1Department of Physiology and 2Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
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Jensen, Kimmo,
Morten Skovgaard Jensen, and
John D. C. Lambert.
Role of presynaptic L-type Ca2+ channels in
GABAergic synaptic transmission in cultured hippocampal neurons.
Using dual whole cell patch-clamp recordings of monosynaptic
GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat
hippocampal neurons, we have previously demonstrated posttetanic
potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic
neuron leads to accumulation of Ca2+ in the presynaptic
terminals. This enhances the probability of GABA-vesicle release for up
to 1 min, which underlies PTP. In the present study, we have examined
the effect of altering the probability of release on PTP of IPSCs.
Baclofen (10 µM), which depresses presynaptic Ca2+ entry
through N- and P/Q-type voltage-dependent Ca2+ channels
(VDCCs), caused a threefold greater enhancement of PTP than did
reducing [Ca2+]o to 1.2 mM, which causes a
nonspecific reduction in Ca2+ entry. This finding prompted
us to investigate whether presynaptic L-type VDCCs contribute to the
Ca2+ accumulation in the boutons during spike activity. The
L-type VDCC antagonist, nifedipine (10 µM), had no effect on single
IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 µM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also
depressed IPSCs during the stimulation (i.e., they increased tetanic
depression). The L-type VDCC "agonist" ()BayK 8644 (4 µM) had
no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train
of 1 s by 91%. In conclusion, the results indicate that L-type
VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation
during high-frequency activity. This helps maintain vesicle release
during tetanic stimulation and also enhances the probability of
transmitter release during the posttetanic period, which is manifest as
PTP. Involvement of L-type channels in these processes represents a
novel presynaptic regulatory mechanism at fast CNS synapses.
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INTRODUCTION |
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GABA (-aminobutyric acid) is the major
inhibitory neurotransmitter in the mammalian CNS, where it acts on
postsynaptic GABAA (Macdonald and Olsen
1994
) and GABAB receptors (Misgeld et al. 1995
) resulting in fast and slow inhibitory postsynaptic
potentials (IPSPs), respectively. The magnitude of GABAergic inhibition
depends on the history of synaptic activation, and it has been
established that paired-pulse depression of IPSPs is caused by GABA
acting on presynaptic GABAB autoreceptors (Davies et
al. 1993
; Deisz and Prince 1989
), or by
depletion of vesicles at the active zones (Jensen et al.
1998
). We have recently reported that inhibitory postsynaptic
currents (IPSCs) in cultured neurons are potentiated following tetanic
activation of the GABAergic neuron (posttetanic potentiation, PTP)
(Jensen et al. 1998
). Following brief tetanization, IPSCs are enhanced by ~60%, which declines to baseline over the course of 1 min. Further investigations indicated that PTP of IPSCs is
caused by accumulation of Ca2+ ions in the GABAergic
boutons, which raises the probability of release during the posttetanic
period (Jensen et al. 1998
).
Following excitation of the GABAergic neuron, GABA is released as a
consequence of a highly localized and transient Ca2+ influx
through N-type and P/Q-type voltage-dependent Ca2+ channels
(VDCCs) in the presynaptic terminal (Ohno-Shosaku et al.
1994). L-type VDCCs are not thought to participate in
transmitter release at GABAergic (Doze et al. 1995
;
Ohno-Shosaku et al. 1994
) or other synapses mediating
fast synaptic transmission (Dutar et al. 1989
;
Horne and Kemp 1991
; Wheeler et al.
1994
). However, secretion of hormones or neuromodulators has
been reported to depend on L-type VDCCs (Miller 1987
;
Perney et al. 1986
). In the present report we have made
dual whole cell recordings from cultured hippocampal neurons and
elicited monosynaptic GABAergic IPSCs. We evoked PTP of the IPSCs and
studied the effect of decreasing presynaptic Ca2+ influx
using low [Ca2+]o, and the GABAB
receptor agonist, baclofen. Low [Ca2+]o
caused a greater reduction in PTP than baclofen, which prompted us to
hypothesize that L-type VDCCs deliver Ca2+ to the boutons
during repetitive activity and thereby participate in the control of
GABA release, which would represent a novel presynaptic regulatory
mechanism at fast CNS synapses. To test this hypothesis, we have
studied the effect of modulating L-type VDCCs on PTP of IPSCs.
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METHODS |
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Hippocampal culture preparation
Cultures of rat hippocampal neurons were prepared by standard
techniques. Briefly, pregnant Sprague-Dawley rats were
anesthetized by pentobarbital sodium (50 mg/kg ip) at gestational
day 17-18. Fetuses were removed and decapitated, and the
hippocampi were dissected free. The tissue was triturated mechanically
in a HEPES-buffered dissection medium and plated on
poly-D-lysine-coated coverslips in 35-mm Petri dishes.
