Department of Biosciences, Division of Animal Physiology, University of Helsinki, FIN-00014 Helsinki, Finland
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ABSTRACT |
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Autere, Anna-Maija,
Karri Lamsa,
Kai Kaila, and
Tomi Taira.
Synaptic activation of GABAA receptors induces neuronal
uptake of Ca2+ in adult rat hippocampal slices.
Synaptically evoked transmembrane movements of Ca2+ in the
adult CNS have almost exclusively been attributed to activation of
glutamate receptor channels and the consequent triggering of voltage-gated calcium channels (VGCCs). Using microelectrodes for
measuring free extracellular Ca2+
([Ca2+]o) and extracellular space (ECS)
volume, we show here for the first time that synaptic stimulation of
-aminobutyric acid-A (GABAA) receptors can result in a
decrease in [Ca2+]o in adult rat hippocampal
slices. High-frequency stimulation (100-200 Hz, 0.4-0.5 s) applied in
stratum radiatum close (
0.5 mm) to the recording site induced a 0.1- to 0.3-mM transient fall in [Ca2+]o from a
baseline level of 1.6 mM. Concomitantly, a 30-40% decrease in the ECS
volume was seen. Exposure of drug-naïve slices to the
GABAA receptor antagonist picrotoxin (100 µM) first
attenuated and only thereafter augmented the Ca2+ shifts.
Application of ionotropic glutamate receptor antagonists resulted in a
monotonic reduction of the Ca2+ response, but a large
Ca2+ shift persisted (60-70% of the original), which was
attenuated by a subsequent application of picrotoxin or bicuculline. In
the absence of ionotropic glutamatergic transmission, pentobarbital sodium (100 µM), an up-modulator of the GABAA receptor,
strongly enhanced the activity-evoked changes in
[Ca2+]o. We suggest that the underlying
mechanism of GABA-induced Ca2+ transients is the activation
of VGCCs by bicarbonate-dependent GABA-mediated depolarizing
postsynaptic potentials. Accordingly, stimulation-evoked
Ca2+ shifts were inhibited by the membrane-permeant
inhibitor of carbonic anhydrase, ethoxyzolamide (50 µM) or in
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered HCO3-free solution. Neuronal
Ca2+ uptake caused by intense synaptic activation of
GABAA receptors may prove to be an important mechanism in
the modulation of activity-dependent neuronal plasticity,
epileptogenesis, and cell survival in the adult brain.
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INTRODUCTION |
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-Aminobutyric acid (GABA) does not act solely
as an inhibitory transmitter in the adult mammalian CNS. The
depolarizing actions of GABA on central neurons have been known for
some time (cf. Alger and Nicoll 1982
; Kaila
1994
; Kaila and Voipio 1987
), but only recently
has the truly excitatory aspect of GABAA receptor function
in the adult hippocampus been recognized (Grover et al. 1993
; Kaila et al. 1997
; Staley et al.
1995
; Taira et al. 1997
). In rat hippocampal
slice the excitatory actions of GABA become most evident on
high-frequency stimulation of the interneuronal network. As we recently
demonstrated in rat hippocampal pyramidal neurons, under such
conditions the GABAA receptor-mediated depolarization and
associated spike firing can far exceed that provided by the glutamatergic drive (Taira et al. 1997
). The
GABA-mediated depolarizing postsynaptic potentials (hereafter termed
GDPSPs) that are evoked on high-frequency stimulation in hippocampal
pyramidal neurons appear to be due to a network-driven,
bicarbonate-dependent increase in extracellular K+
([K+]o) (Kaila et al. 1997
;
Lamsa and Kaila 1997
).
