Section of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616
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ABSTRACT |
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Jinks, Steven L. and E. Carstens. Activation of Spinal Wide Dynamic Range Neurons by Intracutaneous Microinjection of Nicotine. J. Neurophysiol. 82: 3046-3055, 1999. Nicotine evokes pain in the skin and oral mucosa and excites a subpopulation of cutaneous nociceptors, but little is known about the central transmission of chemogenic pain. We have investigated the responses of lumbar spinal wide dynamic range (WDR)-type dorsal horn neurons to intracutaneous (ic) microinjection of nicotine in pentobarbital-anesthetized rats. Nearly all (97%) units responded to nicotine microinjected ic (1 µl) into the low-threshold region of the hind-paw mechanosensitive receptive field in a concentration-related manner (0.01-10%). Responses to repeated injections of 10% nicotine exhibited tachyphylaxis at 5-, 10-, and 15-min interstimulus intervals. Significant tachyphylaxis was not seen with 1% nicotine. All nicotine-responsive units tested (n = 30) also responded to ic histamine (1 µl, 3%) and did not exhibit tachyphylaxis to repeated histamine. However, there was significant cross-tachyphylaxis of nicotine to histamine. Thus 5 min after ic nicotine, histamine-evoked responses were attenuated significantly compared with the initial histamine-evoked response prior to nicotine, with partial recovery over the ensuing 15 min. Neuronal excitation by ic nicotine was not mediated by histamine H1 receptors because ic injection of the H1 receptor antagonist, cetirizine, had no effect on ic nicotine-evoked responses, whereas it significantly attenuated ic histamine-evoked responses in the same neurons. The lowest-threshold portion of cutaneous receptive fields showed a significant expansion in area at 20 min after ic nicotine 10%, indicative of sensitization. Responses to 1% nicotine were significantly reduced after ic injection of the nicotinic antagonist, mecamylamine (0.1% ic), with no recovery over the ensuing 40-60 min. These data indicate that nicotine ic excites spinal WDR neurons, partly via neuronal nicotinic acetylcholine receptors that are presumably expressed in cutaneous nociceptor terminals. Repeated injections of high concentrations of nicotine led to tachyphylaxis and cross-tachyphylaxis with histamine, possibly relevant to peripheral analgesic effects of nicotine.
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INTRODUCTION |
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Nicotine and other cholinergic agonists evoke
sensations of irritation or pain when delivered to skin or oral mucosa
(Dessirier et al. 1997, 1998
; Jansco et al.
1961
; Jarvik and Assil 1988
; Keele and
Armstrong 1964
; Magerl et al. 1990
). Cholinergic
agonists excite unmyelinated sensory fibers, including identified
nociceptors (Douglas and Ritchie 1960
;
Fjallebrant and Iggo 1961
; MacIver and Tanelian
1993
; Steen and Reeh 1993
; Tanelian
1991
; Wang et al. 1993
). Little is known about
central nociceptive processes triggered by peripheral nicotine. It
recently was reported that nicotine, as well as a variety of other
irritant chemicals, excites neurons in the dorsomedial or ventrolateral
aspects of trigeminal subnucleus caudalis when applied topically to the
surface of the tongue or cornea, respectively (Carstens et al.
1995
, 1998
). Trigeminal neuronal responses to nicotine
increased in a concentration-dependent manner and exhibited
tachyphylaxis to repeated application (Carstens et al.
1998
).
Although nicotine is associated with cutaneous pain and irritation,
systemic administration of nicotine produces analgesia (e.g.,
Cooley et al. 1990; Yang et al. 1992
).
Peripheral administration of ABT-594, a derivative of the powerful
cholinergic agonist epibatidine, is also analgesic (Bannon et
al. 1998
). In the present study, we have investigated further
nicotinic excitation and possible sensitization of spinal neurons
associated with nociception and determined if subsequent neuronal
responses show tachyphylaxis possibly consistent with a peripheral
analgesic effect. We also investigated the role of neuronal nicotinic
acetylcholine receptors (nAChRs) in the nicotinic excitation of spinal
neurons. An abstract of part of this work has appeared previously
(Jinks and Carstens 1998b
).