Plating medium consisted of MEM with Earle's salts and
L-glutamine (Glutamax-1, GIBCO) supplemented with
horse serum (HS, 10%), fetal calf serum (FCS, 10%), penicillin (50 IU/ml), and streptomycin (50 µg/ml). Cultures were grown in 5%
CO2-10% O2 at 37°C (Brewer and Cotman
1989). Plating medium was fully replaced by 2 ml feeding medium
after 1 day in vitro, and thereafter 1 ml was exchanged twice weekly.
Feeding medium had the same composition as plating medium except that
FCS was omitted and HS was reduced to 5%. The mitosis inhibitors
5'-fluoro-2'-deoxyuridine (FUDR; 15 µg/ml) and uridine (35 µg/ml) were added after 3-4 days when cultures showed a confluent background.
Electrophysiology
Coverslips with the cultured cells (10-30 days in vitro) were
placed in a stainless steel chamber mounted on an inverted Nikon Diaphot 200 microscope, where individual neurons were visualized through ×200 Normarski optics. The chamber was perfused (1 ml/min) with an extracellular (control) medium containing (in mM) 140 NaCl, 3.5 KCl, 2.5 CaCl2, 2.5 MgCl2, 10 glucose, and 10 HEPES, adjusted with NaOH to pH 7.35 and 305 mosM/kg at 22°C.
Patch-clamp electrodes (3-6 M) were fabricated from borosilicate
glass (1.2 mm OD) on a Flaming/Brown P-97 puller (Sutter Instruments).
The presynaptic electrode contained (in mM) 15 NaCl, 140 KOH, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 0.10 leupeptin, and 2 MgATP, adjusted with methanesulfonic acid to pH 7.3, 285 mosM/kg. The postsynaptic patch-electrode contained (in mM) 130 CsCl2, 1 CaCl2, 1 MgSO4, 1 CaCl2, 11 EGTA, 0.10 leupeptin, 4 MgATP, and 5 QX-314,
adjusted with CsOH to pH 7.3, 285 mosM/kg. Dual whole cell recordings
were made using Axopatch 200 and 200A amplifiers in the voltage-clamp mode at a Vh of
70 mV. Excitatory synaptic
interactions between neurons were blocked by
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and
DL-2-amino-5-phosphonovaleric acid (DL-APV; 50 µM). GABAergic neurons were located by screening cultures with whole cell recordings and investigating the presence of autaptic IPSCs. Single control stimulation pulses (to 0 mV for 3 ms) were delivered by
a pulse-generator (Master 8, AMPI) to the presynaptic neuron at 0.2 Hz,
and stimulus trains were delivered at 40 or 80 Hz for 1 or 2 s.
Whole cell currents were low-pass filtered at 10 kHz, monitored on a
penwriter (Servogor 220), digitized using an AD-converter (Instrutech
VR 100 B) and stored on a VTR and a Pentium PC equipped with Clampex
(pClamp v. 6.0. software, Axon Instruments).
Drug application
Active substances were dissolved as stock solutions in distilled
water at 1,000 times the final concentration and frozen. These were
diluted in extracellular medium just before use and perfused through
the bath (exchange time 2-3 min). Nifedipine was prepared as a stock
solution dissolved in DMSO. When added to the control solution at its
final concentration of 0.1%, DMSO had no effect on the synaptic
transmission. ()BayK 8644 and isradipine were prepared as stock
solutions in 50% ethanol. Isradipine was applied from a three-barrel
gravity-feed pipette (tip opening ~200 µm), which allowed
application directly onto the neurons with exchange between control and
isradipine solutions in ~1 s. Perfusion of 10 µM bicuculline from
the third barrel rapidly abolished evoked IPSCs, showing that the
perfusion covered the synaptic field completely. Experiments with
dihydropyridines were performed in the dark. All changes in
[Ca2+]o were compensated by changes in
[Mg2+]o to keep the sum of divalent cations
at 5.0 mM.
Drugs and chemicals were purchased from Sigma, except CNQX and
DL-APV (Tocris Cookson), ()BayK 8644 (RBI), and
(±)-baclofen (a gift from Prof. Povl Krogsgaard-Larsen, Royal Danish
School of Pharmacy). Culturing media were purchased from GIBCO, except for FUDR, uridine and poly-D-lysine, which were purchased
from Sigma.
