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SPINAL PHARMACOLOGY OF ANTINOCICEPTION PRODUCED BY
MICROINJECTION OF OR ␦OPIOID RECEPTOR AGONISTS IN THE
VENTROMEDIAL MEDULLA OF THE RAT
R. W. HURLEY,
a,b
P. BANFOR
a
AND D. L. HAMMOND
a,b
*
a
Department of Anesthesia and Critical Care, University of Chicago,
Chicago, IL 60637, USA
b
Committee on Neurobiology, University of Chicago, Chicago, IL
60637, USA
Abstract—This study examined the role of spinal GABAergic,
serotoninergic and ␣
2
adrenergic receptors in the antinoci-
ception produced by the microinjection of equi-antinocicep-
tive doses of selective opioid receptor agonists in the nu-
cleus raphe magnus (NRM) or the nucleus reticularis gigan-
tocellularis pars ␣(NGCp␣) of the rat. Rats were pretreated
with intrathecal administration of either the GABA
A
receptor
antagonist bicuculline, the GABA
B
receptor antagonist
CGP35348, the serotonin
1/2
receptor antagonist methyser-
gide, the ␣
2
adrenergic receptor antagonist yohimbine or
saline. Ten minutes later, either the ␦
1
opioid receptor agonist
[D-Pen
2,5
]enkephalin (DPDPE), ␦
2
opioid receptor agonist
[D-Ala
2
,Glu
4
]deltorphin (DELT) or opioid receptor agonist
[D-Ala
2
,NMePhe
4
,Gly-ol
5
]enkephalin (DAMGO) was microin-
jected into the NRM, NGCp␣or sites in the medulla outside
these two regions. The increase in tail-flick latency produced
by microinjection of DPDPE into the NRM or NGCp␣was
antagonized by intrathecal pretreatment with either methy-
sergide or yohimbine. Intrathecal pretreatment with
CGP35348 antagonized the antinociception produced by mi-
croinjection of DPDPE in the NRM, whereas bicuculline an-
tagonized the antinociception produced by microinjection of
DPDPE in the NGCp␣. The increase in tail-flick latency pro-
duced by microinjection of DELT into the NGCp␣, but not the
NRM was antagonized by intrathecal pretreatment with yo-
himbine or CGP35348. Intrathecal pretreatment with methy-
sergide or bicuculline did not antagonize the antinociception
produced by microinjection of DELT into either the NRM or
the NGCp␣. The increase in tail-flick latency produced by
microinjection of DAMGO in the NRM was antagonized by
intrathecal pretreatment with methysergide or CGP35348, but
not by bicuculline or yohimbine. Taken together, these re-
sults support the hypothesis that the antinociception pro-
duced by activation of ␦
1
,␦
2
or opioid receptors in the
rostral ventromedial medulla is mediated by different neural
substrates. © 2003 IBRO. Published by Elsevier Science Ltd.
All rights reserved.
Key words: spinal cord, nucleus raphe magnus, nucleus re-
ticularis gigantocellularis pars ␣, monoaminergic, GABAer-
gic, descending inhibition.
Neurons in the nucleus raphe magnus (NRM) and nu-
cleus reticularis gigantocellularis pars ␣(NGCp␣), col-
lectively referred to as the rostral ventromedial medulla
(RVM), comprise an important efferent pathway for the
modulation of nociception. Activation of neurons in these
regions can result in either a facilitation (reviewed by
Urban et al., 1999; Porreca et al., 2002) or an inhibition
(reviewed by Millan, 2002) of nociceptive transmission
at the level of the spinal cord. The RVM is also an
important site of action for the production of antinocicep-
tion by opioid receptor agonists. For example, the an-
tinociceptive effect of systemically administered mor-
phine is antagonized by microinjection of opioid receptor
antagonists in the RVM (Dickenson et al., 1979). Micro-
injection of morphine, or of selective agonists for the ,
␦
1
or ␦
2
opioid receptor in the RVM, is also sufficient to
produce antinociception (Jensen and Yaksh, 1986b;
Porreca and Burks, 1993; Rossi et al., 1994; Ossipov et
al., 1995; Thorat and Hammond, 1997; Harasawa et al.,
2000). Considerable evidence obtained in the mouse
indicates that the antinociceptive effects of intracere-
broventricularly administered opioid receptor agonists
are mediated by activation of GABAergic, noradrenergic
and serotonergic receptors in the spinal cord (Wigdor
and Wilcox, 1987; Arts et al., 1991; Rady et al., 1991;
Holmes and Fujimoto, 1994; Rady and Fujimoto, 1995,
1996; Suh et al., 1996). However, few studies have
examined the spinal pharmacology of the antinocicep-
tion produced by microinjection of opioid receptor ago-
nists at specific supraspinal sites in the rat (Yaksh and
Rudy, 1978; Jensen and Yaksh, 1986b; Tseng and Col-
lins, 1991; Grabow et al., 1999). Further, no comparative
analysis of the spinal pharmacology of antinociception
produced by microinjection of ,␦
1
or ␦
2
opioid receptor
agonists in the RVM has been undertaken. To address
these issues, the present study examined the effects of
antagonism of spinal serotonin
1/2
,␣
2
adrenergic,
GABA
A
or GABA
B
receptors on the antinociception pro-
duced by microinjection of the ␦
1
opioid receptor agonist
[D-Pen
2,5
]enkephalin (DPDPE), the ␦
2
opioid receptor ag-
onist [D-Ala
2
, Glu
4
]deltorphin (DELT), or the opioid re-
ceptor agonist [D-Ala
2
, NMePhe
4
, Gly-ol
5
]enkephalin
(DAMGO) in the RVM of the rat. The spinal pharmacology
of opioid receptor-mediated antinociception was not ex-
amined because these agonists are generally without ef-
fect in the tail-flick and hotplate tests after microinjection in
*Correspondence to: D. L. Hammond, Department of Anesthesia,
University of Iowa, 200 Hawkins Drive JCP 6505-2, Iowa City, IA. Tel:
⫹1-319-384-7127; fax: ⫹1-319-356-2940.
