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The Journal of Neuroscience, July 15, 1998, 18(14):5426-5432
Role of the Nucleus Raphe Magnus in Antinociception Produced
by ABT-594: Immediate Early Gene Responses Possibly Linked to
Neuronal Nicotinic Acetylcholine Receptors on Serotonergic
Neurons
R. Scott
Bitner,
Arthur L.
Nikkel,
Peter
Curzon,
Stephen
P.
Arneric,
Anthony W.
Bannon, and
Michael W.
Decker
Neurological and Urological Diseases Research, Pharmaceutical
Products Division, Abbott Laboratories, Abbott Park, Illinois
60064-3500
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ABSTRACT |
Recently, a novel cholinergic channel modulator,
(R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594),
was shown to produce potent analgesia in a variety of rodent pain
models when administered either systemically or centrally into the
nucleus raphe magnus (NRM). The purpose of the present study was to
investigate the possible supraspinal contribution of ABT-594 by
assessing its ability to induce expression of the immediate early gene
c-fos, a biochemical marker of neuronal activation, in the NRM of rats. Putative serotonergic neurons in the NRM, a medullary nucleus proposed
to be involved in descending antinociceptive pathways, were identified
immunohistochemically using a monoclonal antibody (mAb) against
tryptophan hydroxylase. ABT-594 (0.03-0.3 µmol/kg, i.p.) produced a
dose-dependent induction of Fos protein that was blocked by the central
nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (5 µmol/kg, i.p.) but not by the peripheral nAChR antagonist
hexamethonium (15 µmol/kg, i.p.). Immunohistological studies using
mAb 299 revealed the expression of  4-containing nAChRs in the NRM.
The 4 immunostaining was dramatically reduced by pretreating (30 d)
animals with the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which was previously shown to substantially attenuate the
antinociceptive actions of ABT-594. In a double immunohistochemical labeling experiment, coexpression of the serotonin marker tryptophan hxdroxylase and the 4 nAChR subunit in NRM neurons was observed. These results suggest that the analgesic mechanism of ABT-594 may in
part involve the activation of the NRM, a site where 4-containing nAChRs are expressed by serotonergic neurons.
Key words:
ABT-594; nucleus raphe magnus; c-fos expression; nicotinic antagonism; serotonin; 4-containing nicotinic
acetylcholine receptor; antinociception
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INTRODUCTION |
Antinociception produced by nicotine
was first reported >60 years ago (Davis et al., 1932 ), yet the use of
nicotinic acetylcholine receptor (nAChR) agonists as viable analgesics
has received limited consideration. However, the discovery of
epibatidine, a potent nAChR agonist with analgesic properties (for
review, see Sullivan and Bannon, 1996) and the cloning of neuronal
nAChR subunits (Sargent, 1993; McGehee and Role, 1995) have provided a
stronger basis for investigation of analgesic nicotinics that act more
selectively in the CNS. Indeed, the novel nAChR agonist
(R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594) was recently reported to produce antinociception equal in
efficacy to morphine in a variety of rodent pain models after systemic
and central administration (Bannon et al., 1998).
The nucleus raphe magnus (NRM) is a serotonergic nucleus located in the
rostral ventromedial medulla of the brainstem. Axons of the NRM project
to the spinal cord (Bowker et al., 1982), terminating primarily in the
dorsal horn (Jones and Light, 1990). Brainstem nuclei that project to
the dorsal horn of the spinal cord can function to inhibit afferent
nociceptive transmission (Basbaum and Fields, 1979; Willis, 1988;
Sandkuhler, 1996). Activation of these descending antinociceptive
pathways may be triggered by physiological stimuli (Millan et al.,
1980) as well as by pharmacological agents (Gogas et al., 1991).
Antinociception involving the NRM has been studied after either
electrical stimulation or direct administration of pharmacological
agents (Proudfit and Anderson, 1975; Oleson et al., 1978; Brodie and
Proudfit, 1986).
