Chem. Senses 28: 181-189,
2003
© Oxford University Press 2003
Sensitivity and Selectivity of Neurons in the Medial Region of the Olfactory Bulb to Skin Extract from Conspecifics in Crucian Carp, Carassius carassius
Division of General Physiology, Department of Biology, PO Box 1051, University of Oslo, N-0316 Oslo, Norway
Correspondence to be sent to: Kjell B. Døving, Division of General Physiology, Department of Biology, PO Box 1051, University of Oslo, N-0316 Oslo, Norway. e-mail: kjelld{at}bio.uio.no
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Abstract |
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To examine the functional subdivision of the teleost olfactory bulb, extracellular recordings were made from the posterior part of the medial region of the olfactory bulb in the crucian carp, Carassius carassius. Bulbar units classified as type I or type II were frequently and simultaneously encountered at a recording site. Type I units displayed a diphasic action potential (AP) with a relatively small amplitude, a short duration (rise time
1 ms) and high spontaneous activity (2.5 per s). Type
II units exhibited an AP with a rise time of 1.8 ms and low spontaneous
activity (1.5 per s). The AP of this latter unit was nearly always followed by
a slow potential, a characteristic diphasic wave with a rise time of 5
ms. Chemical stimulation of the olfactory organ with a graded series of
conspecific skin extract induced an increased firing of the type I units.
During the period of increased activity of the type I units, the activity of
the type II units was suppressed. Stimulation with nucleotides, amino acids
and taurolithocholic acid did not induce firing of the type I units of the
posterior part of the medial region of the olfactory bulb. These results
indicate that the posterior part of the medial region of the olfactory bulb is
both sensitive to and selective for skin extract from conspecifics, which has
been shown to be a potent stimulus inducing alarm behaviour. The results of
the present study indicate that recording single unit activity from a
particular region of the olfactory bulb is a suitable method for isolating
pheromones or other chemical signals that induce specific activity in the
olfactory system. The projection of the neurons categorized as type II was
determined by antidromic activation of their axons by electrical stimulation
applied to the medial bundle of the medial olfactory tract. The anatomical
basis of the type I and type II units in the fish olfactory bulb is
discussed.
Key words: bulbar neurons, mitral cells, olfactory bulb, ruffed cells, selectivity, sensitivity, skin extract
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thommesen (1978
) first
presented the idea that discrimination of odours in teleosts is based upon a
topological representation of the sensory neurons onto the olfactory bulb.
This concept was confirmed by electrical stimulation of the axons of the
mitral cells that form the olfactory tract in living cod. Discrete stimulation
of the different tract bundles induced different behaviours interpreted as
feeding, alarm and courtship (Døving
and Selset, 1980). Ablation of the medial and the lateral parts of
the medial olfactory tracts in crucian carp eliminated the alarm reaction
(Hamdani et al.,
2000) and reduced courtship behaviour
(Weltzien et al.,
2003), respectively, while ablation of the lateral olfactory
tracts significantly reduced the feeding behaviour
(Hamdani et al.,
2001b). Anatomical studies with a neurological tracer demonstrated
that the olfactory receptor neurons (ORNs) with intermediate dendritic
lengths, bearing microvilli, participated in feeding behaviours
(Hamdani et al.,
2001a) and those with long dendrites and bearing cilia were
correlated with the alarm reaction (Hamdani
and Døving, 2002). Additionally, recent studies using other
techniques have revealed that morphologically different ORNs in rainbow trout
and zebrafish are stimulated specifically by different stimuli: microvillous
ORNs are stimulated by amino acids (Sato
and Suzuki, 2001; Lipschitz
and Michel, 2002) and ciliated ORNs by pheromones
(Sato and Suzuki, 2001).
