GABAB Receptors Couple to Potassium and Calcium Channels on Identified Lateral Perforant Pathway Projection Neurons

Xueyong Wang and Nevin A. Lambert

Department of Pharmacology and Toxicology, Medical College of Georgia, and Medical Research Service, Veterans Administration Medical Center, Augusta, Georgia 30912-2300


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Wang, Xueyong and Nevin A. Lambert. GABAB Receptors Couple to Potassium and Calcium Channels on Identified Lateral Perforant Pathway Projection Neurons. J. Neurophysiol. 83: 1073-1078, 2000. Activation of presynaptic GABAB receptors inhibits neurotransmitter release at most cortical synapses, at least in part because of inhibition of voltage-gated calcium channels. One synapse where this is not the case is the lateral perforant pathway synapse onto dentate granule cells in the hippocampus. The current study was conducted to determine whether the neurons that make these synapses express GABAB receptors that can couple to ion channels. Perforant pathway projection neurons were labeled by injecting retrograde tracer into the dorsal hippocampus. The GABAB receptor agonist baclofen (10 µM) activated inwardly rectifying potassium channels and inhibited currents mediated by voltage-gated calcium channels in retrogradely labeled neurons in layer II of the lateral entorhinal cortex. These effects were reversed by coapplication of the selective GABAB receptor antagonist CGP 55845A (1 µM). Equivalent effects were produced by 100 µM adenosine, which inhibits neurotransmitter release at lateral perforant pathway synapses. The effects of baclofen and adenosine on inward currents were largely occlusive. These results suggest that the absence of GABAB receptor-mediated presynaptic inhibition at lateral perforant pathway synapses is not simply due to a failure to express these receptors and imply that GABAB receptors can either be selectively localized or regulated at terminal versus somatodendritic domains.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of presynaptic G protein-coupled receptors (GPCRs) inhibits neurotransmitter release at many synapses (Miller 1998; Thompson et al. 1993; Wu and Saggau 1997). This inhibition is often mediated by a decrease in presynaptic calcium influx resulting from inhibition of presynaptic voltage-gated calcium channels (Dittman and Regehr 1996; Wu and Saggau 1997). The large majority of central presynaptic terminals contain G protein subunits that can inhibit the calcium channels responsible for neurotransmitter release. This leaves a limited number of mechanisms whereby neurons can avoid presynaptic inhibition mediated by GPCRs. A neuron can simply fail to express a particular GPCR protein, or selective targeting or regulation could allow neurons to use receptors at some membrane domains, but not at presynaptic terminals. The former mechanism appears to be widely used. GPCRs are often expressed in distinct populations of neurons, and inhibition of calcium channels in neuron cell bodies is often predictive of inhibition in presynaptic terminals. In contrast, there are relatively few instances of subcellular utilization of GPCRs in neurons. For example, structural and functional evidence suggests that metabotropic glutamate receptors (mGluRs) can be selectively targeted to or used at subcellular membrane domains (Scanziani et al. 1998; Shigemoto et al. 1996; Stowell and Craig 1999).

GABAB receptors are the G protein-coupled metabotropic receptors for GABA (Bettler et al. 1998; Möhler and Fritschy 1999; Mott and Lewis 1994). Similar to other GPCRs that couple to pertussis toxin-sensitive G proteins, activation of GABAB receptors activates inwardly rectifying K+ (GIRK) channels and inhibits voltage-gated calcium channels. These receptors are ubiquitously expressed in cortical neurons, and activation of presynaptic GABAB receptors inhibits neurotransmission at most cortical synapses (Mott and Lewis 1994). However, a few cortical synapses are unaffected by GABAB receptor agonists. One such synapse is the lateral perforant pathway (LPP) synapse made by neurons in layer II of the lateral entorhinal cortex on the distal dendrites of granule cells in the dentate gyrus (Ault and Nadler 1983; Brunner and Misgeld 1993; Lanthorn and Cotman 1981; Mott and Lewis 1994). Dentate granule cells also receive input from the medial entorhinal cortex through the medial perforant pathway (MPP). These synapses, unlike LPP synapses, are inhibited by activation of GABAB receptors. The purpose of this study was to ask whether lateral perforant path projection neurons express functional GABAB receptors. The results indicate that these neurons express GABAB receptors that couple to potassium channels and voltage-gated calcium channels. Coupling to ion channels in these neurons by GABAB receptors is as effective as coupling by adenosine receptors, activation of which inhibits neurotransmitter release at LPP synapses (Kahle and Cotman 1993). These neurons thus possess the signal transduction machinery required for presynaptic inhibition mediated by GABAB receptors but avoid using this mechanism at their synapses in the dentate gyrus. This suggests that GABAB receptors, similar to other G protein-coupled receptors, are selectively targeted to or regulated at subcellular membrane domains.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Retrograde labeling