Iwamoto (1991) reported that nicotine injected directly into the NRM
produced antinociception in rat models of thermal pain. Similarly, we
demonstrated that an intra-NRM injection of ABT-594 produced
antinociception in a thermal model of acute and a chemical model of
persistent pain (Curzon et al., 1997; Bannon et al., 1998). Mapping
studies in the rat have revealed the expression of different nAChR
subunits in various brainstem regions at the RNA (Wada et al., 1989)
and protein (Swanson et al., 1987) levels, supporting an nAChR-mediated
mechanism of antinociception involving a supraspinal site of action.
However, to date there are no reports specifically identifying nAChR
expression in the NRM.
The purpose of the present study was to further investigate the
possible contribution of the NRM to antinociception produced by
systemic ABT-594. Expression of the immediate early gene c-fos is
triggered by a variety of depolarizing events (Morgan et al., 1987;
Sagar et al., 1988; Morgan and Curran, 1991), including nAChR
activation (Greenberg et al., 1986). We previously demonstrated that an
antinociceptive dose of ABT-594 given systemically produced an increase
in c-fos expression in the NRM of rats (Bannon et al., 1998). Here,
further studies were conducted to determine the ability of ABT-594 to
induce the expression of Fos protein in the NRM as an in
situ marker of nAChR activation. Effects of nAChR antagonism on
ABT-594-induced c-fos expression were also assessed. In addition, the
expression of the 4 subunit of the nAChR in the NRM was
immunohistochemically evaluated. To specifically test the possibility
that 4-containing nAChRs are expressed by serotonin-containing
neurons, we also examined the effects of a local serotonergic lesion on
4 expression in the NRM and assessed coexpression by double
immunohistochemical labeling.
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MATERIALS AND METHODS |
Animals and experimental treatments.Male Sprague
Dawley rats (Charles River Laboratories, Portage, MI) weighing 350-400
gm were used. The animals were group-housed in an American Association for the Accreditation of Laboratory Animal Care-approved facility at
Abbott Laboratories (Abbott Park, IL) in a temperature-regulated environment with lights on between 7:00 A.M. and 8:00 P.M.. All experimental procedures involving animals were conducted under protocols approved by Abbott's Institutional Animal Care and Use Committee.
Rats received a single intraperitoneal injection of either saline (1 ml/kg) or ABT-594 (0.03, 0.1, or 0.3 µmol/kg) or, in other studies,
were pretreated with either the nAChR antagonists hexamethonium (15 µmol/kg, i.p.) or mecamylamine (5 µmol/kg, i.p.) followed 15 min
later by ABT-594 (0.3 µmol/kg, i.p). Two hours after drug treatment
the animals were deeply anesthetized with pentobarbital and perfused
through the aorta with buffered saline (3 min, 25 ml/min) followed by
10% formalin (12 min, 30 ml/min). The brains were removed and
post-fixed in either 10% formalin or 20% sucrose PBS for 12-24 hr
before immunohistochemical assessment.
Rats were anesthetized intraperitoneally with 55 mg/kg sodium
pentobarbital and placed in a David Kopf student stereotaxic instrument
(Tujunga, CA) with the skull on an even horizontal plane. Coordinates
for intra-NRM injections from intra-aural zero were anteroposterior,
 2.5 mm; mediolateral, 0.0 mm; and dorsoventral 0.5 mm. Rats were
then injected with either PBS or the serotonin neurotoxin
5,7-dihydroxytryptamine (5,7-DHT; 3.75 nmol in 0.3 µl over 60 sec)
using a 31 gauge injector. Thirty days after an intra-NRM injection of
5,7-DHT (or PBS), the animals were anesthetized and perfused, as
described above. The ability of an intra-NRM injection of 5,7-DHT to
produce a serotonergic lesion was determined by assessing tryptophan
hydroxylase immunostaining throughout the NRM.
Compounds. ABT-594 was synthesized (Holladay et al., 1997)
at Abbott Laboratories. Mecamylamine hydrochloride, hexamethonium hydrochloride, and 5,7-dihydroxytryptamine creatinine sulfate were
obtained from Sigma (St. Louis, MO).