The ideas expressed by Thommesen have been confirmed in different teleosts
by surface electrode recordings from the olfactory bulb
(Døving et al.,
1980; Hara and Zhang,
1996,
1998) and by optical imaging
(Friedrich and Korsching, 1997,
1998). Recently, the chemotopy
of the teleost olfactory bulb was confirmed by recording activity from single
olfactory bulb neurons (Nikonov and
Caprio, 2001). Because anatomical studies
(Kosaka and Hama, 1979;
Kosaka, 1980;
Alonso et al., 1987;
Arévalo et al.,
1991) and electrophysiological methods (Zippel et al.,
1999,
2000) have revealed the
existence of different types of neurons in the teleost olfactory bulb, it
seems obligatory that forthcoming electrophysiological studies focusing on the
activity of single neurons take into account the knowledge of these different
neuron types.
Several properties make the teleost olfactory bulb different from that of
mammals. The first is the absence of the periglomerular cells. The second is
the existence of a particular type of neurons called ruffed cells (RCs). One
distinctive character of this neuron is a series of protrusions making a ruff
at the initial portion of the axon (Kosaka
and Hama, 1979; Kosaka,
1980; Alonso et al.,
1987; Arévalo et
al., 1991). RCs, which constitute a significant proportion of
the mitral cell region, make few contacts with other neurons and do not seem
to receive direct inputs from the ORNs. The third property is the mitral cells
(MCs), which in teleosts are divided into two types according to the
morphology of their somata, the number and extension of their dendritic
arborization, the origin of the axons and their location in medial and lateral
portions of the olfactory bulb (Alonso
et al., 1988).
Given the histological features of the neurons within the teleost olfactory
bulb mentioned above, it is rewarding to see that by suitable
electrophysiological methods one can observe distinctions between the
different neurons (Zippel et al.,
1999,
2000). However, at present no
histo-physiological combined study has been made to confirm the anatomical
identity of the RCs. Yet, in the present study we confirm the duality of the
appearance of neuron activity from single units and the specificity of the
activity induced by skin extract at the medial part of the bulb. We will
advocate the possibilities of using electrophysiological recordings as a
bioassay for isolating pheromones or other biological active agents important
for the teleost olfactory system.
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Materials and Methods |
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Crucian carp, Carassius carassius L. (20–35 g body wt), were caught in a small lake (Tjernsrud) just outside Oslo city, Norway, and were transported to the aquaria facilities at the Department of Biology where they were fed three times a week. Fish were initially anaesthetized with benzocaine (45 mg/l) and immobilized by i.p. injection of Saffan (24 mg/kg; Schering-Plough Animal Health, Welwyn Garden City, UK). To avoid any unforeseen movement during the experiment, fish were wrapped in a wet cloth and fixed by two steel rods, which fastened to the upper parts of the orbital bones, taking care not to damage the olfactory epithelium. Fish were continuously irrigated through the mouth and over the gills by pond water during the experiments.
Surgery
The skull above the olfactory tracts and the right olfactory bulb was removed under a stereomicroscope. The mesenchymal tissue around the olfactory tract was aspirated by gentle sponging and the anterior part of the brain cavity was filled with paraffin oil. The medial bundle of the medial olfactory tract (mMOT) was separated from the rest of the olfactory tract, care being taken to avoid rupture of the blood vessels running along the olfactory tract. The fish remained in good condition for at least 8 h after surgery, as judged by the blood flow and the nervous activity recorded.
Recordings
Extracellular recordings from single (or a few) units in the posterior part
of the medial region of the olfactory bulb were performed with microelectrodes
made from tungsten wire (125 µm) prepared as described by Hubel
(1957). The position of the
electrode was adjusted by an electrical micromanipulator (SD Instruments MC
1000), and connected to an amplifier (Grass P55). The bandwidth was adjusted
to 0.3–3 kHz. A notch filter of 50 Hz was activated. The reference
electrode was positioned on the border of the brain cavity. Signals from the
amplifier were displayed on an oscilloscope (Tektronix 565; Portland, OR) and
made audible with an audio monitor. The nervous activity was also recorded on
a PC (Dell OptiPlex GX1p) via an analogue to digital converter (µ1401; CED,
Cambridge, UK) for later analysis and display. The nervous activity was stored
on a PC with the aid of a software program (Spike 2, version 4.04; CED).
