Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, Ohio 43614
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ABSTRACT |
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Stojic, Andrey S., Richard D. Lane, and Robert W. Rhoades. Intracortical Pathway Involving Dysgranular Cortex Conveys Hindlimb Inputs to S-I Forelimb-Stump Representation of Neonatally Amputated Rats. J. Neurophysiol. 85: 407-413, 2001. Reorganization of the primary somatosensory cortex (S-I) forelimb-stump representation of rats that sustained neonatal forelimb removal is characterized by the expression of hindlimb inputs that are revealed when cortical GABA receptors are pharmacologically blocked. Recent work has shown that the majority of these inputs are transmitted from the S-I hindlimb representation to the forelimb-stump field via an, as yet, unidentified pathway between these regions. In this study, we tested the possibility that hindlimb inputs to the S-I forelimb-stump representation of neonatally amputated rats are conveyed through an intracortical pathway between the S-I hindlimb and forelimb-stump representations that involves the intervening dysgranular cortex by transiently inactivating this area and evaluating the effect on hindlimb expression in the S-I forelimb-stump representation during GABA receptor blockade. Of 332 S-I forelimb-stump recording sites from six neonatally amputated rats, 68.3% expressed hindlimb inputs during GABA receptor blockade. Inactivation of dysgranular cortex with cobalt chloride (CoCl2) resulted in a significant decrease in the number of hindlimb responsive sites (9.5%, P < 0.001 vs. cortex during GABA receptor blockade before CoCl2 treatment). Results were also compiled from S-I forelimb recording sites from three normal rats: 14.1% of 136 sites were responsive to the hindlimb during GABA receptor blockade, and all of these responses were abolished during inactivation of dysgranular cortex with CoCl2 (P < 0.05). These results indicate that the S-I hindlimb representation transmits inputs to the forelimb-stump field of neonatally amputated rats through a polysynaptic intracortical pathway involving dysgranular cortex. Furthermore the findings from normal rats suggest that this pathway might reflect the amplification of a neuronal circuit normally present between the two representations.
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INTRODUCTION |
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Reorganization of the
forelimb-stump representation in the primary somatosensory cortex (S-I)
of rats with neonatal forelimb removal is characterized by the
expression of unexpected hindlimb inputs, the majority of which are
only revealed when the effects of cortical receptors for -amino
butyric acid (GABA) are pharmacologically blocked (Lane et al.
1997
). These findings led to the following question, what
is/are the source(s) of the hindlimb inputs revealed during GABA
receptor blockade? Earlier work suggested that hindlimb inputs
expressed in the S-I forelimb-stump field of neonatally amputated rats
reflected changes occurring at subcortical levels of the dorsal
column-medial lemniscal pathway. Lane et al. (1995)
demonstrated that neonatal forelimb removal in rats results in the
invasion of cuneate nucleus by sciatic nerve afferents and the
development of cuneate neurons (40%) with receptive fields that
included both the forelimb-stump and hindlimb. Furthermore, Stojic et al. (1998)
reported that 19% of neurons in
the forelimb-stump representation of the ventral posterolateral (VPL)
nucleus had receptive fields that included both a forelimb-stump and
hindlimb receptive field, and that of these cells, 33% projected to
the S-I forelimb-stump representation. However, recent work by
Lane et al. (1999)
suggests that these subcortical
changes may contribute very little to the reorganization of the S-I
forelimb-stump representation. Electrolytic lesions of the S-I hindlimb
representation [or transient inactivation with cobalt chloride
(CoCl2)] of neonatally amputated rats reduces
the number of hindlimb responsive sites in the S-I forelimb-stump
representation by 80% (Lane et al. 1999
).
The neural circuit by which these inputs from the S-I hindlimb
representation reach the forelimb-stump field is not known. Latency
data from Lane et al. (1999) suggest that these inputs might be conveyed via a polysynaptic pathway. In the current study, we
test the possibility that hindlimb inputs to the S-I forelimb-stump representation of neonatally amputated rats are conveyed through a
polysynaptic intracortical pathway between the S-I hindlimb and
forelimb-stump representations which includes a synapse in the
dysgranular cortex interposed between these two regions by transiently
inactivating this dysgranular cortex during GABA receptor blockade.
