Department of Anesthesia and Critical Care, University of Chicago,
Chicago, Illinois (D.L.H.) and Departments of
Anatomy and Physiology,
W.M. Keck Center for Integrative Sciences, University of California at
San Francisco, San Francisco, California (H.W., N.N. and A.I.B.)
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Introduction |
There
is considerable evidence that delta opioid receptor agonists
act in the spinal cord to produce antinociception. This evidence
includes pharmacological investigations of the antinociceptive effects
of i.t. administered delta opioid receptor agonists (Porreca et al., 1984
; Malmberg and Yaksh, 1992; Mattia et
al., 1992; Stewart and Hammond, 1993), electrophysiological
characterization of the effects of these agonists on the response
properties of dorsal horn neurons (Dickenson et al., 1987;
Hope et al., 1990; Kalso et al., 1992; Duggan and
Fleetwood-Walker, 1993) and neurochemical determinations of their
effects on the release of neurotransmitters from the spinal cord (Go
and Yaksh, 1987; Pohl et al., 1989; Collin et
al., 1991; Ueda et al., 1995). Other studies have used
immunocytochemical visualization of Fos, the protein product of the
immediate-early protooncogene c-fos (Curran et
al., 1984; Dragunow and Faull, 1989; Hughes and Dragunow, 1995),
to identify populations of neurons that are activated by noxious
stimuli (Hunt et al., 1987; Bullitt, 1989; Menétrey
et al., 1989; Herdegen et al., 1991) and to
concomitantly examine the ability of opioid receptor agonists to
suppress the expression of Fos-LI in the spinal cord (Presley et
al., 1990; Hammond et al., 1992; Abbadie et
al., 1994; Tölle et al., 1994). However, these
studies nearly exclusively examined the effects of morphine, a
mu opioid receptor agonist (Takemori and Portoghese, 1987;
Corbett et al., 1993). Consequently, the contribution of delta opioid receptors to the regulation of immediate-early
gene expression in the spinal cord remains unknown.
Previous studies of the effects of morphine on the expression of Fos-LI
in the spinal cord routinely used a systemic route of administration.
Because systemically administered morphine distributes to spinal, as
well as to peripheral and supraspinal sites of action, the neural
mechanism by which opioid receptor agonists suppress Fos-LI in the
spinal cord remains unclear. For example, there is considerable
evidence that opioid receptor agonists activate bulbospinal pain
modulatory pathways that originate in the periaqueductal gray and
ventromedial medulla (Gebhart, 1982; Basbaum and Fields, 1984; Gebhart
et al., 1984). Consistent with these data, i.c.v.
administration of morphine or of the more selective mu
opioid receptor agonist DAMGO produces antinociception and a
dose-dependent and naloxone-reversible inhibition of the expression of
Fos-LI in the spinal cord (Gogas et al., 1991, 1996a, b).
There is also evidence for a contribution of peripheral sites,
particularly under conditions of inflammatory nociception. Thus,
injection of morphine or other opioid receptor agonists in the hindpaw
of the rat attenuates the hyperalgesia produced by i.pl. injection of
inflammatory irritants (Joris et al., 1987; Stein, 1993,
1995; Hong and Abbott, 1995). Intraplantar administration of morphine also inhibits the expression of Fos-LI in the spinal cord in a dose-dependent and naloxone-reversible manner (Honoré et
al., 1996). In contrast, despite the well-established ability of
i.t. administered opioid receptor agonists to produce antinociception (Yaksh, 1993), it is not yet known whether this antinociception is
associated with a suppression of noxious-stimulus-evoked Fos-LI in the
spinal cord.
The present study specifically examined the effects of i.t.
administration of two prototypic delta opioid receptor
agonists, DPDPE and DELT, on the expression of Fos-LI evoked by
injection of formalin in one hindpaw of the rat. The ability of these
agonists to inhibit formalin-induced flinching behavior in the same
animals was also determined. For comparison, the effects of an
equiantinociceptive dose of the mu opioid receptor agonist
DAMGO were also examined. The results indicate that i.t. administration
of DAMGO produces a strong reduction in both formalin-induced flinching
behavior and in Fos-LI in the spinal cord. In contrast, neither the
delta-1 agonist DPDPE nor the delta-2 agonist
DELT produce a strong suppression of Fos-LI in the spinal cord despite
a significant reduction in formalin-evoked pain behaviors. These data
indicate that inhibition of Fos expression can be dissociated from the
antinociceptive effects of different opioid receptor agonists.
Furthermore, these results provide new evidence, at the level of a
"third messenger," that the antinociception produced by i.t.
administration of delta and mu opioid receptor
agonists is mediated by different mechanisms. A preliminary report of
this work has appeared (Hammond et al., 1995b).
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Methods and Materials |
Animals.