Electrical stimulation of the mMOT
The projection of the RCs to the olfactory tracts is still uncertain. For
this reason, in some experiments (n = 6) of the present study the
mMOT was mounted onto a pair of platinium wires connected to an electronic
pulse generator that gave electric pulses with variable duration and intensity
(Grass SD9).
Chemical stimulation
The olfactory organ ipsilateral to the recording site (the right side) was
exposed to a continuous flow of artificial pond water (APW): 2.9 x
10-2 g/l NaCl, 3.7 x 10-3 g/l KCl, 5.8 x
10-2 g/l CaCl2, 1.6 x 10-2 g/l
NaHCO3. The flow could be interrupted by a series of miniature
valves to give exposure to solutions of different compositions prepared in
APW. This part of the study was divided into two subparts according to the
aims addressed. The first one, where the olfactory organ was stimulated by
conspecific skin extract made up at different dilutions, addressed the
sensitivity of the medial part of the olfactory bulb to skin extract. In the
second one, the olfactory organ was exposed, besides the skin extract, to a
series of potent stimuli of fish olfactory neurons made at 10-4 M:
a mixture of L-arginine, L-methionine and
L-alanine; a single amino acid (L-alanine); the
nucleotides adenosine 5'-triphosphate (ATP) and inosine
5'-triphosphate (ITP); taurolithocholic acid (TAUR).
Most of the experiments were performed in the following way. The stimulation series was tested from the lowest to the highest concentration of skin extract. The olfactory epithelium was not stimulated for a second time until the spontaneous activity returned to the pre-stimulus level. The stimulus was injected into the anterior naris of the olfactory organ through a polyethylene tube at a flow of 0.3 ml/min with minimal mechanical stimulation of the olfactory receptor cells. Inspections of the arrival of stimulant at the outlet of the tubing revealed that the onset of the increased activity of the bulbar cells occurred in conjunction with the arrival of the stimulus at the olfactory epithelium. These responses were termed excitatory.
Preparation of skin extract
Crucian carp were killed by decapitation and skin was taken from the sides
of the fish. Total weight was 2 g. The skin samples were placed in 100 ml
of distilled water and homogenized in a blender. The homogenate was filtered
through glass wool. The amount of dry material in the filtrate was 1.6
g/l. The filtrate was frozen immediately and fresh concentration series were
made before each experiment at dilution steps of 10-6,
10-5, 10-4, 10-3, 0.5 x 10-2
and 10-2 from stock solution.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extracellular recordings were made from 175 recording sites in 135 electrode penetrations of the olfactory bulb in 75 fish. When the microelectrode was advanced into the posterior part of the medial region of the olfactory bulb (the poserior medial quadrant), unitary discharges of different amplitudes were regularly observed. The appearance of the spikes was diphasic, with the initial deflection either positive or negative.
The amplitudes of the spikes depend upon the relation between the
localization of the recording electrode and the cell structure. Bulbar unit
activity was usually encountered at a depth of between 150 and 300 µm
corresponding to the MCs layer. Electrode penetration was usually stabilized
to a depth which allowed high amplitude of the spikes. Two distinct types of
unit activity could be distinguished at one particular recording site based on
their shapes and sizes. Type I units were characterized by a diphasic action
potential (AP) of short duration (rise time 1 ms). Such units correspond
presumably to MCs (Figure
1A,B).
Type II units displayed an AP with long duration (rise time 1.8 ms)
(Figure
1C,D).
The AP of this latter unit was nearly always followed by a slow potential
(SP), characteristic diphasic wave with a rise time of 5 ms. The delay
between AP and SP (measured as indicated in
Figure 1C) varied between 8 and
8.5 ms. Both types of units were activated by electrical stimulation of the
mMOT, and the conduction velocities calculated for different APs recorded from
type II units varied between 0.34 and 0.55 m/s (n = 6) at room
temperature (Figure 2). The
appearance of the AP and the following SP indicated a particular type of unit.