Immunohistochemistry of paraffin sections. After
post-fixation in 10% formalin, brains from saline-treated animals were
processed and embedded in paraffin, sectioned (6 µm), and mounted on
aminosilane-coated glass slides (Newcomer Supply). Specifically,
coronal sections from the brainstem that contained the NRM (9.3 mm to
11.0 mm from bregma; Paxinos and Watson, 1997) were mounted. The
immunohistochemical procedure used consisted of a three-step
avidin-biotin complex (ABC)-peroxidase technique. Sections were first
deparaffinized through a xylene and graded alcohol series, followed by
a 20 min incubation in blocking serum. The sections were then incubated for 60 min with a primary antibody (Ab) against tryptophan hydroxylase (mouse monoclonal IgG, 2.3 µg/ml; Sigma) or the 4 subunit of the
nAChR [rat monoclonal IgG, monoclonal antibody (mAb) 299, 2 µg/ml;
purchased from J. Lindstrom, University of Pennsylvania]. Sections
were next washed in PBS and incubated in a biotinylated secondary Ab
solution (10 µg/ml) for 30 min, washed in PBS, and then incubated
with ABC reagent (Vectastain Elite, Vector Laboratories, Burlingame,
CA). The sections were visualized by incubation for 2-8 min in a
peroxidase substrate solution (diaminobenzidine) and counterstained
with hematoxylin. Sections were examined and photographed with a DMRB
light microscope (Leica, Nussloch, Germany).
Double immunohistochemical labeling using Abs against tryptophan
hydroxylase and the 4 nAChR subunit, described above, was performed
using a fluorochrome and biotinylated secondary Ab, respectively.
Sections from the NRM of a saline-treated rat were first incubated with
the anti-tryptophan hydroxylase Ab for 90 min and then washed in PBS
and incubated with a fluorescein-conjugated secondary Ab (20 µg/ml,
Vector Laboratories) for 60 min. The sections were then examined and
photographed under a fluorescence microscope (excitation filter BP,
450-490 nm; Leica, DMRB). Next, the same sections were incubated with
the anti-4 Ab (mAb 299) overnight at 4°C. The following day the
sections were washed in PBS and incubated for 30 min with a
biotinylated secondary Ab (5 µg/ml, Vector Laboratories) that was
mouse- adsorbed and affinity-purified to prevent cross-reactivity with
the primary Ab used in the previous incubation against tryptophan
hydroxylase. Finally, the sections were incubated with ABC reagent,
visualized by incubation for 8 min in a peroxidase substrate solution
(diaminobenzidine), and counterstained with hematoxylin. The sections
were again examined and photographed under a light microscope (Leica,
DMRB) and compared with the previous fluorescent micrographs for double
labeling.
Immunohistochemistry of free-floating sections. After
overnight incubation in 20% sucrose-PBS, brains from saline- and
ABT-594-treated rats were cut on a cryostat (40 µm coronal sections).
Free-floating sections were immunostained for Fos protein using a
three-step ABC-peroxidase technique beginning with a 30 min incubation
with blocking serum. Sections were next incubated with anti-Fos Ab (sheep polyclonal IgG, 1:2000; Genosys) for 48 hr at 4°C, washed with
PBS, and incubated for 1 hr with a biotinylated secondary anti-sheep Ab
solution (1:200). Finally, sections were washed in PBS, incubated with
ABC reagent (Vector Laboratories), and then developed in a peroxidase
substrate solution. The sections were mounted, coverslipped, examined,
and photographed with a light microscope (Leica, DMRB). Fos-like
immunoreactivity (FLI) was quantified using an image analysis system
(Leica, Quantimet 500) that identified and counted immunostained
neurons according to a gray level that was empirically determined
before analysis.
Statistics. Data were analyzed using ANOVA followed by
Fisher's protected least significant difference (PLSD) post
hoc test. Statistical analyses on data were performed using
StatView (Abacus Concepts, Calabasas, CA).