R. W. Hurley and P. Banfor contributed equally to this work.
E-mail address: Donna-Hammond@uiowa.edu (D. L. Hammond).
Abbreviations: DAMGO, [D-Ala
2
, NMePhe
4
, Gly-ol
5
]enkephalin; DELT,
[D-Ala
2
, Glu
4
]deltorphin; DPDPE, [D-Pen
2,5
]enkephalin; i.t.,
intrathecal; NRM, nucleus raphe magnus; NGCp␣, nucleus reticularis
gigantocellularis pars ␣; RVM, rostral ventromedial medulla.
Neuroscience 118 (2003) 789–796
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0306-4522(03)00041-1
789
the RVM (Rossi et al., 1994; Pan et al., 1997; Ackley et al.,
2001).
EXPERIMENTAL PROCEDURES
These experiments were approved by the Institutional Animal
Care and Use Committee of the University of Chicago. All proce-
dures were conducted in accordance with the “Guide for Care and
Use of Laboratory Animals” published by the National Institutes of
Health and the ethical guidelines of the International Association
for the Study of Pain. Every effort was made to minimize animal
suffering and to limit the number of animals used.
Animals
Male Sprague–Dawley rats (Charles River Laboratories; Sasco
stock, Kingston, NY, USA) weighing 300–400 g were anesthe-
tized with halothane and prepared with an intrathecal (i.t.) catheter
that terminated at the L4 or L5 segment of the spinal cord (Yaksh
and Rudy, 1976). Rats that exhibited motor impairments such as
hindlimb or forepaw paresis were killed. Four to 6 days later, the
rats were reanesthetized with a mixture of ketamine hydrochloride
(85 mg/kg i.p.) and xylazine (9 mg/kg i.p.), and implanted with an
intracerebral guide cannula (26 gauge, Plastic One, Inc.,
Roanoke, VA, USA) that terminated 3 mm dorsal to either the
NRM or the NGCp␣in the RVM. The cannula was secured to the
skull with stainless steel screws and dental acrylic. A 30-gauge
stainless steel stylet was placed in the guide cannula to maintain
its patency. Rats were housed individually after surgery under a
12-h light/dark cycle with food and water available ad libitum.
Seven to 10 days elapsed before behavioral testing began. Rats
received only one dose combination and were used only once in
this study.
Behavioral tests
Nociceptive threshold was assessed by the radiant-heat tail flick
and 55 °C hotplate tests. In the tail-flick test (D’Amour and Smith,
1941), the rat’s blackened tail was positioned under an intense
light beam and the time for the rat to remove its tail from the
thermal stimulus was recorded. This test was performed twice at
each time point on two different regions of the distal tail. The
results of the two trials were averaged and recorded as the
tail-flick latency. In the event that the rat did not withdraw its tail
from the stimulus by 14 s, the test was terminated to prevent
tissue damage, and the rat was assigned this cutoff latency. In the
hotplate test (Woolfe and MacDonald, 1944), the rat was placed
on an enclosed copper plate heated to 55 °C. The time between
placement of the rat on the hot plate and the occurrence of either
a hind-paw lick or a jump off the surface was recorded as the
hotplate latency. Hotplate latency was measured once per time
period. In the absence of a hind-paw lick or a jump by 40 s, the test
was terminated to prevent tissue damage and this cutoff latency
was recorded. Motor function was evaluated using the inclined-
plane test (Rivlin and Tator, 1977). The tail-flick, inclined-plane
and hotplate tests were performed in succession.