In contrast to its conventional inhibitory role in the adult brain,
during the early stages of postnatal development (up to postnatal
day 8-10 in the rat), GABA may act as an excitatory transmitter
in the CNS (Ben-Ari et al. 1989; Cherubini et al. 1991
). Consistently with this, it has been shown that both
synaptic and pharmacological stimulation of GABAA receptors
leads to an elevation in intracellular Ca2+
([Ca2+]i) through activation of voltage-gated
calcium channels (VGCCs) in pyramidal cells and interneurons in the
immature rat hippocampus (Leinekugel et al. 1997
), thus
contributing to the neurotrophic actions of GABA during the neonatal
period (Barbin et al. 1993
). In adult hippocampal
slices, synaptically induced transmembrane Ca2+ shifts have
almost exclusively been attributed to the stimulation of ionotropic
glutamate receptors (see Heinemann et al. 1990
), and
most of the ensuing calcium uptake appears to be mediated by
plasmalemmal VGCCs (Heinemann et al. 1990
;
Miyakawa et al. 1992
; Paalasmaa and Kaila
1996
). Because activation of the GABAA receptors
can result in conspicuous depolarization and spike firing in the
postsynaptic neuron, an obvious question to ask is whether GDPSPs are
linked with a significant neuronal uptake of calcium. In the present
study we provide compelling physiological and pharmacological evidence
for this hypothesis.
Because stimulation-induced changes in the extracellular space (ECS) can affect local interstitial ion accumulations, in some of the experiments we also investigated the changes in the ECS volume paralleling tonic activation of the inhibitory network to better appreciate the magnitude of the accompanying Ca2+ shifts.
Part of the results has appeared in an abstract form (Autere et
al. 1997).
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METHODS |
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Hippocampal slices (400 µm) from 30-40-day-old Wistar rats
were prepared using established procedures (Taira et al.
1993). The experiments were carried out in an interface-type
recording chamber (volume, 0.6 ml; flow rate, 1.0 ml/min; temperature
32°C), and the slices were allowed to recover for ~1 h before
recording began.
The physiological solution contained (in mM) 124 NaCl, 3.0 KCl, 2.0 CaCl2, 25 NaHCO3, 1.1 NaH2PO4, 2.0 MgSO4, and 10 D-glucose. It should be noted that in the
bicarbonate-buffered solution the concentration of extracellularly
available Ca2+ is 1.55-1.6 mM (see Heinemann et al.
1990). The solution was equilibrated with 95%
O2-5% CO2 to yield a pH of 7.4. A stream of
the same gas mixture was (following warming and humidifying) continuously passed over the preparation. In some of the experiments, NaHCO3 was replaced by 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and the solution was gassed with 100% O2.
The pH was adjusted to 7.4 with NaOH. For the ECS volume measurements, 0.5 mM tetramethylammonium ion (TMA+) was added into the
perfusion solution.
Double-barreled microelectrodes were pulled from borosilicate glass
(2GC150FS, Clark Electromedical, Pangbourne, Reading, UK). The
nonfilamented barrel was silanized by exposure to vapor of
dimethyl-trimethyl-silylamine (Fluka) followed by baking in an oven at
200°C. The following procedure was applied for
Ca2+-sensitive microlectrodes. After dry beveling,
the silanized barrel was back-filled with a solution containing 1 mM
CaCl2, 100 mM NaCl, and 1 mM HEPES (pH 7.6), and a short
column of the Ca2+-selective ionophore (Fluka membrane
cocktail 21048) was taken into the tip by suction. The reference barrel
was filled with 150 mM NaCl. The outer diameter of these
microelectrodes was 2-8 µm, and the resistances of the
Ca2+ and reference barrels were 15-20 G and 20-40
M
, respectively. The electrodes had a slope of 28-30 mV for a
10-fold change in [Ca2+]o, and they were
calibrated in terms of free concentration. Measurements of the
extracellular concentration of bath-applied impermeable ions such as
TMA+ can be used to study transient changes in the ECS
volume (see Nicholson and Phillips 1981
). In principle,
changes in the ECS volume and in the TMA+ concentration are
inversely proportional. TMA+-sensitive electrodes were
manufactured in a manner similar to the Ca2+ electrodes
except that the silanized barrel was filled with a solution containing
(in mM) 150 NaCl, 3.5 KCl, and 0.5 TMA and the ionophore used was
TMA+-selective (Corning 477317). The resistance of the
TMA+-selective barrel was 0.5-1.0 G
, and the electrode
had a slope of 55-59 mV per decade change.
