1 Instituto Cajal, Consejo Superior de Investigaciones Científicas, E-28002 Madrid, Spain; and 2 Laboratoire de Neurobiologie et Mouvements, Centre National de la Recherche Scientifique, F-13402 Marseille, France
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ABSTRACT |
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Araque, Alfonso, Alain Marchand, and Washington Buño. Voltage-gated and Ca2+-activated conductances mediating and controlling graded electrical activity in crayfish muscle. J. Neurophysiol. 79: 2338-2344, 1998. Crayfish opener muscle fibers provide a unique preparation to quantitatively evaluate the relationships between the voltage-gated Ca2+ (ICa) and Ca2+-activated K+ (IK(Ca)) currents underlying the graded action potentials (GAPs) that typify these fibers. ICa, IK(Ca), and the voltage-gated K+ current (IK) were studied using two-electrode voltage-clamp applying voltage commands that simulated the GAPs evoked in current-clamp conditions by 60-ms current pulses. This methodology, unlike traditional voltage-clamp step commands, provides a description of the dynamic aspects of the interaction between different conductances participating in the generation of the natural GAP. The initial depolarizing phase of the GAP was due to activation of the ICa on depolarization above approximately 40 mV. The resulting Ca2+ inflow induced the activation of the fast IK(Ca) (<3 ms), which rapidly repolarized the fiber (<6 ms). Because of its relatively slow activation, the contribution of IK to the GAP repolarization was delayed. During the final steady GAP depolarization ICa and IK(Ca) were simultaneously activated with similar magnitudes, whereas IK aided in the control of the delayed sustained response. The larger GAPs evoked by higher intensity stimulations were due to the increase in ICa. The resulting larger Ca2+ inflow increased IK(Ca), which acted as a negative feedback that precisely controlled the fiber's depolarization. Hence IK(Ca) regulated the Ca2+-inflow needed for the contraction and controlled the depolarization that this Ca2+ inflow would otherwise elicit.
Ca2+-activated K+ conductances shape and control the electrical activity in neurons and muscle fibers (e.g., Barret and Barret 1976 Preparation
Experimental procedures were as described previously (e.g., Araque and Buño 1995 Microelectrodes and recordings
Fibers were impaled with two 1 M KCl-filled micropipettes (1-5 M Synthetic voltage-clamp command waveforms
The GAPs recorded under current clamp in response to 60-ms depolarizing current pulses in control conditions and the ANAP evoked after BAPTA-loading were simulated with steps and ramps, generated with the Clampex program of the pClamp-5.0 software, following smoothing with a 4-pole Bessel low-pass filter (Ithaco, model 4212). Step and ramp amplitudes, durations, slopes, and filter settings were adjusted to closely match the waveform and voltage of GAPs and the ANAP obtained under current clamp (e.g., Fig. 1A). Synthetic GAPs and the ANAP were used as voltage-command waveforms under voltage clamp; they mimicked responses obtained from the same fiber under current clamp. The use of computer-generated synthetic GAP and ANAP enabled the subtraction of linear capacitive and leak currents with the standard pCLAMP procedure. The holding potential was set at the value of the resting potential measured under current clamp in the fiber, usually
Solutions
The control solution had the following composition (in mM): 210 NaCl, 5.4 KCl, 13.5 CaCl2, 2.6 MgCl2, and 10 Tris buffer, pH adjusted to 7.2 with HCl. In Cd2+ solution, 5 mM CdCl2 was added in equimolar exchange with CaCl2. CTX was added to the control solution from a 8-mM stock and superfused at 0.4 mM. Experiments were performed at 21-23°C. All chemicals were purchased from Sigma.