Analysis
IPSC amplitudes were measured on-line in Clampex, and off-line
using Clampfit (pClamp program suite, Axon Instruments). All IPSCs were
inspected visually and rejected if spontaneous activity disturbed the
measurements. Tetanic depression of IPSCs during train stimulation was
calculated as the percentage by which the plateau level was depressed
with respect to the peak, which in numerical terms is given by:
100 (Plateau/Peak · 100). The peak amplitude was taken as the
average of 10 single pretetanic IPSCs, including the 1st in the train.
The plateau was calculated as the average GABAA current
during the train in the interval spanning from 300 to 400 ms for
stimulation at 80 Hz, and 800 to 1,000 ms for stimulation at 40 Hz
(indicated by heavy bars in the figures). PTP is presented as the
percentage change in the amplitude of single IPSCs following the train
in relation to the pretetanic level. All data are presented as
means ± SE, with n indicating the number of pairs of
neurons tested. Paired t-tests were used to assess
differences between control and drug groups, and changes were
considered to be significant at P-values < 0.05.
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RESULTS |
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Paired whole cell recordings were used to record monosynaptic
GABAergic IPSCs in cultured hippocampal neurons in the presence of CNQX
(10 µM) and DL-APV (50 µM). The presynaptic GABAergic neuron and a postsynaptic neuron were both clamped at 70 mV
(Vh). IPSCs were evoked by stimulating the
GABAergic neuron with a step to 0 mV for 3 ms. In spite of inclusion of
high-energy phosphates (MgATP) in the intracellular media, minor
rundown of IPSC amplitudes was seen. We have measured rundown to be
~10% during the first 20 min of recording at low-frequency
stimulation (unpublished observation).
Effects of baclofen on tetanic depression and posttetanic potentiation of IPSCs
The release of GABA from cultured hippocampal neurons is modulated
by presynaptic tetanic stimulation (Jensen et al. 1998). During the tetanus, IPSCs display a use-dependent decrease in amplitude
(tetanic depression). Following the train, GABA release evoked by
low-frequency stimulation (0.2 Hz) is enhanced for ~60 s (PTP). We
examined the effect of lowering the probability of release on tetanic
depression and PTP of IPSCs in four pairs of neurons (Fig.
1). Tetanic stimulation (80 Hz for 1 s) of the presynaptic neuron was delivered in control solution and
subsequently during bath perfusion of baclofen (10 µM). Baclofen
depressed single IPSCs by 68.5 ± 4.7%. Tetanic depression was
20.0 ± 13.5% in the presence of baclofen, which was
significantly less than for the control (55.4 ± 11.8%;
P < 0.05). When IPSCs were normalized to the
pretetanic baseline level, PTP was found to be 116.9 ± 10.6% in
the presence of baclofen, which is 3.3 times larger than in control
solution (35.9 ± 10.4%, P < 0.01; (Fig.
1Bb).
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Effects of changed [Ca2+]o on PTP
Because baclofen inhibits Ca2+ influx through N- and
P/Q-type Ca2+ channels (Hirata et al. 1995),
we tested the effect of changing [Ca2+]o,
which will alter presynaptic Ca2+ influx through all VDCCs.
Perfusion of 1.2 mM Ca2+ (low Ca2+) depressed
the pretetanic IPSCs by 71%, whereas PTP was enhanced from 55.4 ± 15% to 97.1 ± 12% (Fig.
2A, P < 0.05, n = 5). Upon perfusing 4.0 mM Ca2+ (high
Ca2+), the pretetanic IPSCs were enhanced by 16%, whereas
PTP was reduced from 51.8 ± 7% to 29.4 ± 11% (Fig.
2B, P < 0.05, n = 5). These
results demonstrate an inverse relationship between release probability
and PTP. Figure 2C shows a comparison between the effects of
baclofen and low Ca2+ on IPSCs and PTP. Both treatments
depressed pretetanic IPSCs to the same extent (P > 0.05), indicating a similar depression of Ca2+ influx
through the Ca2+ channels coupled to the rapid vesicle
release. However, PTP was enhanced three times more by baclofen than by
low Ca2+ (P < 0.05). In addition to N- and
P/Q-type channels, low Ca2+ will decrease the influx
through L- and T-type VDCCs. Because the voltage dependency and kinetic
properties of T-type channels (Nooney et al. 1997
) make
it unlikely that these would make a marked contribution to presynaptic
Ca2+ influx during repetitive activity, we speculated that
L-type VDCCs could be involved.