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METHODS |
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All protocols described here were developed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Medical College of Ohio.
Neonatal forelimb removal
Neonatal forelimb removals were carried out as previously
described (Lane et al. 1995). Briefly, postnatal day 0 (>12 h old) rats were anesthetized by hypothermia, and the left
forelimb amputated by making a circular incision through the skin and
muscle distal to the shoulder. The shaft of the humerus was exposed and
severed between its proximal and middle thirds. The skin was closed
with cyanoacrylate adhesive following cauterization of the brachial artery and application of 0.7% bupivicaine. The pups were then rewarmed and returned to their mothers.
Recordings from S-I
A total of six rats that sustained neonatal forelimb removal and
three normal rats were used in this study. Rats were initially anesthetized with ketamine (60 mg/kg) and xylazine (15 mg/kg) administered intraperitoneally and prepared for recordings as has been
previously described (Lane et al. 1999). The trachea was
cannulated, and the left brachial plexus and sciatic nerves were
exposed. The animal was then placed in a stereotaxic head holder and
ventilated mechanically. A bipolar stimulating electrode was placed on
the brachial plexus, just proximal to the origins of the median, ulnar,
and radial nerves, and another on the sciatic nerve ~15 mm distal to
the sciatic notch. Rats were then paralyzed with gallamine triethiodide
(30 mg, delivered intraperitoneally) and kept under light anesthesia
during the recording session by administering urethan (1.1 g/kg) as
needed. A midline incision was then made in the scalp. The cisterna
magna was opened to drain cerebral spinal fluid, and a large craniotomy
was made over the right cerebral cortex, contralateral to the amputated
forelimb. Recordings from normal animals were also made from the right
cerebral cortex. The dura and arachnoid were reflected and warm culture medium (Neurobasal medium, Gibco-BRL) was applied to the cortical surface to prevent drying. The surface of the cortex was photographed at ×44 magnification to mark the placement of recording electrode penetrations.
Unit clusters were recorded with parylene-coated tungsten electrodes
(Z = 0.9-1.3 µ). Cutaneous receptive fields were mapped with
tactile stimuli delivered with blunt probes. Electrode penetrations were spaced ~200 µm apart and made in a rectangular array and multi-unit activity was recorded at depths between 600 and 800 µm
below the pial surface. The S-I forelimb-stump and hindlimb representations were identified, and between 40 and 80 recording sites
were evaluated in the forelimb-stump field, while 20-30 sites in the
hindlimb field were examined.
Sequence of mapping experiment
A series of four mappings were made in each animal as outlined in Fig. 1. First, the forelimb-stump and hindlimb representations were mapped under normal anesthetized conditions (Fig. 1A). After this, the GABA receptor blockers bicuculline methiodide (BMI, for GABAA) and saclofen (SAC, for GABAB) were applied to the surface of the cortex (methods described in the following text), and the forelimb-stump representation was remapped to identify recording sites in the S-I forelimb-stump representation that expressed dual forelimb-stump and hindlimb receptive fields (Fig. 1B). Following identification of these dual receptive field sites in the S-I forelimb-stump field, the dysgranular cortex interposed between the hindlimb and forelimb-stump fields was temporarily inactivated with a series of CoCl2 injections as described in the following section and the forelimb-stump representation was remapped (Fig. 1C). Finally, the S-I forelimb-stump recordings sites were reevaluated after the effects of the CoCl2 injections into dysgranular cortex had dissipated (Fig. 1D).
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Application of BMI and SAC to the cortical surface
Blockade of GABA receptors was accomplished using methods
previously described (Lane et al. 1997). In brief, a 30 µl solution containing equal parts of 50 µM BMI and 50 µM SAC was
applied to the cortical surface. Experimental observations have
previously demonstrated that the antagonists are effective by 10 min
following application and continue to be effective at blocking GABA
receptor activity for ~30 min (Stojic et al. 2000
), a
time sufficient to complete mapping of the S-I forelimb-stump
representation. If more than 30 min was required to complete a
particular mapping sequence, additional 30 µl aliquots of the GABA
blockers were applied as needed.
Temporary inactivation of dysgranular cortex with CoCl2
This procedure was modified from a protocol used to inactivate
the S-I hindlimb representation by Lane et al. (1999). A
30 mM solution of CoCl2 dissolved in saline was
injected into 6-10 sites (0.2 µl/site) in dysgranular cortex around
the anterior, posterior, and lateral borders of the S-I hindlimb
representation (as determined by physiologic mappings). The injections
were made at a depth of 600-800 µm below the pial surface.
Inactivation of dysgranular cortex was determined by recording
spontaneous activity and stimulus-driven neuronal activity with a
sentinel recording electrode affixed along side the
CoCl2 injection pipette. Experimental
observations indicate that a single injection was effective in
silencing an area of cortex with a radius of ~250 µm for ~30 min.
The spread of CoCl2 from the injection sites as just described opened the possibility their direct effect could have
extended into parts of the S-I hindlimb and forelimb-stump representations. This issue was resolved by monitoring all recording sites within the S-I hindlimb and forelimb-stump regions during CoCl2 inactivation. If
10% of recording sites
in either representation became unresponsive to stimulation of their
principle cutaneous region of innervation, the mapping experiment was
suspended to allow the effects of CoCl2 to
dissipate, then fresh CoCl2 injections were made
into dysgranular cortex and the mapping experiment resumed.
Identification of recording sites
Specific recording sites in the S-I forelimb-stump and hindlimb
representations, as well as the sites of CoCl2
injection into dysgranular cortex, were marked by making small
electrolytic lesions at each site (1-2 s duration, 3-4 V). After
this, animals were killed with a lethal dose of
CO2 and perfused transcardially, first with a
0.9% heparanized phosphate buffer saline, followed by a 4%
paraformaldehyde solution. The brain was then allowed to postfix
overnight. Following this, the brain was extracted, and the right
cerebral cortex was removed, flattened and sectioned into 50 µm
sections on a freezing microtome. The sections were then processed for
cytochrome oxidase (CO) using the methods of Wong-Riley
(1979).
Analysis of physiologic data
The results from the six neonatally amputated rats and three normal rats were used to compare the percentage of dual forelimb-stump and hindlimb responsive recording sites within the S-I forelimb-stump representation under four conditions: prior to GABA receptor blockade; during GABA receptor blockade, prior to silencing of dysgranular cortex; during GABA receptor blockade and silencing of dysgranular cortex with CoCl2; and after recovery from CoCl2 treatment while maintaining GABA receptor blockade. These data were evaluated statistically using a two-way repeated measures ANOVA. Mean differences in percentages of recording sites with dual forelimb-stump and hindlimb receptive fields under pairs of treatment conditions described just above were evaluated using Scheffe post hoc analysis. The accepted level of significance was P < 0.05.
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RESULTS |
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Effect of CoCl2 inactivation of dysgranular cortex on the expression of hindlimb receptive fields in the S-I forelimb-stump representation of neonatally amputated rats
Inactivation of dysgranular cortex resulted in a significant
reduction in the expression of hindlimb receptive fields in the S-I
forelimb-stump representation revealed during GABA receptor blockade.
An example of this is illustrated in the case from a neonatally
amputated rat shown in Fig. 2. Prior to
GABA receptor blockade, 1 of 57 (1.8%) recording sites in the S-I
forelimb-stump field was responsive to hindlimb stimulation (not
shown). When the effects of cortical GABA receptors were blocked with
BMI and SAC, 35 sites (61.4%) demonstrated responsivity to the
hindlimb (Fig. 2A) in addition to the forelimb-stump. This
is consistent with previous results (Lane et al. 1997,
1999
). CoCl2 inactivation of dysgranular
cortex resulted in the selective loss of hindlimb expression at most of
these sites (31 of 35 sites, 88.5%, Fig. 2B). In this case
as well as all others, CoCl2 inactivation of the
dysgranular cortex caused <10% of the recording sites in either representation to become unresponsive to stimulation of their principle
cutaneous region of innervation.