Synthetic AP voltage-clamp commands
We analyzed the GAPs in crayfish opener muscle preparations under control current-clamp conditions. The amplitude of the initial peak and the steady components of the GAP elicited by 60-ms current pulses increased, and the delay to the initial response decreased with current pulse intensity. Moreover, the GAP evoked by different fibers were different depending on cell characteristics (e.g., Bittner 1968 Synthetic GAP in control condition
The currents evoked by the synthetic GAP in control conditions are an estimate of those elicited by current pulses when the fiber generated the GAP under current clamp. They consisted of a large, brief, inward component ( Synthetic ANAP in BAPTA-loaded condition
The currents evoked by the synthetic ANAP under voltage clamp represent those underlying the response evoked by current pulses in BAPTA-loaded fibers under current clamp. They displayed a larger (78 ± 24.5%) and broader (1.3 ± 0.6 ms) inward component than in the control condition, in agreement with the higher AP amplitude and the IK(Ca) block (Fig. 1B, BAPTA). The inward current activated at Synthetic GAP in BAPTA-loaded condition
BAPTA-loaded cells fired ANAPs when depolarized under current clamp, whereas GAPs were impossible responses. However, the responses evoked by GAPs under voltage clamp provided an estimate of the ionic currents that were either in shortage or in excess in terms of generating the control and BAPTA-loaded current-clamp responses, respectively (see Isolation of ICa and IK(Ca)). The synthetic GAP evoked a large, wide, inward component ( Synthetic ANAP in control condition
Under current clamp in control conditions, GAPs rather than ANAPs were evoked by depolarization. However, the responses elicited by the ANAP in control conditions under voltage clamp enable us to determine the ionic currents either absent or in excess in terms of generating the control and BAPTA-loaded current-clamp responses, respectively (see Isolation of ICa, IK(Ca) and IK and Fig. 3). The synthetic ANAP evoked a relatively small, brief, inward current (
IK remaining under Cd2+
When the synthetic GAP was used as voltage command after Cd2+ superfusion, ICa and IK(Ca) were eliminated while IK remained. IK increased slowly to 135.9 ± 20.3 nA, then decayed gradually to a steady state and eventually dropped to zero (Fig. 1D, left). However, when the voltage command was the synthetic ANAP, the IK was larger (57.2 ± 15.8%) and faster, peaking 2.1 ± 1.2 ms earlier (Fig. 1D, right).
Isolation of ICa, IK(Ca), and IK
The suppression of ICa in the presence of Cd2+ and the abolition of IK(Ca) after BAPTA loading opens the possibility of isolating each current by subtraction (cf. Araque and Buño 1995 Conductances underlying the GAP
Although the current magnitudes and waveforms are key variables in AP genesis (see above and DISCUSSION), it is also of interest to estimate the conductance changes associated with the GAP (Hodgkin and Huxley 1952 Control of GAP amplitude
The amplitude of the synthetic GAP was changed (e.g., Figs. 3 and 4), and the relative contribution of ICa, IK(Ca), and IK to the changes in GAP amplitude that occurred in the natural condition were estimated (e.g., Araque and Buño 1995
The above results show that, with the use of synthetic AP waveforms under voltage clamp and pharmacological agents that block specific conductances, it is possible to examine the contributions of different voltage and Ca2+-gated currents to the genesis of the GAP that typifies the electrical activity of crayfish opener muscle fibers. The procedure depends on the 1) similarities between real and synthetic APs; 2) blocking specificity of the agents used; and 3) absence of voltage-dependent block. The real and synthetic APs do show small differences; however, these were insignificant at depolarized values above IK(Ca) involvement in the regulation of the GAP and dependence on IK(Ca)
In agreement with our previous demonstration using traditional pulse commands under voltage clamp (Araque and Buño 1995 All-or-none activity following IK(Ca) block
BAPTA loading or extracellular CTX evoked the ANAP instead of the GAP variant (Araque and Buño 1995 Functional implications
When the cell is depolarized by excitatory postsynaptic potential (EPSP) barrages, the voltage-dependent ICa is activated and a Ca2+ inflow leads to a Ca2+-induced Ca2+ release, which triggers contraction (Gyorke and Palade 1992
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Blatz and Magleby 1987
; Gorman et al. 1981
; Hille 1992
; Madison and Nicoll 1984
; Marty 1983
; Yarom et al. 1985
). Of the two types of Ca2+-activated K+ channels with small and large unitary conductances (SK and BK, respectively) (e.g., Latorre et al. 1989
; Marty 1983
), the latter controls action-potential (AP) repolarization and generates the early spike after hyperpolarization in invertebrate, sympathetic, and CA1 hippocampal pyramidal neurons (e.g., Adams et al. 1982
; Crest and Gola 1993
; Gola et al. 1990
; Storm 1987
). A Ca2+-activated K+ current (IK(Ca)) also controls the graded electrical activity of crayfish opener muscle fibers through BK channels that show large unitary conductances (Araque and Buño 1995
; Araque and Buño, unpublished observations). The depolarizing phase of the graded AP (GAP) of opener fibers is exclusively mediated by a voltage-gated Ca2+ current (ICa) of the L type (e.g., Araque et al. 1994
; Fatt and Ginsborg 1958
; Mounier and Vassort 1975a
). Therefore opener fibers provide a unique preparation to quantitatively evaluate the relationships between the waveforms and magnitudes of the ICa and IK(Ca) underlying the GAPs that typify these fibers.
), and the force of the contraction is governed exclusively by the degree of membrane depolarization (Bittner 1968
; Orkand 1962
). Consequently, IK(Ca) provides the negative feedback that controls the depolarization induced by ICa activation, thus precisely grading muscle contraction.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Araque et al. 1994
). Briefly, opener muscles from the first walking leg of crayfish (Procambarus clarkii) were isolated and transferred to a superfusion chamber (2 ml). Short muscle fibers (<400 mm) from the middle and proximal portions of opener muscles of small crayfish (<5 cm) were used.
) and recorded under two-electrode voltage and current clamp with a Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). The current electrode was substituted, after performing control recordings, by a new electrode filled with 0.16 M BAPTA neutralized with KOH (pH 7.2). BAPTA was ionophoretically injected with 500-ms, 100-nA current pulses, delivered at 0.2/s during 15 min. The final intracellular BAPTA concentration was estimated to be 2.5 mM (see Araque and Buño 1995
). Pulse generation, data acquisition, and analysis were performed with a PC/AT personal computer (IBM) and pClamp-5.0 software (Axon Instruments) through a LabMaster TM-40 interface board (Scientific Solutions, Solon, OH). Data were obtained from six different fibers and expressed as means ± SD, unless stated otherwise.
60 to
70 mV. It should be emphasized here that the traditional voltage-clamp methodology, as used by us with pulse commands and estimation of the voltage and time dependencies of the different isolated currents (cf., Araque and Buño 1994
, 1995
), does not give the time course and magnitude of the different ionic currents as they occur during the generation of GAPs. However, that information was obtained with the synthetic AP commands under voltage clamp used here.
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FIG. 1.
Voltage- and Ca2+-gated currents evoked by synthetic action-potential (AP) voltage commands. A: current-clamp responses (···) evoked by depolarizing current pulses (protocol shown above) in control [graded AP (GAP)] and bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-loaded conditions [all-or-none AP (ANAP)], superimposed with corresponding synthetic voltage commands ( ). B: currents evoked in Control and BAPTA conditions (left and right, respectively) by the corresponding GAP and ANAP, respectively. C: same as B, but currents evoked by GAP in BAPTA (left) and ANAP in Control conditions (right), respectively. D: IK evoked by GAP (left) and ANAPs (right) after Cd2+ superfusion, as indicated. To simplify the analysis, the 1st ANAP in the current-clamp response in A was simulated as if the pulse terminated during the AP rising phase and the Vm returned to the resting value after the AP falling phase; this was the synthetic ANAP.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
; Orkand 1962
). Therefore, to simplify our analysis, the transmembrane current pulse stimulus was adjusted (107 ± 31.5 nA, mean ± SD) to evoke similar GAPs in the six fibers selected. The GAPs evoked above a membrane potential (Vm) threshold of
43.2 ± 4.5 mV, showed an initial peak within22.3 ± 5.2 ms (19 ± 5.8 mV take-off to peak amplitude,14.1 ± 5.7 ms duration), and slowly declined reaching a steady state within 41.8 ± 14.8 ms (e.g., Fig. 1A, GAP, discontinuous record). The results described below were essentially identical in the six fibers studied, but only one was selected to illustrate the results.
42.1 ± 4.4 mV threshold (Fig. 1A, ANAP, discontinuous record). Neither GAPs norANAPs were observed in 5 mM Cd2+ or Ca2+-free solutions, indicating their Ca2+-dependent nature (not shown, but see Araque and Buño 1995
).
) were used under voltage clamp in control and Cd2+ solutions and after BAPTA loading. The reasons for using Cd2+ solutions were that they block voltage-gated Ca2+ conductances and therefore also Ca2+-activated K+ conductances. BAPTA loading was used because it chelates intracellular Ca2+, thus inhibiting Ca2+-activated K+ conductances without affecting voltage-gated Ca2+ conductances (however, see Synthetic GAP in BAPTA-loaded condition) (see Araque and Buño 1995
).
425.6 ± 102.7 nA; 3.5 ± 0.7 ms) that activated at
43.0 ± 7.5 mV, followed by a smaller (98.3 ± 18.5 nA) outward current (Fig. 1B, Control). The outward component activated rapidly, emerging directly from the decay phase of the inward current (see Isolation of ICa, IK(Ca) and IK), and then decayed slowly toward a low-magnitude steady state (80.8 ± 10.5 nA), which lasted throughout the slowly decaying phase of the GAP before falling to zero. It should be emphasized that the control current included ICa, IK(Ca), and IK (see Isolation of ICa, IK(Ca) and IK). Indeed, it has been previously demonstrated that an early inward current ICa, an early outward component mainly due to IK(Ca), and a late outward component primarily originated by IK were mixed in the total current evoked by traditional voltage command pulses (Araque and Buño 1994
, 1995
; Araque et al. 1994
; Hencek et al. 1978
; Mounier and Vassort 1975a
,b
).
39.1 ± 5.3 mV. The ensuing outward current was also larger (65.0 ± 16.5%), although its activation was slower peaking 2.5 ± 0.5 ms later, and it returned to zero more rapidly.
1,420.5 ± 200.2 nA; 4.5 ± 1.1 ms) followed by a slowly rising outward current that decayed gradually to a low steady state (Fig. 1C, BAPTA). Interestingly, the inward component was larger and lasted longer than in controls, whereas the peak outward current was 36 ± 7.3% smaller and displayed a lower steady-state value (Fig. 1B, Control). The differences can be attributed to the presence of ICa, IK(Ca), and IK in the control and the lack of IK(Ca) in the BAPTA-loaded case.
).
502.3 ± 50.6 nA; 2.2 ± 0.3 ms) followed by a large, outward component in control conditions (Fig. 1C, Control). Interestingly, whereas the outward component was much larger (94.4 ± 12.2%) and peaked 2.7 ± 0.6 ms earlier than that evoked by the synthetic ANAP in BAPTA-injected fibers, the inward current was considerably smaller (34.6 ± 6.3%) and 2.6 ± 0.2 ms briefer (see Fig. 1B, BAPTA). Again, the differences agree with the presence of ICa, IK(Ca), and IK in control and the lack of IK(Ca) in BAPTA-loaded conditions, respectively.
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FIG. 3.
ICa, IK(Ca), and IK evoked by different GAP amplitudes. A: GAP voltage commands. B-D: ICa, IK(Ca), and IK, respectively.
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FIG. 2.
Voltage- and Ca2+-gated currents and conductances during the synthetic AP peaks. A: synthetic voltage commands; dashed lines indicate analysis epoch. B: currents evoked by GAP (left) and ANAP (right) in Control, BAPTA, and Cd2+ conditions (thick solid, dotted, and thin solid lines, respectively). C: ICa, IK(Ca), and IK components (dotted, thick solid, and thin solid lines, respectively) elicited by GAP and ANAP (left and right, respectively). D: conductance modifications (gCa, gK(Ca), and gK) during the GAP. Individual conductances were calculated from the equation gx = Ix/(Vm Ex), where × denoted the pervading ion and Ex its equilibrium potential. The estimated ECa and EK were +40 and
70 mV, respectively; see RESULTS for details. Top trace corresponds to the initial part of the GAP.
). ICa (or BAPTA-Cd2+ current) evoked by both synthetic APs had similar peak values and overall profiles, except notably for the small, late inward component evoked by the sustained depolarizing segment in the GAP that was absent with the ANAP (Fig. 2C). ICa activated fast, peaked at similar values when evoked by the GAP and ANAP (
1,185.6 ± 105.8 nA), immediately before the GAP and the ANAP peaks (3.2 ± 0.7 ms and 2.5 ± 0.3 ms, respectively), and decayed rapidly (<6 ms). IK(Ca) (or control-BAPTA current; Fig. 2C) was larger in response to the synthetic ANAP than the GAP (603.0 ± 25.2 nA and 832.5 ± 46.7 nA, respectively), as expected from its voltage dependence (Araque and Buño 1995
) and the driving force increase. Moreover, mixed with the late steady-state ICa evoked by the synthetic GAP, there was a late sustained IK(Ca) component that was absent with the synthetic ANAP. IK activated late and was important late during the sustained depolarization of the GAP (Fig. 2C; see Fig. 1D).
). Indeed, the participation of driving force variations associated with the GAP voltage command were eliminated when the conductance (g) was calculated, thus the conductance waveforms provide direct information regarding the contribution of channel activation during the GAP in physiological conditions of operation. The corresponding gs were estimated by applying the formula gx = Ix/(Vm
Ex), where x is the permeant ion and Ex its equilibrium potential. The corresponding Exs were estimated from the ICa reversal potential and the IK tail current amplitudes for IK(Ca) and IK (see Araque and Buño 1994
, 1995
; Araque et al. 1994
).
; Bittner 1968
; Orkand 1962
). The ICa (or BAPTA-Cd2+ current; Fig. 3B) increased and peaked at gradually decreasing delays with GAP voltage. The inward peak was followed by a lower amplitude sustained inward component that increased with GAP voltage. A brief decrease of the inward current was sometimes observed between the peak and the steady-state ICa at high AP voltages. This decrease was probably due to an incomplete block of IK(Ca), or to small modifications in the ICa inactivation rate in BAPTA-loaded conditions (see Araque and Buño 1995
). The IK(Ca) (or Control-BAPTA current; Fig. 3C) gradually increased and peaked at briefer delays as the GAP voltage augmented. The outward peak was followed by a steady-state current that lasted throughout the depolarization and increased with GAP voltage. The IK increased gradually with GAP voltage (Fig. 3D), and it lasted throughout the GAP depolarization and activated slowly at long delays that did not change with GAP voltage.
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FIG. 4.
ICa, IK(Ca), and IK during GAP steady-state depolarization; effects of GAP amplitude. A: GAP waveforms (dashed lines indicate the epoch analyzed). B: ICa and IK(Ca) (dotted and solid lines, respectively) evoked at different GAP amplitudes. C: current-voltage relationships of ICa, IK(Ca), and IK ( ,
, and
, respectively) measured at the beginning of the analysis epoch (vertical bars indicate SE; n = 6).
; Orkand 1962
). All three currents were active during the steady-state depolarization and increased with GAP voltage (Fig. 4; see also Fig. 3). ICa and IK(Ca) increased with GAP voltage (Fig. 4, B and C,
and
, respectively), and the similarity of both current magnitudes (i.e., ICa and IK(Ca)) at all GAP voltage values should be emphasized (see DISCUSSION). IK also increased with GAP voltage (Fig. 4C,
; see also Fig. 3D).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
40 mV, which corresponded to the activation threshold of voltage-gated currents. Thus the synthetic APs appear to approximate well to the current-clamp situation. Although BAPTA may exert an effect on ICa magnitude, the predominant, if not exclusive, effect of BAPTA was the suppression of IK(Ca). The results obtained from CTX experiments are qualitatively similar to those obtained with BAPTA injection (e.g., generation of ANAPs) confirming the validity of BAPTA results. However, because the CTX-mediated IK(Ca) block is voltage dependent (Mackinnon and Miller 1988
), the quantitative analysis presented here was based on the effects of BAPTA. For a more complete discussion of possible problems associated with ICa measurement after BAPTA injection, and the efficacy of CTX block, see Araque and Buño (1995)
.