Male Sprague-Dawley rats (Sasco, Madison, WI;
250-350 g) were anesthetized with halothane. One end of a PE-10
catheter was introduced through a slit in the atlantooccipital membrane
and threaded caudally for 8 cm in the subarachnoid space, which
positioned the tip of the catheter at the L2 segment of the spinal
cord. The other end was tunneled subcutaneously and externalized at the
top of the head (Yaksh and Rudy, 1976; Hammond, 1988). The rats were
housed individually after surgery and allowed 5 to 6 days to recover
before testing.
Formalin test and experimental design.
Animals were placed
individually in Plexiglas testing chambers and allowed to acclimate for
at least 15 min. A mirror was situated behind the chamber and another
was situated below the floor of the chamber to allow an unobstructed
view of the rat's paws. The first study examined the effect of i.t.
pretreatment with DPDPE, DELT or DAMGO on formalin-induced flinching
and the expression of Fos-LI in the spinal cord. Either vehicle, 10 or 60 µg of DPDPE, 3.0 or 30 µg of DELT or 0.3 µg of DAMGO was
injected i.t. 10 min before the s.c. injection of 100 µl of 5%
formalin in the plantar surface of one hindpaw. These doses and the
pretreatment time were based on previous studies of the efficacy of
these agonists in the tail-flick, hot-plate and carrageenan-inflamed
paw-flick tests (Malmberg and Yaksh, 1992; Stewart and Hammond, 1993,
1994). The rats were returned to the testing chamber and the number of flinches that occurred during the next 60 min was recorded in 5-min
epochs. Three to five rats were selected from each group for
immunocytochemical analysis based on their behavioral scores approximating the mean of the group.
The second study established the pharmacologic specificity of the
effects of DPDPE, DELT and DAMGO on formalin-induced flinching and the
expression of Fos-LI in the spinal cord. Rats were injected i.t. with a
mixture of either 1.0 µg of BNTX and 60 µg of DPDPE, 3.0 µg of
NTB and 30 µg of DELT, or 3.0 µg of CTOP and 0.3 µg of DAMGO.
These doses of BNTX, NTB and CTOP antagonize the antinociceptive effects of DPDPE, DELT and DAMGO at delta-1,
delta-2 and mu opioid receptors, respectively, in
the rat (Stewart and Hammond, 1993, 1994; Hammond et al.,
1995a; Tseng et al., 1995). Ten minutes later 100 µl of
5% formalin was injected s.c. in the plantar surface of one hindpaw.
The rats were returned to the testing chamber and the number of
flinches that occurred during the next 60 min was recorded in 5-min
epochs. Three to five rats were selected from each group for
immunocytochemical analysis based on their behavioral scores
approximating the mean of the group. CTOP and NTB were coadministered
with their respective agonists so that the effects of the agonist and
antagonist would coincide (Malmberg and Yaksh, 1992; Guirmand et
al., 1994). Although BNTX is more effective when administered
shortly before DPDPE, it was coadministered in this study to ensure its
action throughout the observation period (Hammond et al.,
1995a).
The third study examined the effect of post-treatment with the highest
effective dose of DPDPE or DELT. These rats received a s.c. injection
of 100 µl of 5% formalin in the plantar surface of the left hindpaw.
After measurement of the first phase response to formalin
(i.e., 0-5 min), the rats were injected i.t. with either
vehicle, 60 µg of DPDPE or 30 µg of DELT, and returned to the
testing chamber. The number of flinches that occurred during the
subsequent 50 min was recorded in 5-min epochs. Immunocytochemical analysis was not conducted on these animals.
A final study evaluated whether injection of the agonists, by
themselves, induced an expression of Fos-LI in the absence of formalin.
This study controlled for possible nonspecific stimulatory effects of
the agonists that could mask a suppression of Fos-LI. For this study,
the rats were injected i.t. with either 60 µg of DPDPE
(n = 2) or 30 µg DELT (n = 2) and
returned to the testing chamber for 60 min. Tissue from these rats was
processed for immunocytochemical visualization of Fos-LI.
Immunocytochemistry.
Sixty minutes after the injection of
formalin, the rats were deeply anesthetized with 60 mg/kg i.p.
pentobarbital and perfused intracardially with 50 ml of 0.05 M PBS, pH
7.4 at 37°C followed by 500 ml of 4% formalin in 0.1 M phosphate
buffer pH 7.4 at 4°C. The spinal cord was postfixed in
situ for 90 min, removed from the vertebral canal and placed in
fresh fixative at 4°C for an additional 90 min. The tissue was then
cryoprotected in phosphate-buffered 30% sucrose buffer for at least 48 hr.
Fos-LI was visualized by ABC/glucose oxidase immunocytochemistry with
use of commercially available kits (Elite Vectastain; Vector
Laboratories, Burlingame, CA). The tissue was coded so that the person
performing the immunocytochemistry had no knowledge of the treatment
condition. Fifty-micron frozen serial sections were cut through the
lumbar enlargement of the spinal cord and collected in 0.05 M PBS.