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METHODS |
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Preparation, recording, and unit characterization
Experiments were conducted with approval from the local animal
care and use advisory committee. The methods were the same as in our
previous studies (Carstens 1997; Jinks and
Carstens 1998a
) and are summarized here. Single-unit
recordings were made in 44 adult male Sprague-Dawley rats anesthetized
with pentobarbital sodium (induction: 65 mg/kg iv, maintenance: 10-20
mg · kg
1 · h
1 iv via the
jugular vein). Anesthesia was sufficient if animals were areflexic and
did not exhibit tachycardia (monitored by electrocardiogram) in
response to noxious stimulation. The lumbar spinal cord was exposed by
laminectomy and stabilized in a frame, and the dura was opened to allow
extracellular single-unit recordings with a tungsten microelectrode.
Units were searched using innocuous mechanical stimulation of the
ipsilateral hind paw, and only those that additionally responded to
noxious skin heating (48 or 50°C for 5 s) were tested with
chemical stimulation. Unit receptive fields were mapped using von Frey
monofilaments with three different bending forces (0.7, 1.5, and 4 g) (see Jinks and Carstens 1998
).
Chemical stimulation
A 30.5-gauge needle attached to PE 50 tubing filled with
chemical solution was inserted just beneath the epidermis in the lowest-threshold portion of the mechanical receptive field (see Carstens 1997). Separate needles were used for different
chemicals or concentrations. There was no evidence of inflammatory
response to needle placements over the <1-h recording period.
Chemicals were injected in a 1 µl volume over ~1 s. The following
chemicals were used: nicotine (0.01-10% = 6 × 10
4
6 × 10
1 M
in 0.9% NaCl; free base, Sigma), histamine dihydrochloride (3% = 3 × 10
1 M in 0.9% NaCl; Sigma), mustard
oil (allyl-isothiocyanate, 50% = 5 M, diluted in paraffin oil; Merck),
mecamylamine (0.1% = 4.9 × 10
5 M, in
0.9% NaCl; Sigma), and cetirizine HCl (0.1% in 0.9% NaCl; Pfizer).
Experimental design and data analysis
Action potentials were discriminated and sampled using Spike
software (Forster and Handwerker 1990). Neuronal
responses to thermal or chemical stimuli were quantified as the total
number of action potentials per 60 s after stimulus application.
Responses are illustrated in peristimulus time histogram (PSTH) format
(binwidth 1 s).
Histamine was tested before nicotine in 24 units, whereas nicotine was
tested first in the remainder. Usually only one unit, or two units (1 on each side of the spinal cord), were tested per animal. In a few
cases, two units were tested on the same side; they had spatially
separate receptive fields (heel vs. toe) and 1 h elapsed between recordings.
The following sets of experiments were made in separate groups of
units. 1) Graded concentrations of nicotine were injected in
ascending order. Mean responses at different concentrations were
compared using repeated measures ANOVA followed by a Newman-Keuls multiple comparisons test. 2) Tachyphylaxis: nicotine at a
fixed concentration (1 or 10%) was microinjected at a 5-, 10-, or
15-min interstimulus interval (for a total of 5 groups) to test for
tachyphylaxis. Mean responses across injection trials within each group
were compared using a paired t-test. 3)
Cross-tachyphylaxis: histamine was injected first, followed 5 min later
by nicotine, and followed thereafter by repeated injections of
histamine at 5-min interstimulus intervals. Mean responses to histamine
injections at various times postnicotine were compared with the
prenicotine response to histamine using a paired t-test.
4) H1 antagonist: the effect of ic microinjection of the H1
histamine receptor antagonist, cetirizine, on neuronal responses evoked
by ic nicotine and ic histamine was tested. Mean neuronal responses to
nicotine or histamine postcetirizine were compared with those before
cetirizine using a paired t-test. 5) Receptive
field expansion: cutaneous mechanical receptive fields were mapped
before and 20 min after ic nicotine (10%). Vehicle (saline) controls
were not presently conducted because we previously reported that they
do not result in any change in receptive field areas (Carstens
1997; Jinks and Carstens 1998a
). Mean receptive field areas pre- and postnicotine were compared using a paired t-test. And 6) mecamylamine: responses to 1%
nicotine were recorded before and at 10-min interstimulus intervals
after ic injection of mecamylamine as close as possible to the site of
ic nicotine injections. Nicotine at 1% was chosen because this
concentration does not cause significant tachyphylaxis, which otherwise
might confound interpretation of postmecamylamine data. Mean responses at different times postmecamylamine were compared with the mean premecamylamine response using an unpaired t-test. In all
cases, P < 0.05 was considered to be statistically significant.