All recordings were made in stratum radiatum of area CA1. The tip of
the bipolar stimulus electrode was positioned close to (within 0.5 mm)
the recording site. Stimulus intensity was supramaximal (10-30 V) and
pulse duration 0.1 ms. High-frequency trains of pulses (100-200 Hz,
40-100 pulses) were given at 3-min intervals. Typically, after the
first train the responses remained very stable (cf. Kaila et al.
1997).
D-2-Amino-5-phosphonopentoate (AP5, 40 µM) and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX, 10 µM, Tocris Cookson, Bristol, UK), picrotoxin (PiTX, 100 µM) and pentobarbital sodium (PB, 100 µM, Sigma, St. Louis, MO), ketamine (50 µM, Parke-Davis, Barcelona, Spain), bicuculline methiodide (10 µM, Research Biochemicals International, Natick, MA), and ethoxyzolamide (EZA, 50 µM, a gift from prof. Gerolf Gross, Univ. Hannover, Germany) were applied in the perfusion solution.
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RESULTS |
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We first investigated the GDPSP-linked changes in the ECS volume and in [Ca2+]o employing simultaneously TMA+ and Ca2+-selective microelectrodes. Upon high-frequency stimulation and in the presence of the ionotropic glutamate receptor antagonists, there was a 143 ± 14% (mean ± SE, n = 6) increase with time-to-peak 3.5 ± 0.9 s in the extracellular TMA+ concentration, thus indicating a corresponding decrease in the ECS volume (Fig. 1). Concomitantly, a 0.2-0.4 mM transient fall in [Ca2+]o, which reached its peak amplitude in 1-1.5 s and a field potential transient consisting of 1-3 mV negative deflections with a time-to-peak ranging between 0.7 and 1.0 s were observed. If the shrinkage of the ECS volume at the time of the peak fall in [Ca2+]o is taken into account, the net loss of Ca2+ from the ECS was 15-35% higher. In further measurements of the ECS volume changes, we noted that application of the ionotropic glutamate receptor antagonists diminished the ECS shrinkage to 51 ± 11% (n = 3, not illustrated) of the control values. Thereafter, upon exposure to PiTX, a further 15 ± 4% (n = 3) attenuation in the ECS volume change was seen. This will correspondingly accentuate the drop in [Ca2+]o, and therefore the shifts in the ECS volume cannot underlie the observed alleviation of the [Ca2+]o transients by the drug treatments.
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Upon application of the GABAA receptor antagonist PiTX (100 µM) in the absence of the ionotropic glutamate receptor blockers, there was first a decrease in the GDPSP-induced
[Ca2+]o shift to 74 ± 2% of the
control value (P < 0.05, paired t-test, n = 4; within 3-6 min from start of wash-in) and only
subsequently did the [Ca2+]o response start
to gain size (Fig. 2A). If the
sole function of the GABAA receptors was to suppress
neuronal activity, one would expect that exposure to the receptor
antagonist would result in an immediate increase in synaptically evoked
[Ca2+]o shifts. This is probably true when
stimulation paradigms not reaching the threshold for triggering GDPSPs
are used (cf. Hamon and Heinemann 1986). However, the
rather untypical nature of the PiTX effect in the present study is
consistent with our recent observation of a similar, biphasic effect of
PiTX on postsynaptic spike firing evoked on high-frequency tetanus
(Taira et al. 1997
). Subsequent application of the
ionotropic glutamate receptor antagonists AP5, NBQX, and ketamine
effectively attenuated the [Ca2+]o shift.
Exposure to the glutamate blockers alone decreased the tetanus-evoked
fall in [Ca2+]o to 66 ± 3% of the
control value (P < 0.005, paired t-test, n = 9, Figs. 2B and
3).
