Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
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ABSTRACT |
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Evans, D. Ieuan,
Roland S. G. Jones, and
Gavin Woodhall.
Activation of Presynaptic Group III Metabotropic Receptors
Enhances Glutamate Release in Rat Entorhinal Cortex.
J. Neurophysiol. 83: 2519-2525, 2000.
The role of group
III metabotropic glutamate receptors (mGluRs) in modulating excitatory
synaptic transmission was investigated in the rat entorhinal cortex
(EC) in vitro. AMPA receptor-mediated excitatory postsynaptic currents
(EPSCs) were recorded in the whole cell configuration of the
patch-clamp technique from visually identified neurons in layers V and
II. In layer V, bath application of the specific group III mGluR
agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 500 µM) resulted
in a marked facilitation of both spontaneous and activity-independent
"miniature" (s/mEPSC) event frequency. The facilitatory effect of
L-AP4 (100 µM) on sEPSC frequency prevailed in the presence of
DL2-amino-5-phosphonopentanoic acid (100 µM) but was
abolished by the group III antagonist
(RS)-cyclopropyl-4-phosphonophenylglycine (20 µM). These data
confirmed that group III mGluRs, and not
N-methyl-D-aspartate (NMDA) receptors were
involved in the response to L-AP4. Bath application of the specific
mGluR4a agonist (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid
(20 µM) also had a facilitatory effect on sEPSC frequency, suggesting
involvement of mGluR4a. In layer II neurons, L-AP4 caused a reduction
in sEPSC frequency but did not affect mEPSCs recorded in the presence
of tetrodotoxin. These findings suggest that a group III mGluR with
mGluR4a-like pharmacology is involved in modulating synaptic
transmission in layer V cells of the EC. The effect on mEPSCs suggests
that this receptor is located presynaptically and that its activation
results in a direct facilitation of glutamate release. This novel
facilitatory effect is specific to layer V and, to our knowledge, is
the first report of a direct facilitatory action of group III mGluRs on
synaptic transmission. In layer II, L-AP4 had an inhibitory effect on
glutamate release similar to that reported in other brain regions.
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INTRODUCTION |
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There is evidence from both basic and clinical
studies that the entorhinal cortex (EC) may be an important site of
seizure generation in temporal lobe epilepsy (e.g., Bear and
Lothman 1993; Bertram et al. 1990
; Du et
al. 1995
; Jones and Lambert 1990a
,b
; Jones et al. 1988
; Lothman et al. 1990
;
Siegel et al. 1990
; Spencer and Spencer
1994
; Sperling et al. 1996
). Experimental
studies of acutely provoked epileptogenesis in brain slices have
provided evidence that epileptiform discharges were likely to be
initiated among cells of the deep layers (IV-VI) and to propagate from
there into the superficial layers (II-III) and into the hippocampus (Jones and Lambert 1990a
,b
). This has led to the
suggestion that the deep layers may be "seizure sensitive" and the
superficial layers relatively "seizure resistant" (Jones
1993
; however, see Dickson and Alonso 1997
). A
major focus of interest in this laboratory has been to attempt to
identify characteristics of glutamate and GABA-mediated synaptic
transmission in the different layers that could underlie these
differing susceptibilities.
One approach we have taken is to examine spontaneous release of the
excitatory and inhibitory transmitters using whole cell patch-clamp
recordings. These studies have demonstrated that there are differences
in both spontaneous excitation (Berretta and Jones 1996a) and inhibition (Wood and Jones 1999
),
which could contribute to an increased susceptibility of the deep
layers to epileptogenesis. As a progression from these studies, we have
become interested in the potential relationship between the complement
of presynaptic receptors and control of transmitter release in deep and
superficial layers. For example, we have demonstrated the presence of
presynaptic N-methyl-D-aspartate (NMDA)
autoreceptors in the EC, which tonically facilitate glutamate release
(Berretta and Jones 1996b
). We have also shown that
spontaneous release of GABA in the EC is controlled by presynaptic
GABAB receptors (Wood et al.