After immersion in PBS containing 3% normal goat serum and 0.3%
Triton X-100 for 1 hr, the sections were incubated for 48 hr at 4°C
in a rabbit polyclonal antiserum directed against an in
vitro translated protein product of the c-fos gene
(courtesy of Dr. Dennis Slamon, Department of Hematology and Oncology,
UCLA) at a dilution of 1:20,000 in PBS containing 1% normal goat serum
and 0.3% Triton X-100. This antiserum does not recognize the
Fos-related antigens and had been preabsorbed with acetone-dried rat
liver powder for 1 hr at 37°C and 2 hr at 4°C to reduce background
staining. After incubation in the primary antibody, the tissue was
transferred to a goat anti-rabbit biotinylated secondary IgG complex
for 1 hr at room temperature and then exposed to the ABC
Elite complex for 1 hr at room temperature. Tissue sections
were thoroughly rinsed with Tris buffer, mounted from tap water onto
gelatin-coated slides, air dried, dehydrated in alcohol in a graded
manner, cleared in xylenes and coverslipped.
Quantitation of Fos-LI.
Four sections from the L4 or L5
segments of the spinal cord of each rat were randomly selected for
quantitation of Fos-LI. The sections were photographed at low power
(4×) using Kodak technical pan film and a Nikon Microphot-FXA
microscope. The film was developed with HC110 Dilution E developer,
stopped with Kodak stop bath and fixed with Kodak rapid fixative. The
individual sections were printed at 60× enlargement and overlaid with
an acetate sheet on which the distribution of Fos-LI neurons was then
plotted by a person with no knowledge of the treatment condition. For
quantitation, we divided the spinal cord into four regions of interest:
(1) the superficial laminae (laminae I, IIo and IIi); (2) the nucleus proprius (laminae III and IV); (3) the neck of the dorsal horn (laminae
V and VI); and (4) the ventral horn (laminae VII-X). The number of
Fos-LI neurons in each region was determined by averaging the counts
made in the four sections for each rat. The number of Fos-LI neurons in
a treatment group was then expressed as the mean ± S.E.M. of
these values.
Statistical analysis.
The number of flinches was expressed
as the mean ± S.E.M. Phase 1 was defined as the 5-min period
immediately after the injection of formalin. Phase 2 was defined as the
period 20 to 60 min after the injection of formalin. Two-way analyses
of variance for repeated measures, in which one factor was drug
treatment and the other factor was time, were used to compare the
effect of drug and vehicle treatment on the number of flinches evoked
by formalin. Comparisons of the number of Fos-LI neurons in the drug
treatment groups and in the vehicle treatment group were made by
one-way analysis of variance. Post hoc comparisons of
individual mean values were made by Newman-Keuls test.
Drugs and injections.
DPDPE (lot no. 13H58451), DELT (lot
no. 63H06631) and DAMGO (lot no. 121H58152) were purchased from Sigma
Chemical Co. (St. Louis, MO). BNTX hydrochloride (lot no. WY-III-69B)
was obtained courtesy of Research Biochemicals (Natick, MA) and the
NIDA Technology Branch. CTOP (lot no. FRY-297A) was purchased from
Research Biochemicals (Natick, MA). NTB was a gift from G.D. Searle
(lot no. XXI-146.3; Skokie, IL). DPDPE, DAMGO and BNTX were dissolved
in saline, which served as their respective vehicle control. DELT and
NTB were dissolved in 5% Molecusol (2-hydroxypropyl-
-cyclodextrin;
Pharmatec; Alachua, FL), which served as their respective vehicle
control. Drug solutions were made fresh and injected i.t. in a volume
of 10 µl followed by a 10-µl volume of saline. The location of the catheter was verified by direct visualization of the tip of the catheter after laminectomy.
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Results |
Effects of intrathecally administered DPDPE on formalin-induced
flinching and expression of Fos-LI in the spinal cord.
Intrathecal
pretreatment with DPDPE dose-dependently decreased the number of
flinches in both phase 1 and phase 2 (fig.
1A). Intrathecal administration of 60 µg of DPDPE inhibited formalin-induced flinching behavior to a
greater extent and for a longer duration than did 10 µg of DPDPE. The
inhibition of flinching produced by 60 µg of DPDPE was attenuated by
coadministration of 1.0 µg of BNTX (fig. 1A). In these rats, the
number of flinches did not differ from the number of flinches observed
in rats pretreated with 10 µg of DPDPE. The antinociceptive effect of
60 µg of DPDPE was completely antagonized by coadministration of 3 µg of BNTX (data not shown). However, because this dose of BNTX has
additional effects at delta-2 and mu receptors
(Hammond et al., 1995a) it was not suitable for further
study.
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Fig. 1.