Zippel et al. (1999)
proposed that these units represent the activity of the so-called RCs.
However, because of the absence of a correlative histological identification
of the units recorded, we categorize these units as type I and type II in this
study.
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Spontaneous activity
The range of spontaneous activity of the bulbar neurons varied greatly from
0.01 to 4 per s. The mean spontaneous frequency varied for each particular
unit within a recording session without any obvious external cause. In all the
recordings made from the bulb, the mean spontaneous spike frequencies of the
two types of units were different. In general, the activity of type I units
was higher than that of type II units and reached a mean of 2.5 per s.
The range of spontaneous activity of type II units varied greatly during all
experiments, with a mean of 1.5 per s. The characteristic pattern of
discharge displayed by a given type I cell usually remained constant for
observation periods of several hours.
Effect of chemical stimulation
General effect of skin extract
Figure 3 shows a typical
recording from the posterior part of the medial region of the olfactory bulb
when stimulating the olfactory organ with skin extract. A remarkable increase
in the spontaneous activity of the bulbar units was noticed, i.e. the mean
frequency of a unit categorized as type I increased from 0.03 to 15 per s. It
is pertinent to note that the response was seen to begin somewhat later than
the opening of the miniature valve. This was due to the delay between the
opening of the valve and the arrival of the stimulus at the olfactory
epithelium. The pre-and the inter-stimulus activities were characterized by a
low frequency of the type I unit (mean value 0.03 per s) and a slightly higher
frequency of the type II unit (mean value 0.2 per s). During the period of
increased activity of the type I unit, which usually persisted after the end
of the stimulus, the type II unit was silent. These types of responses were
the most frequently observed effects evoked by skin extract.
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Sensitivity
Because the recordings showed that only neurons categorized as type I in
the medial part of the olfactory bulb reacted to skin extract with increased
firing rate, recordings with stimulation of different dilutions of skin
extract have been done only from this type of unit. Fifty-three recording
sessions were made from the posterior part of the medial region of the bulb in
25 different fishes when stimulating the olfactory organ with a series of
increasing concentrations of skin extract (10-6, 10-5,
10-4, 10-3 and 10-2 dilutions from stock
solution) and a mixture of amino acids (L-arginine,
L-methionine and L-alanine) (10-4 M). In all
recordings, the activated neurons (type I units) responded with increasing
frequencies to increasing concentrations of skin extract. No reaction to a
high concentration of a mixture of amino acids was noticed
(Table 1 and
Figure 4).
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Effect of L-alanine
Figure 5 shows the effect of
stimulation of the olfactory organ with L-alanine on the activity
of single units in the posterior part of the medial region of the olfactory
bulb. Three consecutive stimuli were applied: a 10-2 dilution of
skin extract (from stock), 10-4 M L-alanine and a 0.5
x 10-2 dilution of skin extract (from stock). As seen, a type
II unit displayed a spontaneous discharge rate of 1.1 per s and the type I
unit was silent. The stimulation with skin extract caused an increase in the
activity of the type I unit to 9.5 per s for a dilution of 10-2 and
to 1.75 per s for a dilution of 0.5 x 10-2 of the stimulus.
During firing of the type I unit, the type II unit ceased to fire. Application
of L-alanine caused a slight increase in the activity of the type
II unit from 1.5 to 4 per s. In a total of 17 observations, it was shown that
L-alanine induced an increase in the activity of the units
categorized as type II in the posterior part of the medial region of the
olfactory bulb.