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Effects of L-type Ca2+ channel modulators on PTP
The selective L-type VDCC antagonist nifedipine (10 µM) had no effect on pretetanic IPSCs (which decreased by 3.9 ± 7% and is comparable with the usual run-down), but tended to reduce tetanic depression, which was 56.9 ± 9% in nifedipine, and 38.1 ± 14% in control (P = 0.08, n = 6, Fig. 3). Nifedipine reduced maximal PTP to 46.8 ± 11% compared with 86.0 ± 24% in control (P > 0.05, Fig. 3Ca). Because the duration of PTP was shortened by nifedipine, we calculated the area under the curve for the 1st 10 posttetanic pulses (i.e., 45 s in the posttetanic period). Nifedipine depressed the PTP curve area to 40.5 ± 11% of the control area (P < 0.01; Fig. 3Cb). The area was still slightly depressed after washing (by 16.7 ± 9%).
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We also tested the effects of another selective L-type VDCC antagonist,
isradipine, which has been reported to have different effects on
hippocampal synaptic plasticity than nifedipine (Christie et al.
1997; Mulkey and Malenka 1992
). Isradipine (5 µM) had no effect on pretetanic IPSCs, which shows that neither
nifedipine nor isradipine had nonspecific blocking effects on N- and/or
P/Q-type VDCCs. When local perfusion with isradipine was started before the tetanic stimulation, the PTP curve area was depressed by 65.3 ± 7%, (P < 0.01, n = 5, Fig.
4Cb), which was
similar to nifedipine. This effect was due to a reduction in peak PTP
and a shortening of PTP duration. Although it is quite likely that
L-channel-mediated influx of Ca2+ occurs during the train
stimulation, this was ascertained by applying isradipine immediately
after the train. Isradipine then had no effect on PTP compared with the
control (P > 0.05, n = 2, Fig.
4Cb).
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Perfusion of isradipine during the tetanic stimulation caused tetanic depression to increase to 63.4 ± 5% compared with 45.0 ± 8% for the control (P < 0.01, n = 5). When results from isradipine and nifedipine experiments were pooled, L-channel blockade was found to enhance tetanic depression by 46% (from 41.2 ± 8% in control, to 60.0 ± 5% in the presence of L-channel blockers, P < 0.005).
Finally, the effect of the L-type Ca2+ channel
"agonist" ()BayK 8644 was tested on eight pairs of neurons.
(
)BayK 8644 (4 µM) had no effect on pretetanic IPSCs, or on PTP
evoked by a train of 40 Hz for 2 s (n = 3, not
shown). Because the process underlying PTP is nearly saturated when
evoked by 80 pulses (Jensen et al. 1998
), we halved the
tetanization to 40 Hz for 1 s (40 pulses), which evoked PTP of
31.6 ± 25%. (
)BayK 8644 then enhanced peak PTP to 60.2 ± 24% (P < 0.05, n = 5), without
causing major changes in its time course (Fig.
5B).
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DISCUSSION |
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PTP depends on presynaptic Ca2+ influx
During high-frequency stimulation, IPSCs show tetanic depression,
which is probably due to depletion of GABA-containing vesicles at the
active zones (Jensen et al. 1998). Upon cessation of the tetanus, there was sometimes an increased rate of spontaneous IPSCs
that lasted for 1-2 s (e.g., the neuron in Fig. 4A). This component of release has been analyzed in detail elsewhere
(Jensen et al. 1998
) and is caused by increased
[Ca2+]i in the boutons (Cummings et
al. 1996
). The increased [Ca2+]i
raises the probability of vesicle release and results in PTP of the
GABAAergic IPSCs (Jensen et al. 1998
),
similar to that seen at other central (Cummings et al.
1996
; Griffith 1990
) and peripheral
(Delaney and Tank 1994
; Tang and Zucker
1997
) synapses. PTP is related to the pretetanic release
probability and is correspondingly modulated by changes in
[Ca2+]o: in low Ca2+, single
pretetanic IPSCs are depressed relatively more than the posttetanic
IPSCs, thereby enhancing PTP (Fig. 2A). The reverse is found
on increasing [Ca2+]o, which enhances single
IPSCs and reduces PTP (Fig. 2B). Presynaptic GABAB receptors are negatively coupled by a G-protein
mechanism to Ca2+ channels that trigger vesicle release
(Lambert and Wilson 1996
; Thompson et al.
1993
). When these are activated by baclofen, a qualitatively
similar effect to that of low Ca2+ was observed. These
results demonstrate that PTP of IPSCs is inversely related to the
probability of release.