Panel A depicts the effects of i.t. pretreatment
with saline ( ; n = 6), 10 µg DPDPE ( ;
n = 7), 60 µg DPDPE ( ; n = 7) or a combination of 60 µg DPDPE and 1.0 µg BNTX ( ;
n = 5) 10 min before the s.c. injection of 5%
formalin into the plantar surface of one hindpaw of the rat. Panel B
depicts the effects of i.t. post-treatment with saline (;
n = 5) or 60 µg of DPDPE (;
n = 5) 7 min after the injection of formalin. The
arrowhead in panel B indicates the time at which the DPDPE or saline
was administered. Each symbol represents the mean ± S.E.M. The
arrows indicate the time at which formalin was injected. Values that
differ from the corresponding vehicle control are indicated by
asterisks (P < .05) or daggers (P < .01).
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Intrathecal post-treatment with 60 µg of DPDPE, administered 7 min
after the injection of formalin, also significantly inhibited flinching
behavior in phase 2 (fig. 1B). The magnitude of the inhibition was
similar to the magnitude of inhibition observed in rats in which this
same dose of DPDPE was administered 10 min before the injection of
formalin (P > .6 for the 15- to 60-min period).
Figure 2, A to C, illustrates the
distribution and numbers of formalin-evoked Fos-LI neurons in the
spinal cords of rats pretreated with saline, 60 µg of DPDPE or a
mixture of 60 µg of DPDPE and 1.0 µg of BNTX. As reported
previously (Presley et al., 1990; Gogas et al.,
1991), s.c. injection of formalin induced the expression of large
numbers of Fos-LI neurons in the medial aspects of ipsilateral laminae
I, IIo and IIi and laminae V-VI, with fewer numbers present in laminae
III-IV and the ventral horn of saline-pretreated rats (figs. 2A and
3A). This concentration of formalin also
induced an expression of Fos-LI in the contralateral spinal cord, but the numbers of Fos-LI neurons were significantly less than in the
ipsilateral spinal cord in each region (data not shown).
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Fig. 2.
Photomicrographs of transverse sections of the
lumbar spinal cord illustrating the distribution of
formalin-evoked Fos-LI neurons in rats pretreated i.t. with either (A)
saline, (B) 60 µg of DPDPE or (C) a mixture of 60 µg of DPDPE and 1 µg of BNTX 10 min before the s.c. injection of 5% formalin in one
hindpaw, or with (D) 60 µg of DPDPE in the absence of formalin. In
panels A to C, the right side of the spinal cord is ipsilateral to the site of formalin injection. Scale bar, 200 µm.
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Fig. 3.
Effects of i.t. pretreatment with (A) DPDPE, (B)
DELT or (C) DAMGO alone or in the presence of its respective antagonist
on the number of Fos-LI neurons evoked by the s.c. injection of 5% formalin in one hindpaw of the rat. Data are the mean ± S.E.M. number of Fos-LI neurons in the different laminar regions on the side
of the spinal cord ipsilateral to the site of formalin injection. The
number of animals in each treatment group ranges from 3 to 5. Values
that are significantly different from the vehicle control are indicated
by * (P < .05) and ** (P < .01).
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In rats pretreated with 10 µg of DPDPE, the distribution and number
of Fos-LI neurons did not differ from that of saline-pretreated rats
(data not shown). Intrathecal administration of 60 µg of DPDPE
produced a modest decrease in the number of Fos-LI neurons in the
ipsilateral laminae I, IIo, IIi, as well as in laminae V-VI and the
ventral horn, but did not decrease the number of Fos-LI neurons in
laminae III-IV (figs. 2B and 3A). The number of Fos-LI neurons was
reduced to 80.1 ± 7.6% of control in ipsilateral laminae I, IIo
and IIi, to 70.4 ± 12.3% of control in laminae V-VI and to
62.6 ± 9.0% of control in the ventral horn. Thus, 60 µg of
DPDPE suppressed Fos-LI to a similar extent in each of these three
regions (P > .4). This dose of DPDPE also significantly decreased
the number of Fos-LI neurons in the contralateral laminae V-VI and
ventral horn (data not shown). Coadministration of 1.0 µg of BNTX
completely prevented the decrease in numbers of Fos-LI neurons produced
by 60 µg of DPDPE (figs. 2C and 3A).
Effects of intrathecally administered DELT on formalin-induced
flinching and expression of Fos-LI in the spinal cord.
Intrathecal
pretreatment with DELT dose-dependently decreased the number of
flinches in both phase 1 and phase 2 (fig.
4A). The 30-µg dose of DELT inhibited
formalin-induced flinching behavior to a greater extent and for a
longer duration than did the 3.0-µg dose. The inhibition of flinching
produced by 30 µg of DELT was completely antagonized by i.t.
coadministration of 3.0 µg of NTB (fig. 4A). Moreover, beginning 40 min after the injection of formalin, the number of flinches in these
rats significantly exceeded those in Molecusol-pretreated rats.
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Fig. 4.