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Selectivity
The variation in activity observed during chemical stimulation of the
olfactory epithelium by skin extract, a mixture of three amino acids
(L-arginine, L-methionine and L-alanine), a
single amino acid (L-alanine), adenosine 5'-triphosphate,
inosine 5'-triphosphate and taurolithocholic acid could be categorized
in two classes. Stimulation caused either an increased activity of the bulbar
units or an inhibition of their spontaneous discharge. Chemical stimuli were
applied at fixed durations and separated by long inter-stimulus intervals in
order to ensure the recovery of epithelial receptor neurons.
Effect of amino acids mixture, nucleotides and taurolithocholic
acid
Figure 6 shows a typical
recording from the posterior part of the medial region of the bulb when
stimulating the olfactory organ with a series of stimuli. Note that only skin
extract induced firing of type I units, which showed an increase in frequency
from 1.5 to 12 per s, while other stimuli did not induce an increase in
activity. No type II unit was active during this recording session, excluding
the possibility of observing the interactions between these two types of
interneurons. This type of response was the most common effect evoked by these
stimuli in the medial part of the bulb
(Figure 7).
Figure 7 also shows that the
order in which stimuli were delivered during one recording session had no
effect on the activity of the neurons.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of the present study demonstrate four features of the neurons in the olfactory bulb of crucian carp: (i) only type I units in the medial part of the olfactory bulb react exclusively to conspecific skin extract with increased activity; (ii) this reaction is concentration-dependant, i.e. the higher the concentration the higher the frequency of the response; (iii) the activity of the type II units is inhibited during the increased activity of the type I units; (iv) the type II units do probably project to the olfactory tracts.
Principally, the olfactory system in vertebrate animals possess the same
neural circuitry; ORNs expressing a particular receptor converge into
restricted areas within the olfactory bulb where they, together with dendrites
of MCs, form spherical structures called glomeruli
(Ressler et al.,
1994; Vassar et al.,
1994; Mombaerts et
al., 1996). The information received through receptors
expressed on ORNs triggers electrical signals travelling towards the brain via
synaptic connections in the glomeruli. However, many differences could be
noticed in different species. In teleosts, three morphologically different
ORNs are scattered throughout the olfactory epithelium: ciliated neurons,
microvillous neurons and crypt neurons
(Ichikawa and Ueda, 1977;
Thommesen, 1983;
Hansen et al., 1997;
Hansen and Finger, 2000).
Recently, we have shown that each morphological type of ORN sends axons to a
restricted area in the olfactory bulb. Microvillous neurons project to the MCs
that form the lateral olfactory tract (LOT) and participate in feeding
behaviour (Hamdani et al.,
2001a), whereas the ciliated neurons project to the MCs that form
the mMOT and participate in the alarm reaction
(Hamdani and Døving,
2002). In the olfactory bulb, the two types of relay neurons (MCs
and RCs) lie close to each other in the same layer and make synaptic
connections via granule cells. Consequently, when recording nervous activity
from the bulb by extracellular means, one should take into account the
presence of these two types of neurons, which, although located in the same
layer, could be easily distinguished. The distinction between MCs and RCs has
been done anatomically in different species
(Kosaka and Hama, 1979;
Kosaka, 1980;
Alonso et al., 1987;
Arévalo et al.,
1991). Furthermore, Zippel et al.
(1999,
2000) have suggested a
correlation between two different APs recorded from the bulb and these
anatomically different neurons (MCs and RCs).
In the present study, recordings from both bulbar neurons (type I and II
neurons) were made either separately or simultaneously in the posterior part
of the medial region of the olfactory bulb. We made recordings from this part
of the bulb because previous studies have shown that this part contains
neurons that project to the mMOT (Satou
et al., 1979;
Dubois-Dauphin et al.,
1980). In accordance with previous findings, our recordings show
two distinct types of nervous activity. One type of unit had a relatively fast
diphasic AP. The other had a longer diphasic AP of long duration and was
followed by a SP. The delay between the peak of the AP and the peak of the SP
was 8 ms. Zippel et al.