Sprague-Dawley rats (10-14 days old) were placed in a stereotaxic frame under barbiturate anesthesia. Approximately 0.5-1.0 µl of a 5% solution of 3,3'-dioctadecyloxacarbocyanine perchlorate (diO; dissolved in dimethylformamide) or of a 5% suspension of fluorescent polystyrene microspheres (40-nm diam; Molecular Probes) was injected into the dorsal hippocampi bilaterally through a glass pipette. Wounds were closed with sutures, and animals were allowed to recover from anesthesia (body temperature maintained with a heating pad) and then returned to their home cages. Fluorescently labeled neurons were evident as early as 24 h after the injection. Neurons labeled with diO contained punctate fluorescent inclusions (Fig. 1). Relatively fast, punctate labeling suggests that diO-labeled membrane was internalized and transported to the cell body by retrograde transport (Honig and Hume 1989).



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Fig. 1. Recording from identified lateral perforant pathway projection neurons. Left: low-power image of a coronal slice of lateral entorhinal cortex showing a band of labeled neurons in layer II ventral to the rhinal sulcus (RS) several days after injection of 3,3'-dioctadecyloxacarbocyanine perchlorate (diO) into the ipsilateral dorsal hippocampus. This slice was fixed in paraformaldehyde and cleared with glycerol before imaging; fluorescent and transmitted images are shown superimposed. Top right: higher power image of fluorescently labeled neurons in the same region of another slice during recording. Bottom right: same high-power field imaged using oblique transillumination with near-infrared light. A patch pipette is attached to one of the retrogradely labeled neurons. Punctate fluorescence of labeled neurons after 24 h indicates that diO was internalized and transported by retrograde transport, rather than by diffusion within the lipid bilayer.

Electrophysiology

Between 3 and 14 days after injection, coronal slices of entorhinal cortex (400 µm) were prepared and maintained at 22°C in artificial cerebrospinal fluid (ACSF) that contained (in mM) 125 NaCl, 25 NaHCO3, 3.3 KCl, 2 MgCl2, 2 CaCl2, and 20 D-glucose and was oxygenated with 95% O2-5% CO2. This solution was used for recording with various additions or substitutions as indicated: 2 mM BaCl2, 0.5 µM TTX, and 100 µM CdCl2. Slices containing the lateral entorhinal area were placed on the stage of an Olympus BX50WI microscope and epi-illuminated with a fluorescein filter set (XF22, Omega Optical). Retrogradely labeled neurons were imaged using a ×40 water-immersion objective, a cooled charge-coupled device (CCD) camera (LSR Olympix) and a CCD video camera (Dage-MTI). Images taken with fluorescence illumination and near-infrared transillumination were superimposed to ensure that labeled neurons were selected for recording (Fig. 1). In control experiments we found that prolonged illumination with the excitation wavelength (475-500 nm) had no obvious effect on the physiological properties of labeled neurons. Nonetheless, we illuminated neurons only briefly (a few seconds) before recording. Whole-cell recordings were made using pipettes containing (in mM) either 120 potassium gluconate, 20 KCl, 10 HEPES, 10 EGTA, 14 phosphocreatine, 4 MgATP, and 0.3 tris GTP, or the same solution with cesium salts substituted for potassium gluconate and KCl (pH adjusted with KOH or CsOH, respectively). Series resistance compensation was not used. Voltage commands were delivered and data were acquired using a multifunction input-output board (National Instruments) and WCP software (John Dempster, Strathclyde University, Strathclyde, UK). Slices were constantly perfused with ACSF (~2 ml/ min). Drugs were added directly to the perfusing solution. All chemicals were from Sigma, with the exception of TTX (Calbiochem) and CGP 55845A, which was a gift from Novartis Pharma.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of lateral perforant pathway projection neurons