1999
). In the current paper, we have turned our attention to
the control of glutamate release at EC synapses by metabotropic
glutamate receptors (mGluRs).
Metabotropic glutamate receptors comprise a family of at least eight
receptors coupled to heterotrimeric G proteins that, in turn, are
linked to a variety of second-messenger systems. They have been
classified into three groups based originally on their amino acid
sequence homology. Group III contains mGluRs 4, 6, 7, and 8, and
activation of these receptors has been reported to depress
glutamatergic transmission in cultures of rat olfactory bulb
(Trombley and Westbrook 1992), hippocampus
(Baskys and Malenka 1991
; Koerner and Cotman
1981
), hippocampal cultures (Forsythe and Clements
1990
), spinal cord (Davies and Watkins 1982
),
locus coeruleus (Dube and Marshall 1997
), and visual
cortex (Jin and Daw 1998
). The inhibitory effect of the
group III mGluRs is thought to arise through a negative coupling to
adenylyl cyclase. This, in turn, causes inhibition of calcium currents
in the presynaptic terminal (Glaum and Miller 1995
;
Takahashi et al. 1996
) and subsequent inhibition of
glutamate release. An alternative model has been provided by
O'Connor et al. (1999)
, in which group III mGluR
activation releases
subunits to mediate glutamate autoinhibition.
Given the widespread demonstration that group III mGluRs exert an
inhibitory effect on glutamate release in the CNS, we hypothesized that
these receptors also may depress release in the EC and that their
influence might be less in the seizure sensitive deeper layers. In
fact, although we found the expected inhibition of release in layer II,
in layer V activation of group III receptors caused a powerful
facilitation of glutamate release. This unexpected effect, which we
linked to the mGluR 4 receptor, could contribute to the observed
susceptibility of layer V to epileptogenesis. Some of this work has
been presented in abstract form (Evans et al. 1999).
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METHODS |
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Combined hippocampal-EC slices were prepared from male Wistar
rats (50-110 g) as previously described (Jones and Heineman 1988). In brief, rats were anesthetized with an intramuscular injection of ketamine and decapitated. The brain was removed rapidly and immersed in oxygenated artificial cerebrospinal fluid (ACSF) chilled to 4°C. Slices (400 µm) were cut using a vibroslice
(Campden Instruments), and stored in ACSF continuously bubbled with
95% O2-5% CO2, maintained
at room temperature. After a recovery period of
1 h, individual
slices were transferred to a recording chamber mounted on the stage of
an upright Olympus BX50WI microscope. The chamber was perfused
continuously with oxygenated ACSF at 30°C at a flow rate of ~2
ml/min. The perfusate contained the following (in mM): 126 NaCl, 4 KCl,
1.25 NaH2PO4, 24 NaHCO3, 2 MgSO4, 2.5 CaCl2, and 10 D-glucose. The solution
was continuously bubbled with 95% O2-5%
CO2 to maintain a pH of 7.4.
Patch-clamp electrodes were pulled from borosilicate glass (1.2 mm OD,
0.60 ID, Clark Electromedical) and had open tip resistances of 4-5
M. They were filled with a solution containing the following (in
mM): 130 Cs-methanesulphonate, 10 HEPES, 5 QX-314, 0.5 EGTA, 1 NaCl,
0.34 CaCl2, 1 MK801, 4 ATP, and 0.4 GTP. The
solution was adjusted to 290 mOsmol and pH 7.3 with CsOH. Neurons lying within the medial EC were visualized in slices using differential interference contrast optics and an infrared video camera. No distinction was made between slices taken from more dorsal or ventral
areas. Whole cell voltage-clamp recordings were made using an Axopatch
200B amplifier (Axon Instruments) and neurons were clamped at
60 mV.