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RESULTS |
Neurochemical-immunohistochemical identification of the NRM
Studies were conducted to immunohistologically identify the
serotonin-containing neurons of the NRM using an antibody against the
rate-limiting enzyme of serotonin synthesis, tryptophan hydroxylase. Positive immunostaining was observed centered above the left and right
pyramidal tracts in the medial ventral medulla, the location of the NRM
(Fig. 1). A similar pattern of staining
in the NRM was also observed using a polyclonal Ab against serotonin
that was prevented by preincubation of the primary Ab with 20 µg/ml serotonin (data not shown).
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Figure 1.
Immunohistochemical identification of the nucleus
raphe magnus (NRM). The rat NRM is located (open square)
in the rostral ventromedial medulla and is centered just above the
pyramidal tract at the base of the brainstem (A)
(adapted from Paxinos and Watson, 1997). Serotonergic neurons of the
NRM (B) were immunohistochemically identified
using an mAb against tryptophan hydroxylase in a saline-treated rat.
Scale bar, 100 µm.
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ABT-594-induced Fos expression in the NRM
The ability of ABT-594 to induce the expression of Fos protein in
the NRM was used as an in situ measure of neuronal
activation. Two hours after systemic administration of ABT-594 (0.03, 0.1, and 0.3 µmol/kg, i.p.), an increase in Fos immunostaining was observed in the NRM (Fig. 2). Fos-like
immunoreactivity was quantitated using a Quantimet 500+ image
analysis system. ABT-594 produced a dose-dependent induction of FLI in
the NRM (F(3,20) = 7.336; p = 0.0017), as illustrated in Figure 3. At
the higher 0.3 µmol/kg dose, ABT-594 produced a fourfold increase in
Fos expression compared with saline-treated controls
(p < 0.01).
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Figure 2.
Photomicrographs of Fos protein in the NRM after
ABT-594. Induction of c-Fos expression in the NRM of rats is shown 2 hr
after systemic administration of ABT-594 at either 0, 0.03, 0.1, or 0.3 µmol/kg intraperitoneally. The nuclear immunostaining associated with
Fos expression is highest in the 0.3 µmol/kg-treated rat.
White hatched triangle represents area of NRM analyzed
for FLI. Scale bar, 100 µm.
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Figure 3.
Graphs of ABT-594 Fos protein induction. Systemic
administration of ABT-594 (0.03-0.3 µmol, i.p.) in rats
(n = 6 per group) produced a dose-dependent
induction of Fos expression in the NRM
(F(3,20) = 7.336; p = 0.0017). Fos-like immunoreactivity (FLI) was
quantified and expressed as the percent of saline-treated control FLI
mean ± SEM [hatched lines represent ± control SEM; *p < 0.01 vs saline-treated rats;
protected least significant difference (PLSD) post hoc
test].
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The effects of nAChR antagonism on ABT-594-induced c-fos expression are
summarized in Figure 4. In these
experiments, ABT-594 (0.3 µmol/kg, i.p.) produced a significant
increase in FLI (p < 0.05) that was blocked by
pretreatment with the nAChR antagonist mecamylamine (5 µmol/kg,
i.p.). In contrast, pretreatment with the peripheral nAChR antagonist
hexamethonium (15 µmol/kg, i.p.) did not prevent the expression of
Fos produced by ABT-594 (0.3 µmol/kg).
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Figure 4.
Graphs of ABT-594 Fos protein induction and the
effects of nicotinic antagonism. Administration of the nAChR antagonist
mecamylamine (5 µmol/kg, i.p.) 15 min before ABT-594 (0.3 µmol/kg,
i.p.) prevents ABT-594-induced Fos expression in the NRM of rats
(n = 4-6 per group) 2 hr later
(A). Administration of the peripheral nAChR
antagonist hexamethonium (15 µmol/kg, i.p.) 15 min before ABT-594
(0.3 µmol/kg, i.p.) did not prevent ABT-594-induced Fos expression in
the NRM of rats (n = 4-6 per group) 2 hr later
(B). FLI is expressed as the percent of saline
control FLI mean ± SEM (*p < 0.05 compared
with saline-treated rats; +p < 0.05 as compared
with ABT-594-treated rats; PLSD post hoc test).