Panel A depicts the effects of i.t. pretreatment
with 5% Molecusol (; n = 5), 3.0 µg of DELT
(; n = 8), 30 µg of DELT (; n = 6), or a combination of 30 µg DELT and 3.0 µg NTB (; n = 6) 10 min before the s.c.
injection of 5% formalin into the plantar surface of one hindpaw of
the rat. Panel B depicts the effects of i.t. post-treatment with 5%
Molecusol (; n = 5) or 30 µg of DELT (;
n = 6) 7 min after the injection of formalin. The
arrowhead in panel B depicts the time at which DELT or Molecusol was
administered. Each symbol represents the mean ± S.E.M. The arrows
indicate the time at which formalin was injected. Values that differ
from the corresponding vehicle control are indicated by asterisks
(P < .05) or daggers (P < .01).
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Intrathecal post-treatment with 30 µg of DELT, administered 7 min
after formalin, also inhibited formalin-induced flinching behavior in
phase 2 (fig. 4B). However, the inhibition was modest and was much less
than that observed when this dose was administered 10 min before
formalin (compare fig. 4, A and B; P < .01 for the 15- to 60-min
period). The magnitude of this inhibition was equivalent to the
magnitude of inhibition produced by pretreatment with 3.0 µg of DELT,
a 10-fold lower dose (P = .6).
Figure 5, A to C, illustrates the
distribution of Fos-LI neurons in the spinal cord of rats pretreated
with either Molecusol, 30 µg of DELT or a mixture of 30 µg of DELT
and 3.0 µg of NTB. The distribution of Fos-LI immunoreactive neurons
in the spinal cord of Molecusol-pretreated rats was similar to that
observed in saline-pretreated rats, although the numbers of neurons in laminae I, IIo and IIi and in laminae III-IV tended to be greater in
Molecusol-pretreated rats (fig. 3, A and B). Neither 3.0 µg nor 30 µg of i.t. administered DELT significantly decreased the number of
Fos-LI neurons in any of the four regions of the spinal cord as
compared with values in Molecusol-treated rats (figs. 3B and 5B).
Coadministration of 3.0 µg of NTB with 30 µg of DELT was without
effect.
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Fig. 5.
Photomicrographs of transverse sections of the
lumbar spinal cord illustrating the distribution of formalin-evoked
Fos-like immunoreactive neurons in rats pretreated i.t. with either (A) Molecusol, (B) 30 µg of DELT or (C) a mixture of 30 µg of DELT and
3.0 µg of NTB 10 min before the s.c. injection of 5% formalin in one
hindpaw, or (D) with 30 µg of DELT in the absence of formalin. In
panels A to C, the right side of the spinal cord is ipsilateral to the
site of formalin injection. Scale bar, 200 µm.
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Effects of intrathecally administered DAMGO on formalin-induced
flinching and expression of Fos-LI in the spinal cord.
Intrathecal
pretreatment with 0.3 µg of DAMGO significantly decreased the number
of flinches in both phase 1 and phase 2 (fig. 6). This dose of DAMGO inhibited
flinching to the same extent as did either 30 µg of DELT or 60 µg
of DPDPE in the 45 min after injection of formalin (P > .3). The
inhibition of flinching produced by 0.3 µg of DAMGO was completely
antagonized by coadministration of 3.0 µg of CTOP (fig. 6).
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Fig. 6.
Effects of i.t. pretreatment with saline (;
n = 6), 0.3 µg of DAMGO (;
n = 6) or the combination of 0.3 µg of DAMGO and 3.0 µg of CTOP (; n = 6) 10 min before the
s.c. injection of 5% formalin into the plantar surface of one hindpaw
of the rat. Each symbol represents the mean ± S.E.M. Values that
differ from the corresponding vehicle control are indicated by
asterisks (P < .05) or daggers (P < .01).
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Unlike either DELT or DPDPE, i.t. pretreatment with DAMGO produced a
very profound suppression of Fos-LI in each of the four regions of the
spinal cord (figs. 3C and 7B). The
numbers of Fos-LI neurons were reduced to 32.7 ± 7.2% of control
in laminae I, IIo and IIi, to 44.2 ± 9.3% of control in laminae
III and IV, to 22.0 ± 7.0% of control in laminae V-VI and to
19.6 ± 5.3% of control in laminae VII-X ipsilateral to the
injection site. These values did not differ from one another (P > .1), which indicates that this dose of DAMGO produced an equivalent
suppression of Fos-LI in each region of the spinal cord (P > .1).
DAMGO also reduced the numbers of Fos-LI neurons in each region of the
contralateral spinal cord to a similar extent (data not shown).
Coadministration of 3.0 µg of CTOP completely prevented the decrease
in number of Fos-LI neurons produced by 3.0 µg of DAMGO in all
laminae, with the exception of laminae VII-X in which the antagonism
was only partial (figs. 3C and 7).
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Fig. 7.