Signals were filtered at 2 kHz and digitized at 20 kHz. Access
resistance was monitored at regular intervals, and cells were rejected
if this parameter changed by 15%. Data were recorded directly to
computer hard disk using Axoscope software (Axon Instruments). Under
the conditions of these experiments, EC neurons exhibited excitatory
postsynaptic currents (EPSCs), mediated by spontaneous release of
glutamate acting at AMPA receptors (see Berretta and Jones
1996a
). Analysis of spontaneous events was carried out off-line
using Minianal software (Jaejin). EPSCs were detected automatically
using a threshold-crossing algorithm, and their frequency, amplitude,
and kinetic parameters were analyzed. At least 100 events were analyzed
for each cell under each condition. A paired Student's
t-test was used for comparison of EPSC amplitudes, and the
nonparametric Kolmogorov-Smirnoff test was used to assess the
significance of shifts in cumulative probability distributions of
interevent interval (IEI) (Van der Kloot 1991
). For
comparison of amplitude distributions, histograms were constructed with
events categorized into 2-pA bins. All error values stated in the text refer to ±SE.
(+)-MK801 maleate, L(+)-2-amino-4-phosphonobutyric acid (L-AP4),
(RS)-cyclopropyl-4-phosphonophenylglycine (CPPG),
DL2-amino-5-phosphonopentanoic acid (2-AP5), and
(1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1) were
obtained from Tocris Cookson. Tetrodotoxin (TTX) was obtained from
Alomone Labs, Jerusalem, Israel. All drugs were applied by inclusion in
the bath perfusion medium.
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RESULTS |
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Results reported here are based on recordings obtained from 43 layer V neurons and 8 layer II neurons. With MK-801 in the pipette
solution to block postsynaptic NMDA receptors, spontaneous AMPA-receptor-mediated EPSCs (sEPSCs) were recorded as inward currents
in neurons voltage-clamped at 60 mV. sEPSCs recorded from layer V
neurons had a mean frequency and amplitude of 1.1 ± 0.01 Hz and
10.4 ± 0.3 pA, respectively, whereas sEPSCs occurred in layer II
neurons at a frequency of 1.20 ± 0.04 Hz and had a mean amplitude
of 7.5 ± 0.1 pA.
L-AP4 increases sEPSC frequency in layer V
In layer V neurons, bath application of the group III mGluR agonist L-AP4 (500 µM) caused a marked increase in sEPSC frequency. An example of this effect in one neuron is shown in Fig. 1A. Analysis of pooled data from five cells indicated that mean EPSC frequency was 1.11 ± 0.01 Hz before and 2.20 ± 0.01 Hz during application of the agonist. Figure 1B shows pooled data illustrating a leftward shift in the cumulative probability curve for IEIs, reflecting the increase in frequency, and this change was shown to be statistically significant (P < 0.001, KS). In the presence of L-AP4 there was a shift toward larger events, such that mean peak amplitude was 10.4 ± 0.3 pA before and 12.8 ± 0.3 pA after the application of L-AP4 (P < 0.001, t-test). The amplitude distribution plots in Fig. 1C show an increased "tail" region to the right of the peak in the presence of L-AP4. Analysis of the kinetics of sEPSCs in the presence of L-AP4 revealed no significant change in rise time. The decay time was very slightly decreased, and this change just reached significance (Table 1).
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L-AP4 increases frequency of mEPSCs
Spontaneous synaptic release of transmitters consists of two components; some vesicles are released as a result of action potentials invading the synaptic terminals, whereas others are released independent of such activity. To determine if the change in frequency of events induced by L-AP4 reflected a direct modulation of release at terminals or an effect that altered the frequency of spontaneous action potentials in the presynaptic neuron, we assessed its effect on activity-independent miniature EPSCs (mEPSCs) recorded in the presence of the voltage-activated sodium channel blocker, TTX (1.25 µM). mEPSCs result from the random monoquantal release of glutamate from presynaptic terminals, and thus any change in the frequency of these events indicates a direct effect on the presynaptic terminal.