Injection of diO or fluorescent polystyrene microspheres into the dorsal hippocampus reliably labeled a distinct band of neurons in the superficial layers of the entorhinal cortex. As previously reported (Dolorfo and Amaral 1998; Hjörth-Simonsen and Jeune 1972; Steward and Scoville 1976), two groups of retrogradely labeled neurons could be identified with this injection site: one group just ventral to the rhinal sulcus in the lateral entorhinal area (Fig. 1) and a second group in the medial entorhinal area, which were most apparent when slices were made in the horizontal plane (not shown). Anterograde tracing studies indicate that the labeled neurons in the lateral entorhinal cortex project to the outer molecular layer of the dentate gyrus (Tamamaki 1997) and are thus the cells of origin of the lateral perforant pathway (LPP).

GABAB receptors couple to potassium channels on LPP projection neurons

We first asked whether lateral perforant pathway projection neurons express GABAB receptors that couple to GIRK channels. With 3.3 mM extracellular K+ (140 mM internal K+), bath application of the selective GABAB receptor agonist baclofen (10 µM) reversibly induced an outward current in every neuron tested (46 ± 16 pA; mean ± SE; n = 6) at a holding potential of -70 mV. During steps to a potential of -100 mV, baclofen induced an inward current (-195 ± 37 pA; Fig. 2A). These effects were reversed by washing with control solution, or by coapplication of the selective GABAB receptor antagonist CGP 55845A (1 µM).



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Fig. 2. GABAB receptors couple to inwardly rectifying potassium (GIRK) channels on identified lateral perforant pathway projection neurons. A: whole-cell currents (I; top) evoked by 100-ms, 30-mV hyperpolarizing square commands (V; bottom) in the presence of 3.3 mM external potassium. Application of 10 µM baclofen (arrows) induced outward current at -70 mV and inward current at -100 mV. Scale bars, 50 ms; 200 pA. B: current-voltage relationships recorded using voltage ramp commands (-140 to +40 mV, 500 ms) in the presence of 30 mM extracellular K+, TTX (0.5 µM), and cadmium (100 µM). Recordings under control conditions, in the presence of baclofen (10 µM), in the presence of baclofen plus CGP 55845A (1 µM), and in the presence of adenosine (100 µM) are superimposed. Baclofen and adenosine elicit currents that reverse near the calculated K+ equilibrium potential (-39 mV) and display pronounced inward rectification, consistent with activation of GIRK channels.

We further characterized the GABAB receptor-activated current in these cells by applying voltage ramps (-140 to +40 mV, 500 ms) in the presence of 30 mM extracellular K+, TTX (0.5 µM), and cadmium (100 µM). Under these conditions baclofen induced a current that displayed pronounced inward rectification and reversed polarity near the calculated equilibrium potential of -39 mV for a current carried by K+ ions (Fig. 2B). In six of six neurons, baclofen induced a 12.9 ± 2.4-nS increase in chord conductance (measured at -140 mV). Again, the changes produced by baclofen were reversed by 1 µM CGP 55845A. These results are consistent with activation of GIRK channels by GABAB receptors in LPP projection neurons.

