramidal cells remain at present unclear. A
global hypoxic insult, performed between p10 and p12, induces seizure
activity in CA1 and CA3 areas of the rat hippocampus but not at younger
or older ages (Jensen et al. 1998
). In this model, the
hypoxic insult was not associated with lesion. It is possible that the
change of excitability of CA3 pyramidal cells induced by ischemic
insults in our study is partly a consequence of the lesion of CA1
pyramidal cells and the reactive plasticity that may take place,
because the neurons deprived of their projecting sites or after axotomy
are often hyperexcitable (Chung et al. 1993
;
Prince et al. 1997
).
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ACKNOWLEDGMENTS |
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We are grateful to D. Diabira for technical assistance.
Financial support from Institut National de la Santé et de la Recherche Médicale, Centre National pour la Recherche Scientifique, and Direction des Recherches, Etudes et Techniques is acknowledged.
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FOOTNOTES |
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Address for reprint requests: V. Crepel, INSERM U 29, INMED, Parc Scientifique de Luminy, BP 13, 13273 Marseille Cedex 09, France.
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 17 May 1999; accepted in final form 30 November 1999.
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REFERENCES |
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