In eight layer V cells, perfusion with L-AP4 resulted in a marked increase in mEPSC frequency. In these neurons, the mean frequency was 1.6 ± 0.02 Hz before application of the agonist and 2.7 ± 0.02 Hz after. The traces in Fig. 2A provide a clear illustration of this effect in one neuron. The graphs in Fig. 2, B and C, show pooled data from all eight cells. Again, the cumulative probability plot for IEI was shifted to the left (Fig. 2B), indicating an increase in mEPSC frequency (P > 0.0001, KS). However, in contrast to the situation where action potentials were intact, in TTX, L-AP4 caused no change in event amplitude (9.9 ± 0.2 pA before and 10.2 ± 0.2 pA during perfusion of L-AP4, P > 0.1, t-test). Figure 2C shows that there was no increase in the tail of larger amplitude mEPSCs in the presence of L-AP4. L-AP4 did not significantly affect the rise time of mEPSCs (see Table 1). Again, there was a slight decrease in the decay time of mEPSCs, but in this case the change was not significant.
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NMDA receptor blockade does not prevent the facilitatory action of L-AP4
It has been reported that L-AP4 at concentrations in excess of 250 µM is a weak agonist at NMDA receptors (Davies and Watkins 1982), and we have demonstrated previously that glutamate
release in the EC is facilitated by presynaptic NMDA receptors
(Berretta and Jones 1996b
). To rule out the possibility
that the L-AP4-induced facilitation of EPSCs was due to activation of
NMDA receptors, we repeated the experiments in the presence of the NMDA
receptor antagonist 2-AP5 (100 µM). We also increased the rigor of
these experiments by using a lower concentration of L-AP4 (100 µM). Under these conditions, in 6/6 cells L-AP4 still caused a rapid and
reversible increase in sEPSC frequency that was indistinguishable from
that described in the absence of 2-AP5 (Fig.
3A). In these experiments, the
frequency of events was substantially lower than when 2-AP5 was not
present (0.59 ± 0.02 Hz). This reflects the removal of the tonic
facilitatory effect mediated by the presynaptic NMDA receptor
(Berretta and Jones 1996b
). The addition of L-AP4 increased event frequency (to 2.11 ± 0.15 Hz); analysis of IEI revealed that the increase was significant compared with control (Fig.
3B, P < 0.001, KS). There was no shift in
amplitude distribution, although an increase in the number of small
events resulted in a decreased mean peak amplitude (9.7 ± 0.2 to
8.8 ± 0.1 pA, P < 0.001, t-test).
These data suggest that the effect of L-AP4 in layer V was due to mGluR
activation alone.
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CPPG blocks the facilitatory effect of L-AP4 on mEPSCs
To confirm that the mGluR responsible for the L-AP4-induced
facilitation of mEPSC frequency belongs to group III, L-AP4 was applied
in the presence of the specific group III antagonist CPPG (20 µM,
n = 6) (Toms et al. 1996). With CPPG in
the bath, the mean frequency and amplitude of sEPSCs was 1.10 ± 0.02 Hz and 9.53 ± 0.37 pA. Subsequent addition of L-AP4 failed
to alter either frequency (1.17 ± 0.01 Hz) or amplitude
(8.83 ± 0.28 pA). However, in four of the six cells tested, after
prolonged washout of CPPG, the facilitatory effect of L-AP4 could be
observed. Figure 4A illustrates one study of the effect of CPPG, and B and
C show pooled data from six cells. There was no significant
shift in either IEI or amplitude distribution. These results confirm
that a group III mGluR is responsible for the facilitation of mEPSCs caused by L-AP4. It should also be noted that bath application of CPPG
alone did not cause any significant change in mEPSC amplitude or
frequency, suggesting that there is little tonic activation of group
III mGluRs by ambient glutamate at this synapse.