Photomicrographs of transverse sections of the
ipsilateral lumbar spinal cord illustrating the distribution of
formalin-evoked Fos-LI neurons in rats pretreated i.t. with either (A)
saline, (B) 0.3 µg of DAMGO or (C) a mixture 0.3 µg of DAMGO and
3.0 µg of CTOP 10 min before the s.c. injection of 5% formalin in
one hindpaw. Scale bar, 400 µm.
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Effects of intrathecally administered DELT or DPDPE on the
expression of Fos-LI in the spinal cord in the absence of
formalin.
Low numbers of Fos-LI neurons were observed in the
spinal cord of rats treated with 60 µg of DPDPE or 30 µg of DELT in
the absence of formalin. Numbers of Fos-LI neurons ranged between 13 and 30/region and were uniformly distributed among the four regions of
interest on both sides of the spinal cord (figs. 2D and 5D). Also, the
intensity of staining of Fos-LI neurons was much lighter than in rats
in which formalin had been injected. The pial surface of the spinal
cord often exhibited large numbers of densely stained Fos-LI cells.
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Discussion |
Antinociceptive effects of delta opioid receptor
agonists in the formalin test.
One of the principal findings of
this study was that i.t. administered delta opioid receptor
agonists produce antinociception in the formalin test in the rat.
Previous investigations of the effects of i.t. administered opioid
receptor agonists in this model of persistent, inflammatory nociception
focussed on mu or kappa opioid receptor agonists
(Pelissier et al., 1990; Yamamoto and Yaksh, 1992; Malmberg
and Yaksh, 1993; Fujibayashi and Iizuka, 1995). The only study that
examined delta opioid receptor agonists concluded that they
were without effect in the mouse at doses that did not produce adverse
motor effects (Murray and Cowan, 1991). The present study is therefore
the first to identify a contribution of spinal delta opioid
receptors in the modulation of nociceptive behaviors in the formalin
test. Specifically, it determined that i.t. pretreatment with the
delta-1 opioid receptor agonist DPDPE or the
delta-2 opioid receptor agonist DELT dose-dependently suppressed flinching behavior in both phase 1 and phase 2 of the formalin test at doses that did not adversely affect motor function. Moreover, this antinociception was attenuated by the delta-1
opioid receptor antagonist BNTX and the delta-2 opioid
receptor antagonist NTB, respectively. Finally, each delta
opioid receptor agonist was also effective when administered after the
injection of formalin. These results extend earlier reports of the
antinociceptive efficacy of i.t. administered delta opioid
receptor agonists in the carrageenan-inflamed paw-flick test (Hylden
et al., 1991; Stanfa et al., 1992; Stewart and
Hammond, 1994) to a second model of persistent, inflammatory nociception. They also complement previous investigations of the antinociceptive effects of these two agonists in models of acute nociception, such as the tail-flick and hot-plate tests, in the rat
(Malmberg and Yaksh, 1992; Stewart and Hammond, 1993; Hammond et
al., 1995a).
Additional evidence of a modulation of formalin-induced pain behaviors
by delta opioid receptors is provided by the report that
s.c. administration of the nonselective delta opioid
receptor antagonist naltrindole increased the number of flinches
elicited in phase 2 by i.pl. injection of a submaximal concentration of formalin (Ossipov et al., 1996). These data suggested that
formalin evokes a release of enkephalins that act at delta
opioid receptors in the spinal cord. Our observation that the number of
flinches in rats that received NTB and DELT exceeded those in the
vehicle control group in the last 20 min may reflect additional
antagonism of the effects of endogenously released enkephalins.
Lack of effect of delta opioid receptor agonists on the
expression of Fos-LI in the spinal cord.
Another principal finding
of this study was that i.t. pretreatment with the delta -1 opioid receptor agonist DPDPE only marginally decreased
formalin-induced expression of Fos-LI in the spinal cord and that the
delta-2 opioid receptor agonist DELT was without significant
effect. Yet, each agonist produced a robust decrease in flinching
behavior in the formalin test that was dose-dependent and was
attenuated by coadministration of the appropriate antagonist. The
relative lack of effect of the delta opioid receptor
agonists is in stark contrast to the profound reduction in the number
of Fos-LI neurons in the spinal cord of rats treated with an
equiantinociceptive dose of the mu opioid receptor agonist
DAMGO. The lack of inhibition cannot be attributed to a nonspecific
stimulatory effect of these agonists by themselves because neither
DPDPE nor DELT increased the expression of Fos-LI in the spinal cord in
the absence of formalin.