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RESULTS |
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Unit sample
A total of 71 lumbar WDR-type dorsal horn units responded to low-threshold mechanical as well as noxious thermal stimulation of skin within the cutaneous receptive field on the ipsilateral hind paw. Receptive fields varied in size, spanning as little as one to two toes or as much as the entire ventral hind paw surface (Figs. 1-7). Most units were localized to the mid-dorsal horn [mean depth: 518.8 ± 246.9 (SD) µm].
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Ninety-seven percent of the units responded to ic nicotine, and each of 30 nicotine-responsive units tested also responded to ic histamine. Responses of a typical WDR unit to noxious heat and irritant chemicals are shown in Fig. 1. The majority (60%) of units showed no or little (1-3 Hz) spontaneous firing, whereas a minority (40%) showed spontaneous firing rates from 3 to 20 Hz.
Concentration-response relationship
All units tested exhibited a concentration-dependent increase in firing after ic nicotine. Figure 2A shows an example of a unit that gave progressively larger and longer-lasting responses to increasing concentrations of ic nicotine. In Fig. 2B, individual (thin lines) and mean (thick line) responses of units are plotted against the concentration of ic nicotine. Responses increased significantly with concentration (ANOVA, P = 0.006), with mean responses to 0.01 and 0.1% being significantly less compared with 10%.
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Tachyphylaxis
At the highest concentration used (10%), the initial injection of nicotine evoked a large response and subsequent injections elicited progressively smaller responses. An example is shown in Fig. 3A. Figure 3B plots individual (thin lines) and mean (thick lines) neuronal responses to repeated injections of 10% nicotine at a 5-min interstimulus interval, showing that responses to both the second and third application of nicotine were reduced significantly compared with the first injection. When the interstimulus interval was increased to 10 (Fig. 3C) or 15 min (Fig. 3D), only the mean response to the third, but not the second, injection of nicotine was reduced significantly. Figure 3, E and F, shows individual and mean responses to a lower (1%) concentration of nicotine delivered at 5- and 10-min interstimulus intervals, respectively, which did not exhibit significant tachyphylaxis.
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Cross-tachyphylaxis to histamine
Because repeated application of the higher concentration of
nicotine produced tachyphylaxis, we tested if there was
cross-tachyphylaxis to histamine. An individual example is shown in
Fig. 4, A-E, and mean
responses of all units tested are plotted in Fig. 4F. After nicotine, the mean response to the next application of histamine (His-1) was significantly lower compared with the initial
histamine-evoked response (His) before nicotine. Responses to
subsequent histamine (His-2, His-3 in Fig. 4F) showed a
partial recovery and, while still reduced, were not significantly
different compared with the initial trial with histamine. In separate
units we verified our prior report (Carstens 1997) of a
lack of tachyphylaxis to repeated injection of histamine (mean response
to 1st injection: 1,995 ± 1,536 impulses/min; mean response to
2nd: 1,790 ± 1,395; P > 0.05; n = 7).
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Histamine H1 receptor antagonist
Because nicotine exhibited cross-tachyphylaxis to histamine, we
addressed the possibility that nicotine might act via cutaneous histamine H1 receptors by determining if a selective histamine H1
receptor antagonist, cetirizine, affected neuronal responses evoked by
ic nicotine. We first verified that the cetirizine reduced ic
histamine-evoked responses in the same units. In the example shown in
Fig. 5, the response to ic histamine
(Fig. 5A) was attenuated significantly after ic cetirizine
(Fig. 5B) with recovery (Fig. 5C), confirming an
earlier report (Carstens 1997). Responses of the same
unit to ic nicotine before and after ic cetirizine are shown in Fig. 5,
D and E, respectively, and demonstrate that the histamine antagonist had no effect on the nicotine-evoked response. Figure 5F plots mean responses to histamine and nicotine for
six units and shows that the mean response evoked by histamine, but not
nicotine, was reduced significantly by ic cetirizine, with subsequent
recovery.