Experimental design
Measurements of nociceptive threshold and motor competency
were made before the injection of drug. Those rats that re-
sponded in ⱕ5.0 s on the tail-flick test, ⱕ15.0 s on the hotplate
test, and had inclined-plane angles of 40° or greater were used
in this study. After determination of the baseline nociceptive
threshold and motor competency, rats were injected i.t. with
either saline, 0.3 g bicuculline methiodide, 30 g CGP35348,
30 g methysergide maleate, or 30 g yohimbine hydrochlo-
ride. Intrathecal injections were made over a 60-s period in a
volume of 10 l and were followed by 10 l of saline to flush the
catheter. Ten minutes later, either 2.5 g DPDPE, 0.94
g DELT or 50 ng DAMGO was microinjected in a volume of 0.4
l into the NRM or NGCp␣over a period of 1 min using a
33-gauge stainless-steel injector that extended 3 mm beyond
the tip of the guide cannula. After injection, the cannula was left
in place for another 60 s to allow the drug to diffuse locally and
to limit its diffusion up the injection track. The progress of drug
delivery to spinal and supraspinal sites was monitored by the
movement of an air bubble in the polyethylene tubing that
connected the injector to the syringe pump. Nociceptive thresh-
old and motor competency were re-evaluated 15, 30, 45 and 60
min later. Data on the effects of i.t. yohimbine in rats that
received a microinjection of DELT in the RVM are taken from a
previous study published by this laboratory (Grabow et al.,
1999). The experiments were conducted at the same time by
these authors, and are presented here for the sake of com-
pleteness.
Histology
At the conclusion of testing, the rats were killed by CO
2
inhalation.
The location and patency of the i.t. catheter was determined by
direct visual inspection after a laminectomy and an i.t. injection of
India ink. The brains were removed and fixed by immersion in a
4% formaldehyde and 30% sucrose solution. Transverse sections
of the brainstem (25 m) were cut on a cryostat microtome and
stained with Cresyl Violet. The location of each microinjection site
was plotted on transverse sections of the rat brainstem modified
from those provided by Neurographics (Kanata, Ontario) and was
verified by a person unaware of the treatment. Sites were consid-
ered to lie within the NRM if they were situated in a triangular area
centered on the midline of the RVM. The apex of this triangle was
on the midline and extended no further dorsally than the top of the
facial nucleus. The base was formed by the top of the pyramids
and extended laterally no further than the middle of each pyramid.
Sites were considered to lie within the NGCp␣if they were situ-
ated in the polygonal area surrounding the NRM. This area was
defined laterally by the most lateral edge of the pyramid, extended
dorsally as far as the top of the facial nucleus, and was bounded
medially by the lateral edge of the NRM (Fig. 1).
Statistical analysis
Rats were grouped for statistical analysis according to placement
of the intracerebral cannula and drug treatment. Comparisons of
the effect of i.t. pretreatment with bicuculline, CGP35348, yohim-
bine or methysergide on the increase in response latency pro-
duced by DPDPE, DELT or DAMGO injected into the NRM or
NGCp␣were performed using a two-way analysis of variance for
repeated measures in which the two factors were drug treatment
and time. NRM and NGCp␣were analyzed separately. Post hoc
comparisons of mean values at individual time points were made
using Newman-Keuls test (Keppel, 1973). P⬍0.05 was consid-
ered significant. Data were expressed as the mean⫾S.E.M.
Drugs
Bicuculline methiodide, yohimbine hydrochloride, DPDPE, DELT
and DAMGO were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Methysergide maleate was purchased from
Research Biochemicals, Inc. (Natick, MA, USA). CGP35348 was
a generous gift of Novartis (Basel, Switzerland). The doses of the
receptor antagonists were previously determined to effectively
block their respective receptor (Reddy et al., 1980; Schmauss et
al., 1983; Hammond and Washington, 1993; McGowan and Ham-
mond, 1993a).
R. W. Hurley et al. / Neuroscience 118 (2003) 789–796790
RESULTS
Microinjection sites
Histological analysis revealed a comparable distribution of
injection sites throughout the rostral-caudal extent of the
NRM and NGCp␣in each treatment group. Some sites
that impinged on the rostral edge of the nucleus reticu-
laris paragigantocellularis lateralis were included in the
NGCp␣for purposes of data analysis. Fig. 1 illustrates
the distribution of injection sites for a representative
treatment group. Microinjection of DPDPE, DELT or
DAMGO at sites outside these two nuclei, such as the
pyramids, trapezoid body, medial longitudinal fascicu-
lus, or dorsal or lateral aspects of the nucleus reticularis
Fig. 1. Distribution of sites in the NRM and NGCp␣at which DPDPE was microinjected in rats pretreated with bicuculline. Solid squares depict sites
in the NGCp␣, whereas solid circles depict sites in the NRM. