Georgetown Institute for Cognitive and Computational Sciences, Georgetown University Medical Center, Washington, DC 20007
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ABSTRACT |
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Tsau, Yang,
Li Guan, and
Jian-Young Wu.
Epileptiform Activity Can Be Initiated in Various Neocortical
Layers: An Optical Imaging Study.
J. Neurophysiol. 82: 1965-1973, 1999.
The initiation site for triggering
epileptiform activity was investigated via optical imaging using
voltage-sensitive dyes in the neocortical slice perfused with
artificial cerebral spinal fluid containing nominally zero magnesium.
The neocortical slices (400-µm thick) were harvested from
Sprague-Dawley rats (P21-28). Optical imaging was made by using a high
speed photodiode array. Spontaneous epileptiform activity emerged
20-40 min after the preparation was perfused with zero-magnesium
solution. There was a good correspondence between electrical and
optical signals (n = 46), although the details of
the two recordings were somewhat different. The initiation sites were
measured optically in 11 preparations. Among them, four were found to
be located in superficial layers, two were found in middle layers, and
five were found in deep layers. Repeated recordings revealed that these
initiation sites were relatively stable; shifting of the initiation
site was not observed. Therefore spontaneous epileptiform activity could be initiated in various cortical layers, from layer I to layer
VI. The activation started from a small area <0.04 mm3 and
spread smoothly from the initiation site to adjacent cortical areas,
suggesting that the initiation site is very confined to one of the
cortical layers. The initiation sites were distributed randomly in
various cortical areas, and no higher probability was found in a
special cortical region. Electrical stimulation delivered via a glass
microelectrode filled with 2 M NaCl (2-5 M) could reliably trigger
epileptiform activity that had the same characteristics as the
spontaneous activity. The cortical neurons activated directly by the
stimulation were around the electrode's tip and estimated to be within
a 50-µm area, suggesting that only a few neurons were needed to form
an initiation site. Because the timing for stimulation was arbitrary
and the evoked events were initiated independent of discharges of
neurons in any other layers, it is likely that the initiation site for
epileptiform activity in various cortical layers is independent of the
control of layer V pyramidal neurons. Together these finding suggest
that the epileptiform focus is confined and can be formed in several (probably all) neocortical layers and in many cortical areas. The
initiating neurons may be of different types because neuronal types in
various cortical layers are different.
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INTRODUCTION |
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In a number of epilepsy models, epileptiform
activity can be initiated from cortical structures (McNamara
1994; Traub et al. 1994
), indicating that the
initiation of epileptiform activity can be intrinsic to the cortex.
Studying the process of epilepsy initiation has been attractive not
only because the understanding of its mechanism would benefit more
efficacious prevention and treatment of the disease but also because
the study of neuronal interactions during this process would reveal
important features of the functional neuronal organization in the
cortex. Pyramidal neurons in layer V of the neocortex have been thought
to be responsible for initiation because pyramidal neurons have the
highest excitability in the neocortex (Connors 1984
),
they have little detectable inhibition (Chagnac-Amitai and
Connors 1989b
), and layer V alone is sufficient for generating
epileptiform activity in a zero-Mg2+ epilepsy
model (Silva et al. 1991
). However, it recently has been
reported that isolated superficial layers also can initiate epileptiform activity and may even dominate the initiation process in
the intact cortex perfused with 10-20 µM bicuculline methiodide (Albowitz and Kuhnt 1995
). Field potential recordings
also suggested that epileptiform activity could start in layer II/III
of the cortex (de Curtis et al. 1994
). Because intrinsic
firing properties of cortical neurons may underlie the initiation of
epileptiform activity (Miles and Wong 1983
;
Prince 1967
, 1969
; Traub and Wong 1982
),
cortical neurons with repetitive firing properties might be capable of
initiating epileptiform activity. This implies that various neurons
from different layers may potentially become initiation cells for
epileptiform activity if they possess repetitive and rhythmic firing
properties. In fact, cortical neurons in both superficial and deep
layers can be induced to fire spontaneously (Flint and Connors
1996
), and therefore these neurons potentially should be able
to initiate epileptiform activity. The location of initiation cells for
triggering epileptiform activity in different cortical layers indicates
the involvement of different neuron types in the formation of the
epileptiform focus. To clarify in which layer the initiating cells are
located, it is necessary to simultaneously monitor neuronal activity in
all cortical layers during epileptiform activity.
Examination of the spatial and temporal characteristics of epileptiform
activity in cortical slice preparations using conventional electrophysiological techniques requires an electrode array. The number
of electrodes in such an array has been limited to just a few
(Chagnac-Amitai and Connors 1989b) because of practical difficulties. Current-source density analysis has been attempted in a
study of the origin of epileptiform activity, but the location of
initiation site could not be determined accurately (de Curtis et
al. 1994
). The timing of epileptiform activity onset from the different regions is a good indicator for where the epileptiform activity starts but only can be compared when these regions are simultaneously measured. Because this transient signal tends to be in
the millisecond domain, such measurements need high temporal resolution.
Optical recording has been applied to achieve the measurement of rapid
changes in transmembrane potential, such as action potentials. The
optical signals from neuronal processes stained with voltage-sensitive
dyes are linear with membrane potential (Ross et al.
1977). If multiple neurons are recorded optically by a single
detector, the optical signal represents the average of the change in
transmembrane potential (Tsau et al. 1996
). When multiple regions are simultaneously recorded by a photodiode array, the
timing of activities in these regions can be compared and the
initiation site for epileptiform activity thus can be determined. In
this study, we applied high speed (1 frame per millisecond) optical
imaging using voltage-sensitive dyes to measure epileptiform activity
over a region of the cortex ~4.5 × 1.5 mm2 in an attempt to directly record the
initiation process. We have reported that dominant foci for initiating
epileptiform activity emerge when the preparation is perfused with
zero-Mg2+ artificial cerebral spinal fluid (ACSF)
or normal ACSF containing bicuculline (20-50 µM) (Tsau et al.
1998
). In this report, we image these stationary foci in
neocortical tissue. We ask in which cortical layer these foci are
located. We also use electrical stimulation via a microelectrode to
trigger epileptiform activity in various cortical layers to visualize
the initiation process.
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METHODS |
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The experiments were carried out on neocortical slices from
Sprague-Dawley rats (P21-28) of either sex. The animals were
decapitated under CO2-induced narcosis, following
the guidelines set by the National Institutes of Health. The brains
were removed and placed in normal ACSF consisting of (in mM) 132NaCl, 3 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 dextrose bubbled with 95%
O2-5% CO2 (pH = 7.4),
and chilled in an ice bath. The preparations were 400-µm-thick
coronal sections from the temporal cortex of Bregma 2 to
5 mm and
were incubated for
1 h in normal ACSF at room temperature before
staining. After staining the slice was submerged in a recording chamber
under a Leitz Ortholux II microscope and perfused with (nominally)
zero-Mg2+ ACSF at 28-31°C for electrical and
optical recordings (Fig. 1). Because
optical recordings are sensitive to mechanical vibrations, several
steps were taken to reduce them: an air table (Newport) was used to
reduce the floor vibration; the lamp house was sealed to eliminate the
illumination fluctuation due to air flow around the light bulb; the
perfusion was paused during the optical recording trials; and a cover
slip was placed on top of the recording chamber to eliminate the
fluctuations at the air-water interface.
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Recording electrodes were made from borosilicate glass containing a
capillary fiber (0.75 mm ID, 1.0 mm OD). Extracellular glass electrodes
were filled with 1 M NaCl and had a resistance ranging from 2 to 5 M. In most experiments, the recording electrodes were placed in
layer II of the cortex. The tips of electrodes were inserted ~100
µm into the slice. Recordings were made with the use of two
preamplifiers (IE201, Warner Instrument, Hamden, CT). In the
experiments where we recorded evoked epileptiform activity, one
electrode was used as a stimulating electrode. Stimuli consisted of
rectangular current pulses 2-5 µA (10-30 V) in amplitude and
100-200 µs in duration. The data from electrical recordings were
stored on video tapes through a digital recorder (CRC VR-100) and then
transferred to a PC for further analysis.
The neocortical slices were stained with the voltage-sensitive dye
RH479 (first synthesized by R. Hildesheim and A. Grinvald and kindly
provided by Dr. L. Loew, University of Connecticut, Farmington, CT, as
JPW 1131) solution (0.05 mg/ml in ACSF) for 1-2 h. Transmitted light
was used for optical recording. Light from a Tungsten filament lamp (12 V, 100 W) was passed through a 705 ± 60 nm filter (mean ± SD) and then to the preparation. The image was projected onto a
124 element photodiode array (Centronics, Newbury Park, CA) through a
×2.8 objective lens (Fig. 1). A low-power objective lens was used to
maximize the recording area. Each pixel received light from a 0.3 × 0.3 mm2 area of the objective plane. The optical field
was adjusted so that the epileptiform activity initiation site and
propagation would be best recorded optically because only part of the
cortical slice could be included in the recording field. Each
photodiode signal was amplified in parallel by a set of 128 parallel
amplifiers (purchased for the Department of Physiology, Yale
University, New Haven, CT). The first stage of amplification was
performed with a current to voltage converter circuit using a feedback
resistor of 5 M. The second stage amplified the voltage by an
additional factor of 1,000 or 2,000, with high-pass filtering with a
100-ms time constant and four-pole Bessel analogue low-pass filtering of 300-Hz corner frequency. The signals from the 124-element photodiode array and two electrodes then were multiplexed and digitized at 1,000 frames per second with a DAP3200e/214 12-bit data-acquisition board
installed in a Pentium PC and 2 MSXB expansion boards (Microstar Laboratories, Bellevue, WA) controlled by a program written in BASIC.
Data were acquired into the buffer memory on the data-acquisition board
and saved to a hard drive. Optical recordings were made without
averaging. Four to 20 trials were recorded from each preparation. To
capture spontaneous epileptiform activity, each trial had a duration of
8-16 s. The signal-to-noise ratio was >20, and the optical signal
size (
I/I, change in intensity divided
by resting light intensity) for epileptiform activity was
~0.1-0.5%. Data analysis was performed with a Pentium PC running
the Interactive Data Language (IDL, Research Systems) program NeuroPlex
(OptImaging, LLC, Fairfield, CT). The optical signals were filtered
digitally with 300-Hz low-pass (Butterworth filter) and 1.2-Hz
high-pass (RC filter) and then normalized for the movie display. The
contour mode displayed the areas of equal signal height as the same
color. The spaces between diodes are interpolated; the display is not in discrete pixels. Variable display mode was used where the scale for
each diode is independent and the timing information is displayed more accurately.
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RESULTS |
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Spontaneous epileptiform activity initiation sites revealed by optical recordings
Spontaneous epileptiform activity emerged 20-40 min after the
preparation was perfused with zero-Mg2+ ACSF.
Epileptiform events occurred in episodic bursts, each burst starting
with a large initial spike and followed by 7-10 Hz oscillations lasting 5-30 s (Flint and Connors 1996; Silva et
al. 1991
; also see Fig. 2). We
have validated the optical recordings by comparing simultaneously
recorded electrical and optical signals throughout all experiments
(n = 46). These results indicate a very good
correspondence between electrical and optical signals (Fig. 2). Both
recordings show a large initial spike followed by prolonged 7- to 10-Hz
oscillations. The timing of the initial spike and some peaks of the
oscillation waves was very similar in the two traces. However, details
of the two recordings were not the same probably because electrical recordings can be affected by current from distant areas, whereas optical recordings strictly reflect the neuronal membrane potential changes in the area the image of which is projected to the recording photodiode (Ross et al. 1977
; Salzberg et al.
1973
). For example, the electrical recording is very sensitive
to a stimulus current delivered at a substantial distance, whereas the
optical signal is not affected at all. Therefore it was anticipated
that the components of field potential recording would differ somewhat from those of the optical recording. In this study, we were interested mainly in the timing and location of the initial spike in different cortical laminae to understand the initiation process of epileptiform activity. Because the timing of the initial spike in optical recordings correlated well with that of the local field potential recordings, we
can use the photodiode array to locate the starting focus of the
initial spike. In the following figures, we will only describe the
characteristics of the initial spike of each epileptiform event. The
secondary oscillations have different characteristics and have been
described elsewhere (Wu et al. 1999
).
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Epileptiform activity could be recorded from the entire preparation
when the preparation was perfused with
zero-Mg2+ ACSF (n = 40), even in a small section (2 mm wide horizontally) of a cortical
slice with all cortical layers intact. This was true of neocortical
slices from randomly chosen regions including frontal, temporal, and
occipital areas. This result indicates that initiation sites for
generating epileptiform activity can be formed spontaneously regardless
of the region of cortex. The initiation site in each stained cortical
slice was identified optically and moved to an appropriate place in the
imaging field. Because the resolution of optical imaging data is
relatively poor compared with the cortical architecture due to the
limited number of pixels, more than one cortical layer often was
projected to a single detector, and distinctions between layers could
not always be made based on the imaging data. For example, the image of
six cortical layers was projected to five photodiodes in Fig.
3, and so some
photodiodes detect activity from more than one cortical layer.
Nevertheless, it is clearly shown by traces from the photodiodes and by
the consecutive image display in Fig. 3 that the epileptiform event
started from a middle layer and propagated to superficial and deep
layers as well as horizontally across the cortical slice. The onset of
the epileptiform event started earliest in the trace recorded from the
middle layer and was delayed (by ~10 ms) in the adjacent detectors.
The further the physical location was from the initiating site, the
more delayed the onset (Fig. 3, top). This also is seen in
the consecutive image display where a red spot representing activation
of cortical neurons appeared in a middle layer and then expanded to the
superficial and deep layers as well as in lateral directions (Fig. 3,
bottom). These results indicated that epileptiform activity
was initiated by an epileptiform focus located in layer III or IV in
the parietal cortex. The location of such a focus was quite stable;
repeated optical recordings (n = 10) from the same
preparation revealed the same initiation site and propagation profile.
Figure 4 is a second example from the
same preparation. This is consistent with the finding that there often
exists a dominant epileptiform focus (Tsau et al. 1998).
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In some preparations, the initiation sites were found to be located in deep layers (probably layer V or VI; Fig. 5, top). The epileptiform events started from deep layers and spread out to the adjacent areas. In other preparations, the initiation sites were found to be in superficial layers (probably layer I or II; Fig. 5, bottom). Optical recordings were repeated 4-10 times on each preparation. These initiation sites were stable, and shifting of the initiation site was not observed. Therefore it seemed that spontaneous epileptiform activity could be initiated in any of the cortical layers. All of the imaging data on the epileptiform focus showed that the activation started from a small confined area <0.3 × 0.3 × 0.4 mm, or 0.04 mm3, and spread smoothly from the initiation site to adjacent cortical areas.
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The initiation site across the cortical layers varied from preparation
to preparation. These sites were scattered over the slice and were
observed in neocortical slices from occipital, parietal, temporal, and
perirhinal areas in different preparations (n = 11).
Among the 11 preparations, 5 had initiation sites in deep layers, 2 in
middle layers, and 4 in superficial layers (Fig. 6). These initiation sites seemed to be
distributed randomly and not to have higher susceptibility in any
particular cortical region. Although evidence has been presented that
the temporal cortex may be more likely to originate epilepsy (e.g.,
Wardas et al. 1990), our data suggest that there is no
preference for a particular cortical area or an exclusive cortical
layer in establishment of an initiation site for epileptiform activity
when the cortex is hyperexcitable and capable of conducting traveling
epileptiform events.
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Continuous recordings of spontaneous firing of cortical neurons
Our optical imaging data have shown the dynamic process of epileptiform activity initiation. In all the experiments, no consistent firing of cortical neurons was optically observed before an epileptiform event was initiated. No consistent firing was observed optically even at the initiation site. Because each photodetector covers 0.04 mm3 of cortical tissue, significant firing of a single neuron may be masked by light noise from many other cortical cells. Another limitation of optical imaging is that each trial of recording is limited to a few seconds due to dye bleaching. We used a microelectrode in many locations in the slices to detect constant firing of neurons. This allowed us to record spikes from the neurons close to the recording electrode's tip. In most experiments (68 of 72), no continuous firing was observed. Occasionally (4 experiments), continuous firing from a single neuron was observed before as well as during epileptiform events. However, none of the activity was correlated to the initiation of epileptiform events. These data indicate that spikes in a single cortical neuron may not be able to initiate epileptiform activity. Although the recording period can be as long as several hours, the major limitation of the electrical recording is that it is very difficult to accurately position the electrode at the initiation site of an epileptiform event.
Electrically evoked epileptiform events
We showed in the preceding text that spontaneous epileptiform
events can be initiated in several cortical layers; it seems that
cortical neurons in several layers have this capability. However, it is
possible that an initiation site that appears in middle or superficial
layers is actually initiated by layer V pyramidal neurons the
discharges of which trigger the activation of an initiation site
located in one of the other cortical layers. Optical recordings might
not be able to detect a small initiating discharge from layer V
pyramidal neurons. This implies that even though we have seen the
activity starting from the superficial or middle layers, it actually
might be triggered by undetectable activity in the deep layers, and
neurons in superficial layers might not be capable of initiating
epileptiform activity themselves. To test this possibility, a
microelectrode was used to measure the ability of an electrical
stimulus to initiate epileptiform events in different layers. A single
electrical pulse (10 V, 0.1 ms) was delivered via an electrode (its tip
diameter was <1 µm, usually ~0.1 µm) with impedance of 2-5
M, placed in one of the cortical layers. Assuming that the
electrical impedance of fluid and brain tissue is much lower than that
of the microelectrode and can be ignored, the stimulation current
I = V/R would be 2-5 µA at the
electrode's tip. The current density would be 2,500 times smaller at a
distance of 50 µm from the tip on the assumption of current spread
from a point source in a volume conductor. Therefore only the neurons
close to the tip would be activated directly by the stimulus, and the
number of neurons should be very limited. We found that epileptiform
events were evoked reliably in all cortical layers by the electrode
stimulation. The evoked epileptiform events had the same
characteristics as those of spontaneous events and propagated
vertically and horizontally. The initiation site depended only on the
position of the stimulation electrode's tip (e.g., Fig.
7). The images, made at 2-ms intervals,
show clearly that activation first appeared at the electrode tip with
no delay and then spread smoothly to the neighboring areas. No jumping of activation from the electrode tip to deep layers or other cortical areas was observed in any of the experiments. Various areas of the
cortex including frontal, occipital, parietal, temporal, perirhinal, and hippocampal cortex were tested; all the cortical structures were
found to be capable of generating evoked epileptiform events (n = 36). Because the timing for stimulation was
arbitrarily set, the evoked epileptiform events must have started
independently of any spontaneous pyramidal neuron discharges.
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DISCUSSION |
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Our optical imaging data directly revealed spontaneous initiation
sites. These initiation sites were found to be stable in every
preparation. In different preparations, the initiation sites were in
different cortical layers where different types of neurons are located.
Continuous spontaneous activity correlated with epileptiform events was
not observed in either the focus or other regions between epileptiform
events. We also used extracellular microelectrodes to monitor single
unit activity during as well as between epileptiform events and did not
observe consistently firing neurons in the neocortical slices.
Therefore it is reasonable to assume that most neurons, except those
responsible for initiation, are not continuously firing between
epileptiform events. We previously have hypothesized that a dominant
initiation focus may be organized by a dynamic process in a group of
local neurons (Tsau et al. 1998); our current results
suggest that this kind of process is not laminar-specific, and it can
happen in various cortical layers.
Optical signals reflect the membrane potential change in all membranes stained with voltage-sensitive dyes. Because neurons contribute to the optical signal regardless of their physical size, a change in membrane potential of virtually all the cortical neurons could contribute to the optical signals. As we report in this study, the initiation site can be located in several cortical layers and so would be composed of neurons of different types because each cortical layer has distinctive cell types. Different types of neurons in various layers may become initiation neurons and trigger epileptiform activity.
Previous studies showed that elimination of layer V abolishes
spontaneous activity in cortical slices (Silva et al.
1991). To explain our data of visualizing initiation foci in
superficial layers, one may assume that an epileptiform event may be
triggered by an initiation site located in layer V pyramidal neurons,
but the activities in layer V are too small to be detected by our optical recording technique. We used the microstimulus experiment to
reduce this possibility. When epileptiform events were evoked by
electrical stimulation, the number of neurons activated directly by
stimulation presumably was limited. In this experiment, we always saw
the activity start from the simulation point instead of from layer V. This suggests that an epileptiform event starts at the layers where the
stimulation electrode is placed. The current density of stimulation is
high around the microelectrode's tip, and the effective area is
probably smaller than a distance of 50 µm. This volume of cortical
tissue can contain only 100 neurons with a 10-µm diameter. It is
therefore likely that the neurons directly activated by the stimulation
electrode also start the epileptiform activity. However, our data do
not rule out the possibility that layer V pyramidal neurons provide
background activity for the neurons in other layers; certain patterns
of the spontaneous activities in layer V may trigger other layers to
start all-or-none epileptiform events. It is difficult for our current
experimental setting to demonstrate that pyramidal neurons provide
background activities. However, Demir et al.
(1999)
suggested similar activities, a
"preepileptiform activity" that can trigger an all-or-none
epileptiform event in other places. Cortical neurons in both
superficial and deep layers in the neocortex can have repetitive
discharge properties (Flint and Connors 1996
). Neurons
in isolated superficial layers of entorhinal cortex also can fire
repetitively (Dickson and Alonso 1997
). It long has been
believed that neurons with repetitive discharge properties are capable
of initiating epileptiform activity (Miles and Wong
1983
; Prince 1967
, 1969
; Traub and Wong
1982
). Our data further suggest that neurons in various layers
may be capable of initiating epileptiform activity and that the
initiation process may be less reliant on one specific type of neuron.
The initiation site revealed by our optical imaging data was composed
of a confined volume of <0.04 mm3, indicating that the
initiation of epileptiform events is a localized process and may be
from local neuronal clusters with potentiated connections (Tsau
et al. 1998). As this is the smallest volume a photodiode could
detect in our experiments, the real epileptiform focus could be
smaller. We hypothesize that a neuron pool with repetitive discharge
properties in such a confined epileptiform focus may synchronously
activate its adjacent neurons and thus initiate epileptiform activity.
This is consistent with the stimulation experiment where a single-pulse
stimulus generates synchronized activation of cortical neurons around
the microelectrode tip and results in an all-or-none epileptiform event.
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ACKNOWLEDGMENTS |
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We thank Drs. L. Cohen and N. Hershkowitz for helpful comments, and A. Schaefer for technical assistance with the experiments and manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-31425, Department of Defense Grant 17-93-V3018, and a grant from Epilepsy Foundation of America.
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FOOTNOTES |
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Address for reprint requests: J.-Y. Wu, Institute for Cognitive and
Computational Sciences, WP 24A Research Building, Georgetown University
Medical Center, 3970 Reservoir Rd., N.W., Washington, DC
20007.
E-mail: wuj{at}giccs.georgetown.edu
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 25 January 1999; accepted in final form 10 June 1999.
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REFERENCES |
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