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Receptive field expansion
Although 10% nicotine induced tachyphylaxis, we also tested if it
increased receptive field area consistent with sensitization. We
measured receptive field areas before and again 20 min after ic
microinjection of 10% nicotine. Figure
6A shows mechanosensitive receptive field areas of a unit mapped using three different von Frey
filaments, before (left) and 20 min after, ic nicotine
(right). After nicotine there was expansion of the
lowest-threshold portion (Fig. 6A, ), as well as a
reduction in mechanical sensitivity in a small area surrounding the
injection site (
). Figure 6B plots mean receptive field
areas determined with the three von Frey filaments before and 20 min
after nicotine. There was a significant expansion only in the
lowest-threshold area (Fig. 6B,
). However, when the area
around the injection site showing reduced tactile sensitivity was
subtracted from the low-threshold area, the postnicotine change in
low-threshold receptive area was no longer statistically significant.
Thus 10% nicotine triggers a limited expansion at the fringe of the
low-threshold mechanical receptive field suggestive of sensitization
but also reduces mechanical sensitivity at the injection site.
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Mecamylamine
We addressed the possible role of nAChRs in mediating nicotinic excitation of spinal dorsal horn neurons by testing if responses to nicotine were reduced after local microinjection of its antagonist, mecamylamine. Figure 7A shows an example. Before mecamylamine, ic nicotine (1%) evoked a robust response (Fig. 7A, left). Mecamylamine then was injected ic as close as possible to the site of nicotine microinjection (Fig. 7A, middle) and elicited a small response as seen in 2/10 other units. Nicotine then was injected 1 min later and evoked a response that was reduced markedly (Fig. 7A, middle), followed by partial recovery 40 min later (right). Averaged nicotine-evoked responses for 10 units are plotted in Fig. 7B versus time. Mean responses were significantly lower at 10, 30, and 40-60 min postmecamylamine compared with the premecamylamine level.
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DISCUSSION |
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Nicotine ic excites spinal WDR dorsal horn neurons in a concentration-dependent manner. Excitation by a high concentration of nicotine is associated with expansion at the fringe of the lowest-threshold cutaneous receptive field region. At the same time, the 10% (but not 1%) concentration of nicotine triggered tachyphylaxis to subsequent nicotine, as well as cross-tachyphylaxis to histamine. Nicotine-evoked responses were unaffected by the selective H1 histamine receptor antagonist, cetirizine, indicating that nicotine did not act via cutaneous H1 receptors. Neuronal responses to nicotine were attenuated by local ic injection of mecamylamine, indicating involvement of nAChRs in nicotinic excitation of cutaneous nociceptors that, in turn, excite spinal WDR neurons.
Tachyphylaxis and cross-tachyphylaxis
Repeated application of 10% nicotine led to significant
tachyphylaxis, which may be mediated by a reduction in sensitivity of
peripheral nociceptors and/or a central mechanism. Nicotine excites
nociceptors presumably via nAChRs expressed in their peripheral fiber
terminals in the skin (see following text); the nociceptor afferent
fibers in turn excite dorsal horn neurons. A peripheral desensitization
of nociceptor endings by nicotine might be mediated via influx of
Ca2+ through nAChRs such as 4
2,
7 and
3
2 (Flores et al. 1996
; Galzi and Changeaux
1995
; Gotti et al. 1997
; Holladay et al.
1997
; Lindstrom et al. 1996
; McGehee and
Role 1995
; Puttfarcken et al. 1997
) or by gating
of voltage-sensitive Ca2+ channels.
Ca2+ influx then might trigger intracellular
events leading to a reduction in excitability of the nociceptor
terminal, possibly analogous to the desensitizing effect of capsaicin
on dorsal root ganglion neurons (Caterina et al. 1997
;
Chard et al. 1995
; Cholewinski et al.
1993
; Liu and Simon 1996b
; Malmberg et
al. 1998
). A local anesthetic effect of the high nicotine
concentration also may have contributed to the tachyphylaxis. This
possibility is supported by our observation of increased mechanical
threshold at the site of 10% nicotine injection (Fig. 6A).