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nAChR expression in the NRM
Immunohistochemical analysis of 4-containing nAChRs in the NRM
is summarized in Figure 5. Expression of
the 4 nAChR was observed in the NRM using mAb 299. At higher
magnification, it was evident that the immunostaining was greatest in
the soma of the neurons, with the exception of the nucleus, but
positive staining was also present in the neuropil. Using a polyclonal
Ab raised against the C terminus of the 4 subunit, a similar pattern
of staining compared with mAb 299 was observed and was prevented with
preincubation of the peptide (20 µg/ml) used to raise the Ab (data
not shown). Alternatively, there were neurons in the NRM that did not
show expression of the 4 subunit.
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Figure 5.
Photomicrographs of the 4 nAChR subunit in the
NRM. 4 immunostaining (mAb 299) was observed in the NRM
(A) of a saline-treated rat. At higher
magnification (B), the 4 subunit is seen
primarily in the cytosol of the soma. Expression of 4 is also seen,
but to a lesser extent, in the neuropil. Scale bars: A,
100 µm; B, 10 µm.
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Intra-NRM 5,7-DHT and nAChR expression
To examine the effects of a serotonergic NRM lesion on the
expression of 4-containing neurons in the NRM, rats received a single intra-NRM injection of 5,7-DHT, a serotonin neurotoxin. Immunohistochemical assessment of the expression of both the serotonin marker tryptophan hydroxylase and the 4 subunit of the nAChR was
performed 30 d after 5,7-DHT (or PBS) treatment (Fig.
6). A similar pattern of immunostaining
with regard to size and number of positive cells in the NRM was
observed in a saline-treated rat, using antibodies against tryptophan
hydroxylase (Fig. 6A) and the nAChR 4 subunit
(Fig. 6C). Intra-NRM administration of 5,7-DHT (3.7 nmol)
almost completely reduced both tryptophan hydroxylase (Fig.
6B) and 4 (Fig. 6D)
immunostaining, compared with the saline-treated control rat. However,
staining using an antibody against the neurofilament protein NF-200, a
neuronal marker, was observed in the NRM of both the PBS and
5,7-DHT-treated rats (data not shown).
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Figure 6.
Photomicrographs of tryptophan hydroxylase and the
4 nAChR subunit in the NRM and the effects of a serotonergic
neurotoxin. Expression of tryptophan hydroxylase is shown in the NRM of
rats 30 d after either an intra-NRM injection of 0.3 µl of PBS
(A) or 3.75 nmol of 5,7-DHT
(B). Expression of the 4 nAChR subunit is
shown in the same animals again, 30 d after either a single
intra-NRM injection of 0.3 µl of PBS (C) or
3.75 nmol of 5,7-DHT (D). The serotonin
neurotoxin resulted in almost a complete loss of serotonergic neurons
and expression of 4-containing nAChRs in the NRM. Scale bar, 100 µm.
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Double immunohistochemical labeling of tryptophan hydroxylase
and 4
To provide direct proof that 4-containing nAChRs are located on
serotonergic neurons in the NRM, double immunohistochemical labeling of
tryptophan hydroxylase and 4 was conducted. There was almost a
complete overlap of immunostaining for tryptophan hydroxylase (Fig.
7A) compared with 4 (Fig.
7B) in neurons of the NRM. For control experiments (data not
shown), the primary Ab against 4 during the second incubation was
omitted; no 4 immunoreactivity was observed in these sections.
Furthermore, reversing the order of the primary Ab incubations and
replacing the anti-tryptophan hydroxylase with a polyclonal Ab against
serotonin produced the same type of overlapping pattern of
coexpression.
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Figure 7.
Photomicrographs of double labeling of tryptophan
hydroxylase and the 4 nAChR subunit in the NRM. Using a
fluorescein-conjugated secondary Ab, tryptophan hydroxylase
immunostaining (A) shows almost complete overlap
compared with the DAB chromogenic immunostaining of 4-containing
neurons (B) in the NRM of a saline-treated rat.