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Because we next wanted to study [Ca2+]o transients evoked solely on synaptic activation of GABAA receptors, all experiments described after this point were done in the continuous presence of the ionotropic glutamate receptor antagonists. Blockade of the GABAA receptors by PiTX in the presence of the glutamate receptor antagonists diminished the [Ca2+]o transient, preserving only 20-30% of the original shift (P < 0.005, t-test, n = 8, Figs. 4A and 6). A similar result (48 ± 7% decrease in the [Ca2+]o shift, P < 0.005, t-test, n = 3) was obtained by using a competitive GABAA receptor antagonist, bicuculline (10 µM) thus further confirming the involvement of GABAA receptors in the phenomenon (not illustrated).
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To further elucidate the link between synaptically evoked [Ca2+]o shifts and GABAergic transmission, we examined the effect of PB (100 µM), a widely used potentiator of the GABAA receptor function, on the [Ca2+]o transients. Application of PB resulted in a significant (126 ± 6%) increase (P < 0.05, paired t-test, n = 4) in the peak fall in [Ca2+]o (Figs. 4B and 6). The accentuation of the response was seen even more clearly as an increase in its duration (Fig. 4B).
To demonstrate the causal connection between depolarizing
GABAA receptor-mediated responses and the
[Ca2+]o transients, it was of importance to
examine the [Ca2+]o shifts under experimental
conditions known to suppress GDPSPs. This was done by taking advantage
of the known HCO3 dependency of the depolarizing GABA
responses. GDPSPs can be attenuated upon application of
membrane-permeant inhibitors of carbonic anhydrase (Grover et
al. 1993; Staley et al. 1995
; Taira et
al. 1997
). In the presence of of the membrane-permeant carbonic
anhydrase inhibitor EZA (50 µM), the
[Ca2+]o shift was diminished to 68 ± 2% (P < 0.005, t-test, n = 4, Figs. 5 and
6). A similar kind of result (diminution
of [Ca2+]o shift to 36 ± 6%,
P < 0.005, t-test, n = 4)
was achieved by replacing the perfusion solution by HEPES-buffered
HCO3-free solution (not illustrated).
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DISCUSSION |
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Common knowledge holds that a strong GABAA-mediated
input will result in effective shunting of the postsynaptic membrane, thus suppressing excitatory synaptic responses and attenuating the
activation of the main Ca2+ conductive pathways, the VGCCs
and N-methyl-D-aspartate (NMDA) receptor
channels (for [Ca2+]o responses, see
Hamon and Heinemann 1986; Heinemann et al.
1984
). Yet, it is becoming increasingly evident that GABAergic
interneurons do not merely gate the activity of principal cells, but
upon intense stimulation they can have an auxiliary or even dominating
role in eliciting postsynaptic firing (see Taira et al.
1997
). The rather unconventional idea of GABAA
receptor-mediated excitation in the adult hippocampus has been
established only recently (see introduction), and therefore the
physiological consequences of this phenomenon are largely unknown. We
focused our study to resolve the apparent but still unanswered question
of whether GABAergic excitation might accentuate activity-dependent
changes in [Ca2+]o in the adult CNS.
Synaptic mechanisms of [Ca2+]o shifts evoked by high-frequency stimulation
Even in the presence of PiTX, NMDA receptors contribute
little to the stimulus-evoked transient fall in
[Ca2+]o in the area CA1 (Köhr
and Heinemann 1989; Paalasmaa et al. 1994
),
whereas under such conditions most of the
[Ca2+]o shift can be attributed to activation
of non-NMDA glutamate receptors and a consequent triggering of VGCCs
(Heinemann et al. 1990
; Paalasmaa and Kaila
1996
; Paalasmaa et al. 1994
). Accordingly, upon
tetanic stimulation of the Schaffer collaterals, Miyakawa et al.
(1992)
detected hardly any Ca2+ influx through the
dendritic NMDA receptor channels using intracellularly injected fura-2.
Hence they concluded that the observed rise in intracellular
Ca2+ was mainly due to activation of VGCCs.