In most cortical neurons several GPCRs couple to pertussis toxin-sensitive (Gi or Go) G proteins (Nicoll et al. 1990). In many cases GABAB receptors and A1 adenosine receptors activate a common population of GIRK channels (Sodickson and Bean 1998). Knowing that activation of adenosine receptors inhibits neurotransmitter release at LPP synapses, we tested the relative effects of baclofen and adenosine on GIRK channels in LPP projection neurons. In three neurons 100 µM adenosine activated a current that was nearly identical with that activated by 10 µM baclofen (97 ± 11% of the baclofen-induced current at -140 mV; mean ± SD; Fig. 2).

GABAB receptors couple to calcium channels on LPP projection neurons

In all known instances in which GABAB receptors activate GIRK channels, these receptors also inhibit voltage-gated calcium channels through similar G proteins in the same neurons. However, selective coupling to GIRK channels or calcium channels (but not both) has been reported for other receptors in some neurons (Fernandez-Fernandez et al. 1999; Li and Bayliss 1998), and activation of GIRK channels alone may not produce presynaptic inhibition (Lüscher et al. 1997). Thus if GABAB receptors coupled only to GIRK channels in LPP projection neurons, this could explain the resistance of LPP synapses to GABAB receptor activation.

We therefore tested the ability of baclofen to inhibit currents mediated by voltage-gated calcium channels in LPP projection neurons. Labeled neurons were recorded in voltage-clamp mode using cesium-filled pipettes in the presence of TTX (0.5 µM), and 2 mM barium was substituted for calcium as a charge carrier. Barium was used because it effectively blocks GIRK channels as well as several other potassium channels. Neurons were held at -80 mV and were stepped to a positive potential (-10 or 0 mV). These commands evoked large inward currents that were completely blocked by external cadmium (100 µM; not shown). Because these recordings were made from fairly intact LPP projection neurons, voltage clamp was clearly compromised. Barium currents were slow to activate and deactivate compared with currents in electrotonically compact cells. In some cases small, steplike current components appeared during a command, suggesting the presence of uncontrolled distal compartments. Despite this problem, we were able to qualitatively assess coupling of GABAB receptors to calcium channels in these neurons. Application of 10 µM baclofen inhibited peak inward current by 21 ± 3% in 18 of 18 neurons (Fig. 3A). In the same cells baclofen inhibited current at the end of 200-ms commands by 15 ± 3%. This degree of inhibition is similar to that which has been observed in other relatively intact neuronal preparations (Lambert and Wilson 1996; Scholz and Miller 1991). Baclofen consistently produced a small outward shift in the holding current (7 ± 2 pA; n = 12), and a small in change in leak currents (Fig. 3B, bottom). Linear leak subtraction indicated that these changes were too small to account for the observed change in inward current (Fig. 3C). Moreover, changes in leak appear to have been restricted to negative potentials; baclofen produced no change in currents evoked by large positive square commands (data not shown). Similarly, baclofen inhibited inward currents evoked by depolarizing ramp commands (-90 to +50 mV, 175 ms), but produced no change in whole-cell current at potentials positive to +10 mV (Fig. 3D). These results are consistent with voltage-dependent inhibition of calcium channels (Bean 1989), without significant changes in passive properties. Inhibition of barium currents in LPP projection neurons by baclofen was completely reversed by washing or coapplication of 1 µM CGP 55845A (Fig. 3A).



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Fig. 3. GABAB receptors couple to calcium channels on identified lateral perforant pathway projection neurons. A: individual recordings of barium currents evoked by 200-ms depolarizing commands to -10 mV from a holding potential of -80 mV. Baclofen (10 µM) and adenosine (100 µM) produce similar inhibition of barium currents. Baclofen inhibition of barium current was reversed by coapplication of CGP 55845A (1 µM). Capacitative artifacts are blanked in this panel. B: expanded view of a similar experiment, showing superimposed traces under control conditions and in the presence of baclofen (arrows) during 70-mV depolarizing (top) or 17.5-mV hyperpolarizing (bottom; scaled X4) voltage commands. Baclofen induced a small outward shift in holding current and increase in leak. Scale bars, 500 pA (top) and 125 pA (bottom), 50 ms. C: superimposed traces in control conditions and in the presence of baclofen (arrow) after linear leak subtraction (same cell as in B). Scale bars, 500 pA, 100 ms. D: superimposed currents evoked by depolarizing ramp commands (-90 to +50 mV, 175 ms) under control conditions and in the presence of baclofen (arrow). Baclofen inhibited inward current, but had little effect at negative or positive membrane potentials.