Double-labeled neurons are indicated by arrows. Scale
bar, 100 µm.
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DISCUSSION |
We previously demonstrated that intra-NRM administration of
ABT-594 produced antinociception in models of acute and persistent pain
(Curzon et al., 1997; Bannon et al., 1998). Results of this study
further support a role for the NRM as one potential site of action for
the antinociception produced by ABT-594. Systemic administration of
ABT-594 produced a dose-dependent induction of Fos expression in the
NRM that was blocked by systemic mecamylamine, but not hexamethonium.
Immunohistochemical evaluation of the NRM revealed the presence of the
nAChR 4 subunit, consistent with the possibility that
ABT-594-induced c-fos expression may be attributable to the direct
activation of an 4-containing nAChR within the NRM. Finally, neurons
in the NRM expressing 4 appear to be serotonergic, as evidenced by
the loss of 4 expression after intra-NRM administration of the
serotonergic neurotoxin 5,7-DHT and, moreover, by the coexpression of
tryptophan hydroxylase and 4 revealed by double-labeling
experiments.
Induction of the immediate early gene c-fos can be triggered by various
stimuli that produce depolarization (Morgan et al., 1987; Sagar et al.,
1988; Morgan and Curran, 1991), including the agonist-receptor
interaction resulting in the opening of a cation channel, as seen with
activation of the nAChR (Greenberg et al., 1986). In this regard,
drug-induced expression of c-fos has been used to identify neuronal
activation in given brain nuclei (Sagar et al., 1988), thereby
revealing potential sites of drug action. The ability of ABT-594 to
produce a dose-dependent induction of c-fos expression in the NRM, as
observed in these studies, suggests that the NRM may indeed be involved
in antinociception produced by systemic administration of ABT-594.
Furthermore, antagonism of ABT-594-induced NRM Fos by mecamylamine, but
not hexamethonium, suggests that activation of the NRM, whether direct
or indirect, requires activation of central nAChRs.
The in situ mapping strategy involving drug-induced Fos
expression has been used with systemic administration of ()-nicotine. Ren and Sagar (1992) reported high levels of Fos immunostaining in the
central visual pathways, including the superficial superior colliculus
and the medial terminal nucleus as well as the interpeduncular nucleus,
after a 60 min intravenous infusion of nicotine in rat. However, in
areas known to contain high levels of nicotine-binding sites, such as
the medial habenula, thalamus, substantia nigra, and ventral tegmental
area (Clarke et al., 1985), nicotine-induced Fos expression was not
observed. Moreover, they did not report c-fos induction in the NRM, as
seen in the present study with ABT-594. This may suggest that ABT-594
activates nAChRs differentially from nicotine. Similarly, other studies
demonstrating expression of Fos after systemic nicotine administration
(Salminen et al., 1996; Pich et al., 1997) do not report activation of
the NRM.
Despite the lack of reports that nicotine induces Fos expression in the
NRM, ()-nicotine injected into the NRM has been shown to produce
antinociception in the rat hot-plate and tail-flick models of thermal
pain (Iwamoto, 1991). We have shown that intra-NRM injection of ABT-594
in rats, consistent with studies using nicotine, produces
antinociception in the hot-box test of thermal pain and the formalin
test of persistent pain (Curzon et al., 1997). In these studies,
systemic mecamylamine pretreatment or intra-NRM coinjection with
mecamylamine blocked the antinociceptive effects of ABT-594 injected
into the NRM, supporting an nAChR-dependent mechanism in the NRM. In
contrast, intra-NRM administration of mecamylamine did not block
antinociception produced by systemic injection of ABT-594. This result
may suggest that nicotinic activation of the NRM is not required for
antinociception produced by systemic ABT-594. However, other sites of
action, in addition to the NRM, may be important in mediating the
effects of systemic ABT-594 antinociception. In other studies from our
laboratory, systemic injection of ABT-594 was shown to also increase
Fos expression in the locus coeruleus (Bitner et al., 1997), a
brainstem nucleus known to be involved in descending antinociceptive
pathways (Yaksh, 1985). Another possible explanation for a lack of
antagonism with intra-NRM mecamylamine could lie in the methodology of
the experiment itself. The 0.3 µl intra-NRM injection volume of
mecamylamine used in the above experiment may not have been sufficient
to antagonize all of the nAChRs throughout the entire NRM that would be
activated by systemically administered ABT-594.