In the present study we found that only a part of the
[Ca2+]o shift evoked by stimuli applied at
100 or 200 Hz close to the site of recordings was blocked by the
ionotropic glutamate receptor antagonists. The rather long duration of
[Ca2+]o transients seen in the absence and
presence of the glutamate antagonists is congruent with the long
duration of GDPSPs and the accompanied spike firing and increase in
[K+]o (cf. Kaila et al. 1997;
Taira et al. 1997
).
Application of PiTX under control conditions (before application of the
glutamate receptor antagonists) resulted in an interesting sequence of
events: first, a slight decrease in the
[Ca2+]o shift and, thereafter, an
augmentation of the response. In previous studies employing relatively
weak stimulation protocols or glutamate agonist application, the
GABAA receptor antagonists bicuculline and PiTX have been
shown to monotonically enhance decreases in
[Ca2+]o (e.g., Hamon and Heinemann
1986). The biphasic effect of PiTX on Ca2+ shifts
in the present study is apparently paralleled by changes in
postsynaptic spike firing on high-frequency stimulation (Taira et al. 1997
). Because the GDPSPs are more sensitive to
GABAA receptor antagonists than the hyperpolarizing
inhibitory postsynaptic potentials (IPSPs) (cf. Alger and Nicoll
1982
), PiTX will first attenuate the excitatory GABA responses
(and overall excitation), and, only after continuous wash-in of the
drug, the hyperpolarizing responses will be affected, thus resulting in
an enhancement of the early spiking mediated by ionotropic glutamate
receptors (Taira et al. 1997
). Consequently, regarding
the fact that most of the calcium influx seen on tetanic stimulation of
afferents ensues from the activation of VGCCs, the biphasic effect of
PiTX on Ca2+ transients is easy to understand. As expected,
applying PiTX on top of the glutamate antagonists strongly attenuated
the Ca2+ shift. To exclude the possibility that this
finding was due to a nonspecific effect of PiTX on Ca2+
channels or Cl
transport (cf. Gross et al.
1997
; Kaila et al. 1997
), we used bicuculline
instead of PiTX in some of the experiments. The remaining component of
the Ca2+ response seen in the presence of ionotropic
glutamate and GABAA receptor antagonists is suggested to
arise from a presynaptic Ca2+ influx (see Heinemann
et al. 1990
) and/or direct stimulation of a small population of
neurons (see Paalasmaa and Kaila 1996
).
PB, which is known to potentiate the GABAA receptor
function is known to enhance depolarizing GABA responses (Alger
and Nicoll 1982; Lamsa and Kaila 1997
) and
associated bicarbonate shifts (Kaila et al. 1992
;
Taira et al. 1995b
; Voipio et al. 1995
).
In line with the idea that GABA-mediated depolarization can augment neuronal uptake of Ca2+, this drug potentiated the
tetanus-evoked Ca2+ transients. This finding is again
contradictory to the conventional view that agents enhancing GABAergic
transmission will reduce transmembrane Ca2+ shifts in the
adult brain.
Depolarizing GABA responses evoked by high-frequency stimulation are
dependent on the availability of bicarbonate. Application of the
membrane-permeant carbonic anhydrase inhibitor, EZA, or replacing the
perfusion solution with nominally HCO3/CO2-free solution buffered with HEPES leads to a suppression of the
network-driven GDPSPs (Kaila et al. 1997; Taira
et al. 1997
). Hence it was of much interest that both
treatments also led to a mitigation of GABA-evoked Ca2+
shifts in the present experiments. In our recent study on the excitatory GABA responses in pyramidal neurons, a partial suppression only of posttetanic GABAergic depolarization was achieved by EZA, thus
explaining the incomplete blockade of Ca2+ transients by
the drug (cf. Fig. 3 in Taira et al. 1997
). Evidently then, noncatalyzed (de)hydration of CO2 in the absence of
carbonic anhydrase activity is able to produce bicarbonate in a
sufficient amount for a large part of the GABAA
receptor-dependent interneuronal excitation and its ionic consequences
to persist (Lamsa and Kaila 1997
).