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Figure 3 shows physiologic recordings made from electrodes placed in the S-I forelimb-stump field, dysgranular cortex, and hindlimb representation during CoCl2 inactivation of dysgranular cortex (see Fig. 2C for positions of recording electrodes and sites of CoCl2 injections). Note that the loss of hindlimb receptive field expression in the S-I forelimb-stump representation corresponded with CoCl2-mediated inactivation of neuronal activity in dysgranular cortex while expression of forelimb persisted. Recording sites within the hindlimb representation remained responsive to cutaneous hindlimb stimulation and sciatic nerve activation throughout the experiment (Fig. 3). After the effects of CoCl2 were allowed to dissipate, hindlimb responses returned to all of the recording sites in the S-I forelimb-stump representation that expressed dual receptive fields prior to CoCl2 treatment.
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Of 332 S-I forelimb-stump recording sites from six neonatally amputated
rats that were responsive to forelimb-stump stimulation, 3.1 ± 3.1% were also responsive to hindlimb stimulation prior to GABA
receptor blockade (Fig. 4, ,
before GRB). During cortical GABA receptor blockade,
68.3 ± 21.7% of the forelimb-stump recording sites became dually
responsive to hindlimb as well as forelimb-stump stimulation
(P < 0.0001 vs. unblocked cortices, Fig. 4,
,
during GRB). During inactivation of dysgranular cortex with
CoCl2, only 9.5 ± 5.2% of forelimb-stump
sites remained responsive to stimulation of the hindlimb
(P < 0.001 vs. cortex during GABA receptor blockade and CoCl2 inactivation, Fig. 4,
, GRB + CoCl2). Following recovery from
CoCl2 treatment, most of the forelimb-stump
recording sites (66.6 ± 26.9% of all forelimb-stump recording
sites) that expressed dual (forelimb-stump/hindlimb) receptive fields
prior to CoCl2 treatment again showed hindlimb
responsivity (P > 0.05 versus GABA receptor blocked
cortex prior to CoCl2 treatment, Fig. 4,
,
recovery + GRB).
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A total of 136 S-I forelimb recording sites from three normal adult
rats were tested. Prior to GABA receptor blockade, 1.9 ± 1.7% of
136 recording sites responded to hindlimb stimulation in addition to
the forelimb (Fig. 4, , before GRB). During GABA receptor
blockade, the number of hindlimb responsive sites increased to
14.1 ± 6.0% (Fig. 4,
, during GRB). This sevenfold
increase was not statistically significant primarily due to the small
sample size. In a previous study (Lane et al. 1997
) in
which six normal rats were tested, a significant difference was
observed between the number of hindlimb responsive sites present prior
to versus during GABA receptor blockade. Inactivation of dysgranular
cortex between the S-I hindlimb and forelimb representations of normal rats totally abolished all hindlimb responses within the S-I forelimb field (0%, P < 0.05 vs. GABA receptor blocked cortex
prior to CoCl2 treatment, Fig. 4,
, GRB + CoCl2). Following recovery from CoCl2 treatment (while maintaining GABA receptor
blockade), hindlimb responses returned to all S-I forelimb recording
sites that prior to CoCl2 treatment were
responsive to the hindlimb (Fig. 4,
, recovery + GRB).
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DISCUSSION |
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The results of this study, along with previous results
(Lane et al. 1999; Stojic et al. 1998
)
support the following conclusions. First, the majority of hindlimb
inputs expressed in the S-I forelimb-stump representation of neonatally
amputated rats are conveyed via a polysynaptic, intracortical pathway
that involves at least one synapse in the dysgranular cortex between
the S-I hindlimb and forelimb-stump fields. Second, the results provide
evidence that a similar pathway is present in normal rats which would
account for the small percentage of hindlimb responsive sites within
the normal S-I forelimb field revealed during GABA receptor blockade.
Technical limitations
The results of the physiologic experiments were dependent on the effectiveness of CoCl2 silencing of dysgranular cortex. There was the potential for CoCl2 injections to be ineffective at a particular site of injection or for the spread of the injections to be insufficient for silencing the desired region of dysgranular cortex. The effectiveness of CoCl2 for silencing of dysgranular cortex was monitored at several sites and invariably confirmed the inactivation of dysgranular cortex. By spacing the injections sites ~500 µm within dysgranular cortex, the combined influence of the CoCl2 injections was likely to inactivate much of the desired region of cortex.
The possibility that the CoCl2 injections might directly influence recording sites in the S-I hindlimb and forelimb-stump representations was also a potential problem. However, in each case, <10% of these sites became unresponsive to stimulation of their principle cutaneous region during the CoCl2 treatment phase of the experiment.
Finally, there was the potential for
CoCl2-induced damage of neuronal tissue, as has
been reported by Malpeli and Burch (1982). However,
Lee and Malpeli (1986)
have shown that one or two
CoCl2 injections at a given location could be
performed without causing acute damage to neuronal tissue. Because of
the potential for CoCl2-induced cortical damage,
the recovery of neuronal activity in dysgranular cortex following
CoCl2 injections was carefully monitored. In all
cases presented in this study, neuronal activity in dysgranular cortex
returned to levels that were comparable to those observed prior to injections.
Role of intracortical connections in the expression of hindlimb inputs in the S-I forelimb-stump representation of neonatally amputated rats
The results of this study indicate that the majority of hindlimb
inputs expressed in the reorganized S-I forelimb-stump representation of neonatally amputated rats during GABA receptor blockade are transmitted through a polysynaptic intracortical pathway between the
S-I hindlimb and forelimb-stump representations. These findings are
consistent with other studies that have indicated that intracortical connections play an important role in the functional reorganization of
adult cortical representations that follow nerve injury
(Darian-Smith and Gilbert 1994; Das and Gilbert
1995
; Doetsch et al. 1988
; Florence et
al. 1998
; Jacobs and Donoghue 1991
; Pons
et al. 1991
). However, these results, and those of Lane
et al. (1999)
differ from previous reports in that the
intracortical pathway described here does not involve direct
connections between the S-I hindlimb and forelimb-stump fields (see
Lane et al. 1999
; Stojic et al. 1998
).
Rather, the intracortical circuit described in this study involves
neuronal elements that are not only within the S-I hindlimb and
forelimb-stump representations, but between the two fields in the
region of dysgranular cortex (Chapin et al. 1987
;
Fabri and Burton 1991
). The presence of
hindlimb-forelimb-stump circuit synapses within this region is
supported by the following evidence: the ability of
CoCl2 treatment of dysgranular cortex to
inactivate this circuit; and the fact that CoCl2
blocks activity at synapses (Malpeli 1999
) but does not
disrupt the activity of fibers of passage.
The development of this polysynaptic intracortical pathway through
dysgranular cortex could result from one of two mechanisms. First, this
circuit could represent the maintenance or amplification of an
intracortical pathway normally formed during development (Hirsch
and Gilbert 1993; Jenkins et al. 1990
;
Kolb et al. 1994
; Li and Waters 1996
;
Li et al. 1996
). Conversely this pathway might reflect
the emergence of a new connection as a result of peripheral nerve
injury, perhaps involving axon collateral sprouting
(Darian-Smith and Gilbert 1994
; Florence et al.