The present study examined the effects of the opioid receptor agonists
on Fos-LI 1 hr after the injection of formalin. This time was selected
because it was desirable to assess the effects of the opioid agonists
during their time of peak effect. Also, the sensitivity of the method
for immunocytochemical detection of Fos-LI has increased compared with
previous studies that required a 2-hr interval for optimal detection of
Fos-LI (Presley et al., 1990). Although the relative timing
of drug administration, application of the noxious stimulus and
perfusion of the animal can be important variables in studies of the
effects of drugs on Fos-LI (Tölle et al., 1994;
Honoré et al., 1995), the 1-hr interval used in this
study is unlikely to significantly influence its findings for several
reasons. First, the inhibitory effect of i.t. administered DAMGO on
spinal Fos-LI was readily observed in this study (see below). Second,
the suppression of formalin-evoked Fos-LI in the spinal cord by i.c.v.
administration of DAMGO, morphine or the kappa opioid
receptor agonist CI-977 was also evident at this interval (Gogas
et al., 1991, 1996a, b). Finally, Fos protein detected
immunocytochemically at 1 hr would reflect events of the first 30 min
after formalin injection that influence the transcription and
translation of c-fos. Because DPDPE and DELT significantly decreased flinching behavior to an extent similar to DAMGO throughout the first 30 min, a decrease in Fos-LI should have been readily detectable under these conditions.
Effect of a mu opioid receptor agonist on the
expression of Fos-LI in the spinal cord.
The finding that i.t.
administered DAMGO reduced both formalin-induced flinching behavior and
Fos-LI in the spinal cord establishes the sensitivity of this study and
complements previous reports that the inhibition of formalin-induced
pain behaviors by systemic or i.c.v. administration of morphine or
DAMGO is accompanied by a decrease in formalin-evoked Fos-LI in the
spinal cord (Presley et al., 1990; Gogas et al.,
1991, 1996a). It also suggests that the suppression of
noxious-stimulus-evoked Fos-LI in the spinal cord by systemically
administered mu opioid receptor agonists is likely to result
from a coincident activation of inhibitory bulbospinal pathways, as
well as a direct action of the opioids on local circuits in the spinal
cord. Intrathecal administration of DAMGO suppressed formalin-induced
Fos-LI in laminae I, IIo and IIi to the same extent as in laminae V-VI
and VII-X. By comparison, systemic (Presley et al., 1990;
Tölle et al., 1994) or i.c.v. (Gogas et
al., 1991, 1996a) administration of mu opioid receptor agonists consistently suppressed Fos-LI in laminae V-VI and VII-X to
a greater extent than in the superficial laminae. The ability of i.t.
administered DAMGO (and of DPDPE) to produce an equivalent suppression
of Fos-LI in the superficial as in the deeper laminae is likely to
reflect the "topical" nature of its application in the spinal cord.
Possible bases for the discrepant effects of delta and
mu opioid receptor agonists on formalin-induced
Fos-LI.
Despite producing an equivalent antinociception in the
formalin test, DPDPE and DELT differed markedly from DAMGO in their effects on the expression of Fos-LI in the spinal cord. The most parsimonious explanation for the inability of antinociceptive doses of
either DPDPE or DELT to significantly decrease formalin-induced Fos-LI
in the spinal cord is that c-fos is not regulated by
delta opioid receptors. Whether delta opioid
receptor agonists regulate the transcription and translation of other
immediate early genes such as jun or krox, as for
morphine (Tölle et al., 1994), remains to be
determined. Alternatively, differences in the effects of DPDPE, DELT
and DAMGO on Fos-LI may reflect the different mechanisms by which these
agonists modulate synaptic transmission in the spinal cord. For
example, it is well established that mu receptors comprise
the largest percentage (70-80%) of opioid receptors in the spinal
cord; the percentage of delta receptors is much smaller (10-15%) (Besse et al., 1991; Stevens et al.,
1991). Autoradiographic studies (Besse et al., 1990; Stevens
and Seybold, 1995) and immunocytochemical visualization of the
mu (Arvidsson et al., 1995; Ding et
al., 1996) and delta (Dado et al., 1993;
Arvidsson et al., 1995; Cheng et al., 1995)
opioid receptors indicate that both receptors are located
postsynaptically on dorsal horn neurons, as well as presynaptically on
the terminals of primary afferent neurons. However, the results of
intracellular or whole-cell recordings from dorsal horn neurons in the
superficial laminae of slices of rat spinal cord suggest that
mu and delta opioid receptor agonists modulate
synaptic transmission by different mechanisms. In these latter studies,
mu opioid receptor agonists inhibited both spontaneous and
evoked EPSP/Cs at low concentrations and, at slightly higher
concentrations, also hyperpolarized a portion of the dorsal horn
neurons (Murase et al., 1982; Jeftinija, 1988; Glaum
et al., 1994; Grudt and Williams, 1994). Bath application of
DPDPE or DELT similarly inhibited evoked EPSP/Cs (Glaum et al., 1994). However, even high concentrations of these agonists did not appreciably alter resting membrane potential (Jeftinija, 1988;
Glaum et al., 1994). Taken together, these observations suggest that delta opioid receptor agonists produce
antinociception by a predominantly presynaptic mechanism,
i.e., inhibition of neurotransmitter release, and that they
are unlikely to effectively hyperpolarize and reduce the excitability
of dorsal horn neurons. Conceivably, any excitatory synaptic input that
persists in the presence of the delta opioid receptor
agonists is sufficient to alter the disposition of intracellular
calcium and activate c-fos. In contrast, DAMGO is likely to
not only presynaptically inhibit neurotransmitter release, but to also
hyperpolarize and reduce the excitability of dorsal horn neurons. In
the added presence of this hyperpolarization, the synaptic input that
persists may be unable to depolarize the neuron to a sufficient extent
to increase intracellular calcium and activate c-fos.