To examine the possibility that ABT-594 interacts directly with nAChRs
in the NRM, immunohistochemical studies were conducted to determine
whether the 4 nAChR subunit was expressed in the NRM. It has been
suggested that the 4 2 subtype comprises at least 90% of neuronal
nAChRs in brain (Flores et al., 1992; Lindstrom et al., 1995).
Consistent with Fos mapping, studies using either radioligand
autoradiography (Clarke et al., 1985), immunohistochemistry (Swanson et
al., 1987), or in situ hybridization (Wada et al., 1989)
have identified nAChRs in various brainstem nuclei, including the
superior colliculus, the ventral tegmental area, and the
interpeduncular nucleus. However, there have been no reports that
specifically cite the presence or confirm the expression of
nAChR-containing neurons in the NRM, as seen in the present study, and
thus these data represent a novel finding.
To neurochemically characterize the type of neuron that may express
4, we assessed the effects of a serotonergic lesion on 4
immunostaining in the NRM. Intra-NRM injection of 5,7-DHT reduced the
immunostaining of the rate-limiting enzyme of serotonin biosynthesis tryptophan hydroxylase as well as 4 immunostaining, indicating that
serotonergic neurons in the NRM may express 4-containing nAChRs.
This was directly confirmed by double-labeling experiments demonstrating that neurons in the NRM coexpress tryptophan hydroxylase and 4 protein.
Because of their descending projections to the dorsal horn of the
spinal cord (Bowker et al., 1982), nAChR activation of serotonergic neurons in the NRM by ABT-594 may inhibit afferent nociceptive transmission at the level of the dorsal horn. In support of this proposed mechanism, the antinociceptive effect of systemically administered ABT-594 in a thermal model of pain was reduced by 62% in
5,7-DHT-treated rats, and ABT-594 injected directly into the NRM
produced antinociception (Curzon et al., 1997). Similarly, others have
demonstrated that either electrical stimulation (Oleson et al., 1978;
Brodie and Proudfit, 1986) or pharmacological activation (Proudfit and
Anderson, 1975; Iwamoto, 1991) of the NRM can produce antinociception
in rats. Antinociception produced by intra-NRM injections of the
nicotinic agonist N-methylcarbachol was shown to be blocked
by intrathecal administration of the serotonin receptor antagonists
LY53857 (5-HT1c/2) and S-()-zacopride
(5-HT3) (Iwamoto and Marion, 1993), again supporting
the possibility that nAChR agonists can stimulate serotonergic neurons
in the NRM that descend to the spinal cord to bring about
antinociception.
In conclusion, data from the present study provide further evidence
consistent with the possibility that the antinociceptive action of the
novel nAChR agonist ABT-594 may in part be mediated by activation of
the NRM. Moreover, the involvement of the NRM in ABT-594
antinociception may be attributable to the activation of an
4-containing nAChR located on serotonergic neurons. However, it
should be emphasized that the NRM may be only one of several nuclei
that represent potential sites of antinociceptive action produced by
ABT-594, and other subunit-containing nAChRs besides, or in addition
to, 4 may be involved. Similar studies, as reported here, involving
ABT-594-induced activation of other CNS nuclei as well as the mapping
of additional nAChR subunits are ongoing.
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FOOTNOTES |
Received Jan. 7, 1998; revised April 20, 1998; accepted April 24, 1998.
This research work was supported by Abbott Laboratories.
Correspondence should be addressed to Dr. R. Scott Bitner, Neurological
and Urological Diseases Research, Pharmaceutical Products Division,
Abbott Laboratories, Building AP9A-LL (D-47W), Abbott Park, IL
60064-3500.
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Top
Abstract
Introduction
Compound via MeSH