(Zippel et al., 1999)
have made recordings from the goldfish olfactory bulb and suggested that this
type of unit most probably originates from RCs. The RCs make synaptic contacts
with a large number of granule cells. It is conceivable that the SP is the
summed potential of the activity of a population of granule cells induced by
the particular unit from which one is recording [see also (Zippel et
al., 1999,
2000)]. According to this
suggestion, the amplitude of the slow wave potential depends on the number of
granule cells activated. It is a pertinent observation that the amplitude of
the peak of the SP varied more than the peak amplitude of the AP. The fact
that at a given recording site the amplitude of the AP followed by a SP was
always larger than that of the AP from a MC might reflect that the RCs are
larger than the MCs.
One of the objectives of the present study was to obtain information on the
projection of the type II cells from the bulb to the olfactory tracts. For
that reason, electrical stimulation of the mMOT was made in some recordings.
To ascertain that a neuron projected to mMOT one should ideally use a
collision test. However, in the present study this was impractical because
several units were encountered at a time and isolation of a particular unit
was difficult. Nevertheless, stimulation of the mMOT with electric pulses
evoked distinct recruitment of nervous units so that those with the lowest
firing threshold appeared first and displayed the highest conduction
velocities. The delay between the electrical shock and the appearance of a
particular unit was constant, indicating antidromic invasion and not
activation via synaptic input. These units were most probably MCs. The
conduction velocities of the APs of the type II units, probably RCs, indicate
that these cells had both myelinated and unmylineated axons in the mMOT. These
findings are in accordance with anatomical studies as both myelinated
(Alonso et al., 1987)
and unmyelinated (Kosaka,
1980) axons have been described to originate from the RCs. The
projection of the RCs to the olfactory tract has not been demonstrated in
previous studies and raises questions of the functional significance of these
cells and their central projections.
In our recordings of the nervous activity in the olfactory bulb we
encountered only one type of unit (type I) whose activity increased its firing
rate upon stimulation of the olfactory epithelium with skin extract, the type
I units. The firing rate of these cells increased with increasing
concentration of the skin extract. Thus, our results imply that the medial
part of the bulb reacts specifically to skin extract. No other odorants used
in the present study generated an increased firing rate of the neurons in this
part of the bulb. These results are in accordance with other studies using
other methods suggesting that the olfactory bulb is divided into different
functional zones (Friedrich and Korsching,
1997,
1998;
Nikonov and Caprio, 2001). It
should be noted that during the firing period of the type I units, the type II
units were silent, and vice versa, suggesting functional coupling
between these relay neurons, possibly via granule cells. A specific chemical
stimulation of the olfactory epithelium results in a stimulation of specific
ORNs that project to MCs in a delimited zone of the olfactory bulb. The
activated MCs stimulate granule cells, which in turn inhibit the RCs in the
vicinity. But because the RCs do not receive direct inputs from the ORNs, it
is still unclear how stimulation of the RCs induces inhibition of the MCs.
Zippel et al. (Zippel et
al., 2000) suggested that it is the inhibition of the MCs
that decreases the inhibition of the RCs via granule cells and consequently
induces the activation of the RCs.
The skin extract is a blend of various substances. Chemical analysis of
skin extract pointed out a content of amino acids of 56.6 µmol/g freeze
dried extract (Saglio and Fauconneau,
1985). A comparable concentration in our skin extract would mean
that the highest concentration of amino acids used in our experiments would be
0.56 µM. There were no responses in the medial olfactory bulb to our
amino acid mixture at 100 µM, indicating the specificity of the neurons in
the posterior part of the medial region of the olfactory bulb.
Finally, based on the results provided by the present and other investigations demonstrating a division of the olfactory bulb into different functional zones, one should be convinced that the method used in this study offers an advantage for using it as a bioassay for isolating pheromones or other biological active agents that are important for the fish olfactory system.
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Acknowledgments |
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This study was supported by the Norwegian Research Council. The authors are grateful to Alexander Kasumyan and Finn-Arne Weltzien for comments on earlier versions of this manuscript.
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Accepted January 20, 2003
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