; Wilson and Davies 1965
).
), the BK channels mediating IK(Ca) had extremely brief opening latencies. IK(Ca) activated so fast that it appeared before the peak ICa activation was reached (2-ms lag) during the GAP (Fig. 2, C and D), thus enabling an extremely quick regulation of the depolarization and subsequent Ca2+ inflow. The IK(Ca) of crayfish muscle fibers is faster than other previously reported BK-type currents, and it inactivates partially, declining rapidly to a persistent steady state, probably reflecting temporal intracellular Ca2+ concentration ([Ca2+]i) variations (Araque and Buño 1995
).
). This strict correspondence could account for the precise regulation of the graded electrical activity of opener muscle fibers. With relatively more or less IK(Ca), the Vm would either be clamped at relatively hyperpolarized Vms, at which ICa would be insufficiently activated or not activated at all, or the fiber would fire ANAPs, and a smooth regulation of [Ca2+]i would be impossible (H. Chagneux, A. Araque, W. Buño, and M. Gola, unpublished observations). The similar ICa and IK(Ca) magnitudes could enable the correct control of Vm within the activation range of ICa and thus of the degree of depolarization and of muscle contraction. Therefore the tuning of the fast kinetics and relative magnitudes of ICa and IK(Ca) demonstrated above would be decisive in determining the final [Ca2+]i that set the force of the contraction.
). Similar results were obtained with ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) loading in barnacle muscle fibers (Hagiwara and Naka 1964). However, EGTA was ineffective in crayfish muscle fibers (Araque and Buño 1995
). BAPTA is a much faster Ca2+ chelator than EGTA; therefore the differences in IK(Ca) sensitivity in both muscles probably indicate that Ca2+ and Ca2+-activated K+ channels were more closely localized in the membrane of crayfish muscle fibers (Araque and Buño 1995
).
). The graded contraction depends linearly on the degree of depolarization (Bittner 1968
; Orkand 1962
). We propose that the continuous and rapid feedback provided by IK(Ca) controls the Ca2+ inflow, thereby regulating the depolarization and the ICa activation during GAPs. The precise feedback relied on similar magnitudes of IK(Ca) and ICa, which allows the persistent Ca2+ inflow needed for sustained contraction while preventing the uncontrolled depolarization that this Ca2+ inflow would otherwise evoke. The important contribution of the leak current that tends to stabilize the system due to its high amplitude (see Araque and Buño 1994
) should be emphasized. The leak conductance must be constantly overcome by the EPSPs and other active responses to reach a stable depolarized Vm.
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ACKNOWLEDGEMENTS |
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We thank Dr. Euan Brown for suggestions and the correction of an initial version of the manuscript and Dr. Mark Sefton for the correction of the final version.
This work was supported by Direción General de Investigación Cientifica y Técnica/Ministerio de Educación y Cultura, Fundación Areces (Spain), European Commission CI1*-CT90-0861VY and ERBCHRXT930190, and National Atlantic Treaty Organization grants, and an Hispano-French Integrated Action to W. Buño. A. Araque was a Fundación Areces postdoctoral fellow.
Present addresses: A. Marchand, Laboratoire de Psychophysiologie, CNRS-URA 1295, F-67000 Strasbourg, France; A. Araque, Dept. of Zoology and Genetics, Iowa State University, Ames, IA 50010.
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FOOTNOTES |
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Address for reprint requests: W. Buño, Instituto Cajal, CSIC, Av. Dr. Arce 37, E-28002 Madrid, Spain.
Received 8 July 1997; accepted in final form 21 January 1998.
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REFERENCES |
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