Neuronal versus glial origin of the Ca2+ sink
The cellular elements in the nervous tissue taking up
Ca2+ on tetanic stimulation cannot, of course, be directly
inferred from the data obtained using ion-selective extracellular
microelectrodes. The following question then arises: what are the
relative contributions of neurons and glial cells to the
[Ca2+]o changes seen in the present study. It
is known that a robust increase in [K]o of the kind seen
during some nonphysiological conditions such as ictal activity can
trigger astroglial Ca2+ influx via VGCCs (Duffy and
MacVicar 1994). However, activation of glial VGCCs requires
large elevations (threshold 20-25 mM) in [K]o
(Duffy and MacVicar 1994
). As reported in our recent
paper (Kaila et al. 1997
), GDPSP-linked
[K]o shifts are typically in the range of 7-9 mM.
Moreover, under physiological conditions glial cells do not fire
spikes, a property that underlies much of the neuronal uptake of
Ca2+. Nevertheless, in the light of the existing data, it
seems likely that under the present experimental conditions there is
both neuronal as well as glial uptake of Ca2+, albeit the
relative contributions of neuronal and glial Ca2+ sinks
remain to be elucidated.
Implications of the present findings
As demonstrated in the present work, activation of
GABAA receptors can lead to prominent transmembrane calcium
fluxes adding to the synaptic mechanisms promoting neuronal
Ca2+ entry. Activation of a certain population of dendritic
calcium channels, namely the low-voltage activated (LVA) channels is
enhanced if it is preceded by hyperpolarization (Magee and
Johnston 1995). Apparently, excitatory postsynaptic potentials
(EPSPs) following hyperpolarizing IPSPs would particularly favor the
contribution of LVA Ca2+ channels to postsynaptic
Ca2+ entry. Therefore the sequential postsynaptic
hyperpolarization/depolarization in pyramidal neurons resulting from
intense stimulation of interneurons is especially well-suited for
promoting the activation of LVA Ca2+ channels
(Lambert and Grover 1995
). This raises the interesting possibility that at least part of the elevation of postsynaptic Ca2+ needed for the induction of tetanus-induced forms of
LTP results from the activation of interneurons (cf. Cavus and
Teyler 1996
; Grover and Teyler 1990
;
Taira et al. 1995a
). The present results also suggest
that GABA-mediated excitation might act as a "hand-shaking" signal
(see Marty and Llano 1995
) to promote
Ca2+-dependent Hebbian-type plasticity changes at GABAergic
synapses.
It was reported by Leinekugel et al. (1995) that
synaptic activation of the GABAA receptors leads to
depolarization and rise in intracellular Ca2+ via
activation of VGCCs in 2-5 day-old but not in 12-13 day-old rat
hippocampal slices. Furthermore, in neonates the GABAA
receptor-mediated depolarization is essential for activity-induced
plasticity of GABAA-mediated transmission (McLean et
al. 1996
). However, whereas Leinekugel et al.
(1995)
used only low-frequency (4-5 Hz, 4-5 pulses)
stimulation, we intentionally selected to employ high-frequency (100-200 Hz, 40-100 pulses) trains of pulses to achieve tonic activation of the interneuronal GABAergic network. Thus, depending on
the stimulation/firing pattern of interneurons, a large fraction of the
resulting Ca2+ transient in the area CA1 can be mediated by
GDPSPs originating in pyramidal neurons. Therefore the conclusion that
synaptic activation of GABAA receptors leads to
Ca2+ uptake in immature but not in mature neurons may be
premature.
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ACKNOWLEDGMENTS |
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This study was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, and the University of Helsinki.
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FOOTNOTES |
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Address for reprint requests: T. Taira, Dept. of Biosciences, Division of Animal Physiology, P.O. Box 17, University of Helsinki, FIN-00014 Helsinki, Finland.
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 13 February 1998; accepted in final form 8 October 1998.
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REFERENCES |
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