Finally, the polysynaptic nature of the pathways that transmit
nociceptive information provides several loci for postsynaptic
inhibition by mu opioid receptor agonists and could serve to
"amplify" the inhibitory effects of drugs such as DAMGO and
morphine on the expression of Fos-LI.
Fos-LI as a measure of antinociception.
There is strong
evidence that the expression of Fos-LI in the spinal cord is a function
of the intensity and duration of the noxious stimulus (Hunt et
al., 1987; Williams et al., 1989; Abbadie et
al., 1994). Furthermore, many studies have determined that opioid
receptor agonists suppress Fos-LI in the spinal cord in a
dose-dependent and naloxone-reversible manner (Presley et
al., 1990; Hammond et al., 1992; Abbadie et
al., 1994) and that there can be an excellent correlation between
the magnitude of antinociception and the extent to which Fos-LI is
suppressed (Gogas et al., 1991; Hammond et al.,
1992). However, closer examination of the literature reveals important
disparities. For example, complete behavioral antinociception can be
produced without complete suppression of Fos-LI in the spinal cord.
Similarly, moderate behavioral antinociception can be produced in the
absence of significant decreases in Fos-LI in the superficial laminae
(Presley et al., 1990; Gogas et al., 1991, 1996a;
Jasmin et al., 1994). Conversely, a decrease in the number
of Fos-LI neurons in the superficial laminae has been observed in the
absence of behavioral antinociception (Kehl et al., 1991; Gogas et al., 1996a, b). Thus, although there is strong
evidence that the expression of Fos-LI in the spinal cord is an
appropriate measure of nociception, it is not as clear that
the suppression of Fos-LI in the spinal cord, and
particularly in the superficial laminae, is an equally good measure of
antinociception. The present finding with the
delta opioid receptor agonists is perhaps the most extreme
example to date of the disparity that can exist between behavioral
antinociception and the expression of Fos-LI in the spinal cord.
In conclusion, i.t. pretreatment with agonists of the
delta-1 and delta-2 opioid receptor produced a
dose-dependent and reversible antinociception in phase 1 and phase 2 of
the formalin test. However, neither DPDPE nor DELT produced a robust
decrease in formalin-induced Fos-LI in the spinal cord. This finding
contrasts with the ability of an equiantinociceptive dose of DAMGO, a
mu opioid receptor agonist, to inhibit Fos-LI in the spinal
cord. The efficacy of i.t. administered DAMGO suggests that a direct
spinal action contributes to the inhibition of noxious-stimulus-evoked
Fos-LI in the spinal cord produced by systemic administration of
mu opioid receptor agonists, such as morphine. The relative
lack of effect of DPDPE or DELT suggests that delta opioid
receptor agonists do not regulate the immediate early gene
c-fos. Alternatively, differences in the mechanisms by which
delta and mu opioid receptor agonists modulate
synaptic transmission of nociceptive information in the spinal cord may
underlie the disparate effects of equally antinociceptive doses of
DPDPE, DELT and DAMGO on the expression of formalin-evoked Fos-LI in
the spinal cord. Collectively, these data provide new evidence, at the
"third messenger" level, that the mechanisms by which i.t.
administered delta and mu opioid receptor
agonists modulate nociception differ.
We thank Nadine Pierre, Brian Donahue and Dr. Megumi Kaneko for
their assistance with aspects of this study.
i.t., intrathecal(ly);
i.c.v., intracerebroventricular;
Fos-LI, Fos-like immunoreactive(ity);
i.pl., intraplantar;
DPDPE, [D-Pen2,5]enkephalin;
DELT, [D-Ala2,Glu4]deltorphin;
BNTX, 7-benzylidinenaltrexone;
NTB, Naltriben;
DAMGO, [D-Ala2,NMePhe4,Gly-ol5]enkephalin;
CTOP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Phe-Thr-NH2;
CI-977, (5R)-(5
,7,8)-N-methyl-N-[7-(1-pyrrolindinyl)-1-oxaspiro[4,5]dec-8-yl]-4-benzofurnacetamide
hydrochloride ;
EPSP/C, excitatory postsynaptic potential/current;
PBS, phosphate-buffered saline.
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Abstract
Introduction
and 
opioid binding sites in the superficial dorsal horn, over the rostrocaudal axis of the rat spinal cord.
Brain Res
548: 287-291[Medline]. 
-NTII-sensitive delta receptor subtype.
J Pharmacol Exp Ther
260: 518-525