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ACPT-1 facilitates EPSCs in layer V
With the development of more selective receptor ligands it
has become possible to dissect further the complement of mGluRs present
at a given synapse. In 14 cells, bath application of the specific
mGluR4a agonist ACPT-1 (20 µM) (Acher et al. 1997)
caused a rapid and reversible increase in sEPSC frequency in layer V (Fig. 5A) that was similar to
that observed with L-AP4. Mean frequency was 4.10 ± 0.01 Hz
before and 7.67 ± 0.01 Hz after ACPT-1 application (P < 0.001, KS test). ACPT-1 also caused a significant
change in mean event amplitude from 12.4 ± 0.2 to 13.7 ± 0.2 pA (P < 0.001 t-test). Within a given
epoch, an increase in the number of events was seen in most amplitude
bins such that there was no overall shift in the peak of the pooled
amplitude distribution curve (Fig. 5). Analysis of event kinetics
(Table 1) revealed that ACPT-1 caused a very small but significant
increase in the rise time of events. The magnitude of this change (100 µS) relative to the overall rise time suggests that this may not be
of great importance in functional terms. There was no change in decay
kinetics.
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L-AP4 inhibits sEPSCs in layer II
The weight of evidence from previous studies has suggested that group III mGluRs have a depressant effect on glutamate release, and the facilitatory effect of group III agonists in layer V was surprising. However, in 4/4 neurons recorded in layer II of EC, we saw a decrease in sEPSC frequency on bath application of 500 µM L-AP4. The mean frequency in control conditions was 1.19 ± 0.04 Hz, and this decreased to 0.83 ± 0.03 Hz in the presence of L-AP4. Figure 6A shows a shift to the right of the IEI distribution curve that was significant (P < 0.001, KS). As Fig. 6B demonstrates, there was no significant change in sEPSC amplitude (P > 0.1, t-test). The effect was not observed when L-AP4 was applied in the presence of TTX (n = 4, Fig. 7). Mean mEPSC frequency was 1.23 ± 0.03 Hz under control conditions and 1.24 ± 0.03 Hz after application of L-AP4 (P > 0.1 KS). Again there was no significant change in mean amplitude (P > 0.1, t-test).
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DISCUSSION |
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The principal finding of this study was that group III mGluRs modulate glutamate release from synapses onto cells of layers V and II of the EC. In layer V, activation of these receptors by the group III agonist L-AP4 caused a marked increase in the frequency of spontaneous EPSCs and an increase in the mean amplitude of events. When action potentials were blocked using TTX, mEPSC frequency was increased by L-AP4, but amplitude remained unchanged. These data, where frequency alone was altered, indicate that L-AP4 acts presynaptically to facilitate glutamate release. In the absence of TTX, the shift in amplitude distribution toward larger events suggests that group III mGluRs also might be present on the postsynaptic cell. However, it is equally possible that these observations are related either to an increase in the number of vesicles released per action potential or in the likelihood of synchronous release.
To confirm that a group III mGluR was responsible for the effects of
L-AP4, it was necessary to exclude the action of this ligand at
presynaptic NMDA receptors (Berretta and Jones 1996b) and to attempt to antagonize the effect of L-AP4 using a group III
antagonist. We used the NMDA receptor antagonist 2-AP5 to block NMDA
receptors, and the increased event frequency induced by L-AP4 was
indistinguishable from that observed without 2-AP5 in the bath. The
decrease in basal frequency of events on application of 2-AP5 confirmed
that NMDA receptors were blocked before the application of L-AP4.
The mGluR antagonist CPPG abolished the action of L-AP4, indicating
that the receptor involved belongs to group III. Using the specific
mGluR4a agonist ACPT-1, we have demonstrated a facilitatory effect on
frequency of EPSCs that was similar to that of L-AP4. In both cases,
there was an approximate doubling of the observed frequency. It is
probable then, that L-AP4 exerts its facilitatory effect by activation
of mGluR4a receptors. In support of this hypothesis, there are
immunocytochemical data demonstrating that mRNA for mGluR4 is highly
expressed in the EC (Thomsen et al. 1992), evidence that
these receptors are likely to be located presynaptically
(Forsythe and Clements 1990
) and that they are L-AP4-sensitive (Eriksen and Thomsen 1995
).