Alternatively, the tachyphylaxis may have been mediated centrally by a
reduction in the efficacy of synaptic transmission from
nicotine-sensitive afferent fibers to dorsal horn neurons (see
following text).
Dorsal horn neurons do not exhibit significant tachyphylaxis to
repeated application of histamine at 5- or 10-min interstimulus intervals (Carstens 1997; Carstens et al.
1998
). It has been reported (Stahle-Backdahl et al.
1988
) that human itch sensation and cutaneous flare exhibit
tachyphylaxis to histamine, which, however, was repeated at much longer
interstimulus intervals (90 min or 24 h). Dorsal horn neuronal
responses to ic histamine were reduced significantly by prior injection
of nicotine (Fig. 4) or capsaicin (Carstens 1997
) at the
same skin locus. Nicotine cross-desensitization to histamine might
involve a peripheral mechanism by which nicotine reduces the
sensitivity of nociceptive endings to subsequent histamine, e.g., via
nAChRs or a local anesthetic effect. Failure of cetirizine to affect
nicotine-evoked excitation of dorsal horn neurons indicates that
nicotine cross-desensitization to histamine is not mediated by
intracutaneous histamine H1 receptors. Alternatively,
cross-desensitization could be mediated centrally. Nicotine might
trigger a centrally mediated depression in dorsal horn neuronal
excitability, although this is inconsistent with our observation of
receptive field expansion postnicotine. Subsequently delivered
histamine would activate a subset of chemonociceptive cutaneous
afferent fibers; if such fibers converged onto the same dorsal horn
neurons that were conditioned by prior nicotine, they might evoke a
lesser response. One practical implication of the present findings is
that peripheral application of cholinergic agonists might have value in
reducing itch in addition to any analgesic effect they may have.
It recently was reported that a synthetic derivative of epibatidine,
ABT-594, has powerful nonopioid analgesic effects that are partly
mediated by the CNS (Bannon et al. 1998). Interestingly, however, ic injection of ABT-594 also reduced responses of spinal dorsal horn neurons to noxious stimulation in a manner that was antagonized by local ic mecamylamine (Bannon et al.
1998
). This might conceivably be mediated by desensitization of
peripheral nociceptor fibers. In this regard, nicotine also has
analgesic properties via a central action (e.g., Cooley et al.
1990
; Iwamoto 1991
; Yang et al.
1992
). Nicotine injected into the skin might be taken up into
the circulation and distributed to the brain to exert an
antinociceptive effect on dorsal horn neurons. However, we injected
10% nicotine in a 1 µl volume (=0.1 mg dose of nicotine); even if
all of the nicotine was taken up into the circulation, it would still
be several-fold lower than dosages of nicotine that produce analgesia
in rats (e.g., 0.8 mg/kg) (Cooley et al. 1990
).
Receptive field expansion
After 10% ic nicotine there was an expansion at the fringe of the
lowest-threshold area of the cutaneous mechanical receptive field (Fig.
6). This was accompanied by a reduction in mechanical sensitivity at
the injection site. This decreased sensitivity might be attributable to
a reduction in sensitivity of local mechanoreceptors by nicotine,
although an earlier study reported that the cholinergic agonist,
carbachol, increased mechanical thresholds of C-fiber polymodal
nociceptors but not myelinated mechanoreceptors in rat skin
(Steen and Reeh 1993). No such increase in mechanical
threshold was observed after control ic saline injections
(Carstens 1997
; Jinks and Carstens
1998a
), indicating that it was not due to volumetric or other
nonspecific effects.
The increase in receptive field area may reflect a peripheral and/or
central sensitization. Cutaneous receptive fields of WDR and
nociceptive-specific dorsal horn neurons expand after inflammation
(Ren et al. 1992) or ic injection of capsaicin
(Simone et al. 1991b
) or histamine
(Carstens 1997
; Jinks and Carstens 1998a
). Receptive field expansion after inflammation
(Ren et al. 1992
) or ic histamine (Jinks and
Carstens 1998a
) was prevented by intrathecal pretreatment with
N-methyl-D-aspartate (NMDA) receptor antagonists, consistent with the idea that the receptive field expansion reflects an NMDA-receptor-dependent central sensitization of
dorsal horn neurons (see Jinks and Carstens 1998a
).
Alternatively, however, nicotine might act peripherally to sensitize
intracutaneous fiber terminals of nociceptors within the receptive
field fringe area, which also might manifest as an increase in the WDR
neuronal receptive field area. In either case, an increase in the area of mechanical receptive fields of pain-signaling neurons may be associated with allodynia (pain elicited by a normally nonpainful mechanical stimulus) that normally ensues after peripheral tissue injury. Although ic application of nicotine is painful, it is not
presently known if this is accompanied by hyperalgesia and allodynia.
Role of nAChRs in nociception
nAChRs consisting of heteromeric 4
2 or
3
4 subunit
combinations, or an
7 homooligomer, are distributed widely in the
CNS (Galzi and Changeaux 1995
; Gotti et al.
1997
; Holladay et al. 1997
; Lindstrom et
al. 1996
; McGehee and Role 1995
) as well as in
dorsal root and trigeminal ganglion neurons (Boyd et al.
1991
; Flores et al. 1996
; Liu et al.
1998
; Swanson et al. 1987
; Wada et al.
1989
). A fraction of dorsal root (Sucher et al.
1990
) or trigeminal ganglion neurons (Liu and Simon
1996a
; Liu et al. 1993
) exhibited inward,
depolarizing currents in the presence of nicotine and other cholinergic
agonists. nAChR antagonists hexamethonium and neuronal (
-)
bungarotoxin, but not the muscarinic antagonist atropine, blocked the
currents (Sucher et al. 1990
), suggesting involvement of
the
4
2 nAChR. Another study (Liu et al. 1993
) reported that nAChR antagonists mecamylamine and hexamethonium, and in
some cases also atropine and
-bungarotoxin (consistent with an
7
nAChR), blocked the inward currents. Our present results are also
consistent with a partial role for nAChRs because nicotine-evoked responses of dorsal horn neurons were significantly reduced after ic
mecamylamine. However, we cannot rule out the possibility that other
cholinergic receptors also participate in nociceptive mechanisms. Cutaneous nociceptors with afferent C-fibers were excited by carbachol applied to the skin, and some were excited additionally by nicotine, muscarine, or both (Steen and Reeh 1993
). Nicotine- and
muscarine-evoked excitations were antagonized, respectively, by
hexamethonium and atropine, suggesting involvement of both nAChRs and
muscarinic cholinergic receptors in chemonociceptive transduction
(Steen and Reeh 1993
). Together with these previous
studies, our present data are consistent with a major role for nAChRs
in mediating nicotine-evoked excitation of dorsal horn neurons and
pain, whereas a role for muscarinic receptors is less certain.
Pain and itch
Histamine produces itch sensation in humans (Keele and
Armstrong 1964; Simone et al. 1987
, 1991a
). Some
cholinergic agonists are reported to produce sensations of itch or itch
mixed with pain (Magerl et al. 1990
). It is not clear if
nicotine evokes itch, rather than or in addition to pain. It recently
was reported that acetylcholine excited histamine-sensitive,
mechanically insensitive C-fiber nociceptors in skin (Schmelz et
al. 1998
). These latter histamine-sensitive fibers have been
suggested to be "itch" receptors (Schmelz et al.
1997
). Therefore it is possible that some of the present WDR
units responsive to both ic histamine and nicotine signal itch.
However, the same neurons also responded to noxious heat. Although the
present results do not resolve mechanisms by which pain and itch are
signaled by ascending sensory pathways (see Carstens
1997
; Jinks and Carstens 1998a
), they emphasize the potentially important role of cholinergic mechanisms in pain and
possibly itch and their interactions.
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ACKNOWLEDGMENTS |
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The authors are grateful to Pfizer, Inc. for a generous gift of cetirizine.
This work was supported by California Tobacco-Related Disease Research Program Grant 6RT-0231 and National Institute of Neurological Disorders and Stroke Grant NS-35788.
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FOOTNOTES |
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Address for reprint requests: E. Carstens, Section of Neurobiology, Physiology and Behavior, University of California, 1 Shields Ave., Davis, CA 95616.
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 21 June 1999; accepted in final form 13 August 1999.
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REFERENCES |
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