1998
). The present results suggest that the intracortical
circuit involved in transmitting hindlimb inputs to the S-I
forelimb-stump representation of neonatally amputated rats reflects the
strengthening or amplification of a circuit that is normally present
between the S-I hindlimb and forelimb-stump fields. This view is
supported by the data from the three normal rats in which the
dysgranular cortex was inactivated with CoCl2
causing the hindlimb inputs to the few sites within the S-I forelimb
field of normal rats revealed during GABA receptor blockade to be
completely abolished. Our physiologic data indicate that a polysynaptic
pathway from S-I hindlimb to dysgranular cortex to S-I forelimb is
present in both normal and neonatally amputated rats. This conclusion
is further supported by the results of tracing experiments that showed
that the S-I hindlimb representation projects to dysgranular cortex and
dysgranular cortex sends axons to the S-I forelimb representation in
both normal and neonatally amputated rats (Chapin et al.
1987
; Fabri and Burton 1991
; Stojic et
al. 1998
).
Role of cortical and subcortical change in the development of hindlimb inputs in the S-I forelimb-stump representation of neonatally amputated rats
The degree to which experience-induced functional changes at the
cortical level can be explained by subcortical reorganization has been
a long standing issue in plasticity research (e.g., Darian-Smith and Gilbert 1994; Faggin et al. 1997
;
Florence and Kaas 1995
; Kiss et al. 1994
;
Panetsos et al. 1995
; Rasmusson and Northgrave 1997
; Tinazzi et al. 1997
). Many studies have
documented the capacity for functional reorganization in the brain stem
(Jain et al. 2000
; Kalaska and Pomeranz 1979
,
1982
; Northgrave and Rasmusson 1996
; Panetsos et al. 1995
; Waite 1984
),
thalamus (Alloway and Aaron 1996
; Chiaia et al.
1992
; Davis et al. 1998
; Florence et al.
2000
; Garraghty and Kaas 1991
;
Jones and Pons 1998
; Kiss et al. 1994
; Nicolelis et al. 1991
, 1993
; Rasmusson
1996
; Rhoades et al. 1987
; Verlay and
Onnen 1981
), and cortex (Aglioti et al. 1994
;
Calford and Tweedale 1991
; Diamond et al.
1993
; Florence et al. 2000
; Fox
1994
; Halligan et al. 1993
; Kaas
1994
; Kelahan et al. 1981
; Mezernich et
al. 1983
; Pascual-Leone and Torres 1993
;
Pons et al. 1991
; Rasmusson and Turnbull
1983
; Rasmusson et al. 1982
; Recanzone et
al. 1992a
,b
) in response to changes in sensory experience. The
relationship between changes occurring at subcortical levels to
rearrangements in cortex is not well understood. The data presented in
this study, along with the previous work (Lane et al. 1995
, 1997
, 1999
; Stojic et al. 1998
) strongly
suggest that reorganization of the S-I forelimb-stump representation in
neonatally amputated rats is principally the result of intrinsic
cortical changes, and to a far lesser degree, a reflection of changes
occurring at subcortical levels of the dorsal column-medial lemniscal pathway.
The data presented here, and the study of Lane et al.
(1999) indicate that most (>80%) of the hindlimb inputs
expressed in the S-I forelimb-stump field are derived from a
polysynaptic intracortical pathway between the S-I forelimb-stump and
hindlimb representations involving dysgranular cortex. The remaining
hindlimb responsive sites (<20%) are likely to reflect inputs derived
from VPL neurons that express dual forelimb-stump and hindlimb neurons
(Stojic et al. 1998
). Although the role of VPL in
hindlimb expression in the S-I forelimb-stump field has not been
directly tested, recent work by Stojic et al. (1999)
indicates that the hindlimb inputs that remained following lesioning of
the S-I hindlimb representation reflect dual forelimb-stump and
hindlimb inputs transmitted from the reorganized cuneate nucleus to the
S-I forelimb-stump field via the thalamus.
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ACKNOWLEDGMENTS |
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Special thanks to R. Wynn for excellent technical assistance.
This research was supported by National Institutes of Health Grants NS-28888 and DE-07734.
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FOOTNOTES |
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Address for reprint requests: R. D. Lane (E-mail: rlane{at}mco.edu).
Received 27 July 2000; accepted in final form 21 September 2000.
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REFERENCES |
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