Because adenosine inhibits transmission at LPP synapses, we also tested the effect of adenosine (100 µM) on barium currents in LPP projection neurons. Adenosine reversibly inhibited peak inward currents by 26 ± 3% (n = 16; Fig. 3A). In nine cells baclofen and adenosine were both applied sequentially. In these cells baclofen inhibited peak inward current by 23 ± 2%, and adenosine inhibited peak current by 26 ± 4%. These values were not significantly different (P = 0.57; paired t-test). To determine whether adenosine and GABAB receptors couple to common effector mechanisms in LPP projection neurons, we applied saturating concentrations of baclofen and adenosine in succession. In seven neurons 50 µM baclofen inhibited peak inward current by 29 ± 1%. Addition of 100 µM adenosine in the continued presence of baclofen produced only an additional 2 ± 4% inhibition (Fig. 3B). These results suggest that GABAB and adenosine receptors couple to a common effector mechanism in LPP projection neurons.

To control for the possibility that the injection of retrograde tracer in the hippocampus induced the expression of GABAB receptors in LPP projection neurons, we made recordings from a large number of neurons in layer II of the lateral entorhinal cortex of uninjected control animals. Although some of these neurons may not have projected to the dentate gyrus, the density of labeled neurons in slices from injected animals suggested that a substantial number were, in fact, LPP projection neurons (see also Schwartz and Coleman 1981). Activation of GABAB receptors activated GIRK currents (n = 5) or inhibited barium currents (n = 32) in every neuron tested (data not shown). This result suggests that the retrograde labeling procedure did not induce expression of GABAB responses in neurons that would not otherwise have them. This experiment cannot rule out the possibility, however, that labeling changed the amount of response observed in individual LPP projection cells.

Medial perforant pathway projection neurons

Unlike LPP synapses, MPP synapses on dentate granule cells are susceptible to presynaptic inhibition by activation of GABAB receptors (Ault and Nadler 1983; Lanthorn and Cotman 1981; Mott and Lewis 1994). We therefore tested the ability of baclofen to activate GIRK channels and inhibit barium currents in retrogradely labeled MPP projection neurons in the medial entorhinal cortex (Fig. 4). Under conditions identical with those used for studying GIRK channels in LPP projection neurons (30 mM extracellular K+, TTX, and cadmium), baclofen produced a 12.6 ± 2.0-nS (mean ± SD; n = 4) increase in chord conductance at -140 mV. This value was not significantly different from that observed in LPP projection neurons (P = 0.93; Student's t-test). Under conditions identical with those used for studying barium currents in LPP projection neurons (TTX, 2 mM barium), baclofen inhibited peak inward current by only 9 ± 1% (mean ± SE; n = 5) in MPP projection neurons. This was significantly less inhibition than was observed in a comparable population of LPP projection cells (P < 0.0001; Student's t-test).


    DISCUSSION
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INTRODUCTION
METHODS
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A wide variety of GPCRs couple to Gi-Go proteins in neurons. Activation of these receptors liberates G protein beta gamma dimers, which directly interact with calcium channel alpha 1A, alpha 1B, or alpha 1E subunits, inhibiting channel function (Zamponi and Snutch 1998). This modulation is thought to underlie presynaptic inhibition mediated by GPCRs at many synapses (Wu and Saggau 1997), and parallel inhibition of somatic calcium currents and nerve terminal calcium influx is commonly observed in individual neurons. Several different Gi-Go-coupled GPCRs can inhibit neurotransmission at individual synapses. Therefore, at synapses where one such receptor inhibits calcium channels, other Gi-Go-coupled receptors must either be absent or selectively uncoupled to avoid inhibiting neurotransmitter release.