Our data suggest that L-AP4 exerts its facilitatory action via an
effect on mGluRs located directly on the excitatory presynaptic terminals in layer V. Consistent with a presynaptic locus of action for
L-AP4 are immunocytochemical data, which have also localized group III
mGluRs to the presynaptic terminal (Bradley et al. 1996; Kinoshita et al. 1996
; Shigemoto et al.
1997
). The group III antagonist CPPG had no effect when applied
alone, suggesting that these receptors are not tonically active under
normal conditions. It might be expected then that these receptors
become activated only during periods when the concentration of
glutamate in the cleft is elevated, resulting in a positive feedback
effect to further elevate glutamate release. It has been shown recently
that sufficient glutamate is released during epileptiform bursts to
activate mGluRs (Merlin et al. 1995
). Because it has
been established that layer V is more susceptible to seizure-like
activity than layer II (Jones and Lambert 1990a
;
Nagao et al. 1996
), it is possible that this seizure
sensitivity could be related, in part, to a positive feedback exerted
on glutamate release by group III mGluRs. This possible mechanism for
generation of local network instability and its potential for
propagation to other regions are currently under investigation in our laboratory.
The facilitatory effect in the EC raises questions with respect to the
identity of the receptor, its G-protein coupling and downstream
effectors. It is now becoming clear that the sequelae to mGluR
activation are more varied and numerous than was at first appreciated.
For example, a potential link has been demonstrated between mGluRs and
protein kinase G in medullary neurons (Glaum and Miller
1995), and mGluR1 has been linked to non-G-protein effects via tyrosine kinases (Heuss et al. 1999
). Thus
mGluR actions are not limited to the classical changes in
IP3 and cAMP levels observed when cloned mGluRs
first were characterized. Indeed, mGluRs inserted into expression
systems may couple nonspecifically to signal transduction pathways
available within that system (Gabellini et al. 1994
). In
this scenario, the fact that the L-AP4 receptor described here
enhances, rather than depresses, glutamate release is perhaps less
surprising. For example, there is good evidence that G-protein
subunits are involved in group III mGluR mediated inhibition of
glutamate release (O'Connor et al. 1999
). This
mechanism depends on activation of calmodulin, which binds in a
mutually exclusive manner with
subunits at a critical subdomain
in the carboxy-terminal tail of group III mGluRs. It is well known that mGluRs are heavily edited, and thus quite plausible that a modification of the carboxy-terminal tail of mGluR4a could alter receptor-effector linkage and hence overall receptor function.
The novelty of the facilitatory effect of L-AP4 in layer V was
reinforced by our data from layer II neurons where L-AP4 had the
inhibitory effect on sEPSCs seen in many other areas. This suggests
that the group III mGluRs demonstrated in layer V are a different
subtype to those in layer II or that they differ in terms of the
specific intracellular coupling cascade. The lack of an inhibitory
effect of L-AP4 on release in layer II in the presence of TTX is
consistent with the previously described mechanism by which group III
mGluRs suppress presynaptic calcium channels activated during
spontaneous transmitter release (Takahashi et al. 1996).
To summarize, we have presented data that show that group III mGluR activation results in a novel facilitatory effect on synaptic transmission in neurons of layer V of the EC and that this action is mediated by a receptor with mGluR4a pharmacology. This novel action suggests that the receptor involved has a different G-protein/effector coupling than those previously described or that the effect of receptor activation is dependent on its location within the CNS. It is plausible that activation of this receptor plays a role in epileptogenesis in the EC and therefore could be an important therapeutic target with potential for further investigation.
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ACKNOWLEDGMENTS |
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The authors thank The Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and the Taberner Trust for financial support.
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FOOTNOTES |
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Address reprint requests to G. Woodhall.
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 27 October 1999; accepted in final form 13 January 2000.
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REFERENCES |
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