Involvement of presynaptic L-type Ca2+ channels in PTP
In neuronal somata, baclofen preferentially depresses N-type
Ca2+ channels (Lambert and Wilson 1996;
Scholz and Miller 1991
), whereas the release-depressant
action of baclofen on GABAergic nerve cells involves an inhibition of
N- and P/Q-type Ca2+ channels located at the secretory
apparatus (Doze et al. 1995
; Ohno-Shosaku et al.
1994
). Our experiments on L-type VDCCs were prompted by the
quantitative discrepancy between the effect of low Ca2+ and
baclofen on PTP (Fig. 2C), which indicated that
baclofen-insensitive Ca2+ channels also participate in PTP.
That presynaptic L-type VDCCs contribute to the generation of PTP was
demonstrated by the finding that both nifedipine and isradipine reduced
peak PTP to a similar extent and shortened its duration. Neither
blocker affected pretetanic IPSCs, confirming that L-type channels do
not contribute to the Ca2+ influx responsible for
transmitter release in response to a single stimulus (Wheeler et
al. 1994
). Accordingly, it has been suggested that L-type
Ca2+ channels are located at some distance from the
neurotransmitter release site (Miller 1987
). We have
also shown that the Ca2+ influx through L-channels
supporting PTP occurs during, and not after, the train. This correlates
well with the finding that the L-channels blockers increased tetanic depression.
The involvement of L-type channels was further substantiated by the
finding that the selective L-type VDCC "agonist," BayK 8644 increased PTP. BayK 8644 increases the probability of L-type channel
openings, causes a negative shift in the voltage dependency so that
channels open at more hyperpolarized potentials, and increases the
number of available channels (Fisher et al. 1990). BayK
8644 increased PTP evoked by a submaximal stimulation (40 pulses), but
not PTP evoked by a stronger stimulation (80 pulses). A likely explanation is that PTP evoked by 80 pulses is already saturated (Jensen et al. 1998
), and cannot be potentiated further
by increased L-type VDCC activity or Ca2+ influx in general.
L-type Ca2+ channels therefore make a substantial
contribution to the Ca2+ influx during tetanic stimulation.
Despite the presence of endogenous and exogenous [i.e., EGTA or
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA)] Ca2+ buffers in the presynaptic neuron
(unpublished observations), this raises the Ca2+ level in
the boutons and thereby the subsequent probability of release. L-type
Ca2+ channels have a relatively large unitary conductance
(25 pS) (Fisher et al. 1990), and, although these
channels seem to be located mainly at neuronal somata and dendrites
(Westenbroek et al. 1990
), a small number of channels at
or near the synaptic boutons could give rise to an appreciable increase
in [Ca2+]i, because of the high surface
area:volume ratio.
Because hippocampal GABAergic terminals are not accessible for direct patch-clamp recording, it is not possible to record single L-type Ca2+ channel activity at this location. However, our data would indicate that functional L-type channels are present in GABAergic boutons and indeed play an important role in the regulation of transmitter release. Our results also indicate that presynaptic L-type VDCCs are not, to any large extent, inhibited by baclofen, because we found that baclofen-insensitive Ca2+ channels participated in PTP.
Other evidence supporting the presence of L-type VDCCs in cultured
nerve terminals is that stimulus-evoked Ca2+ rises are only
reduced by 40-70% by a combination of -conotoxin GVIA,
-agatoxin IVA, and
-conotoxin MVIIC, which together block N-, P-,
and Q-type channels (MacKenzie et al. 1996
). If L-type channels make a substantial contribution to the residual 30-60% of
the Ca2+ rise, L-type antagonists would cause a
corresponding reduction in the total Ca2+ influx. We are
currently using high resolution imaging techniques to see whether this
can be detected.
Conclusions
The data presented here indicate that L-type Ca2+ channels contribute to Ca2+ accumulation in the presynaptic terminals during high-frequency activity in cultured GABAergic neurons, and thereby enhance transmitter release in posttetanic period. This represents a novel presynaptic regulatory mechanism at a fast CNS synapse. At the ultrastructural level, it will be interesting to search for L-type Ca2+ channels at GABAergic and glutamatergic synaptic boutons.
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ACKNOWLEDGMENTS |
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We are grateful to P. Krogsgaard-Larsen (Royal Danish School of Pharmacy) for a gift of baclofen. We thank K. Kandborg for preparation of the cultures and S. Kristensen for technical help.
We thank the Danish Medical Research Council and Aarhus Universitets Forsknings Fond for financial support.
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FOOTNOTES |
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Address for reprint requests: J.D.C. Lambert, Dept. of Physiology, University of Aarhus, Ole Worms Alle 160, DK-8000 Aarhus C, Denmark.
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 28 May 1998; accepted in final form 8 November 1998.
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REFERENCES |
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