One possible mechanism of regulating presynaptic inhibition mediated by GPCRs is subcellular localization of these receptors. Recent studies have indicated that mGluRs can be selectively localized at somatodendritic and axonal sites (Shigemoto et al. 1996; Stowell and Craig 1999) and can selectively inhibit neurotransmitter release at a subset of synapses along a single axon (Scanziani et al. 1998). This prompted us to ask whether GABAB receptors, which are structurally related to mGluRs, can also be used at discrete subcellular domains. We examined lateral perforant pathway projection neurons, which are known to make synapses in the dentate gyrus that are insensitive to activation of GABAB receptors (Lanthorn and Cotman 1981) but are sensitive to activation of other Gi-Go-coupled receptors (Kahle and Cotman 1993). We found that LPP projection neurons express functional GABAB receptors that activate GIRK channels and appear to inhibit voltage-gated calcium channels. Identical effects were produced by activation of adenosine receptors. This suggests that GABAB receptors couple to somatodendritic voltage-gated calcium channels in these neurons, but presumably do not couple to presynaptic calcium channels at terminals.



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Fig. 4. Baclofen occludes inhibition of barium current by adenosine. Baclofen (50 µM; applied as indicated by the horizontal bar) inhibited barium current and largely occluded inhibition by adenosine (100 µM), applied in the continued presence of baclofen. Inset: raw traces from this experiment, with control, baclofen, and adenosine traces superimposed; capacitative artifacts are truncated.

The experiments presented here are not without limitations. For example, it is possible that the somatodendritic channels inhibited by baclofen in LPP projection neurons are not involved in neurotransmitter release at LPP terminals. However, Gi-Go proteins typically inhibit calcium channels that participate in neurotransmission (i.e., channels that contain alpha 1A, alpha 1B, or alpha 1E subunits) (Zamponi and Snutch 1998). In addition, occlusion of adenosine inhibition of barium current by baclofen suggests that this possibility is unlikely, unless adenosine receptors couple to different calcium channels in terminals and cell bodies. Direct measurements of presynaptic calcium transients in LPP terminals will be required to confirm that calcium influx is unaffected by activation of GABAB receptors (and inhibited by activation of adenosine receptors). It should also be emphasized that the limited space clamp of intact neurons prevented us from documenting characteristic features of Gi-Go inhibition of calcium channels, such as kinetic slowing and voltage-dependent relief of inhibition. Recordings from acutely isolated LPP projection neurons are necessary to demonstrate these features, which would make a more convincing case for direct inhibition of calcium channels.

Whatever the mechanism that prevents inhibition of neurotransmitter release by activation of GABAB receptors at LPP terminals, it will be of interest to determine whether a similar mechanism is used at other excitatory and inhibitory synapses that do not respond to activation of these receptors (Colbert and Levy 1992; Gil et al. 1997; Lambert and Wilson 1993; Tang and Hasselmo 1994). The finding that GABAB receptors are present on LPP projection neurons raises a number of questions. Which GABABR subunits are expressed in LPP projection neurons? Are GABAB receptors excluded from LPP terminals, or are they present and in some way selectively uncoupled? Do the local collaterals of LPP projection neurons (Lingenhöhl and Finch 1991; Tamamaki and Nojyo 1993) make synapses that are sensitive to activation of GABAB receptors? What provides the signal that ultimately prevents GABAB modulation of calcium channels at LPP terminals? If this signal is provided by the postsynaptic granule cell, is it restricted to the distal dendrites? Answering these questions should lead to a greater understanding of how neurons use GPCRs to modulate synaptic function.


    ACKNOWLEDGMENTS

We thank J. Dempster for providing data acquisition and analysis software, the Imaging Core Facility of the Institute for Molecular Medicine and Genetics for use of their facilities, and Novartis Pharma AG for supplying CGP 55845A.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-36455 and a Merit Award from the Department of Veterans Affairs.


    FOOTNOTES

Address for reprint requests: Nevin A. Lambert, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300.

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 1 June 1999; accepted in final form 8 October 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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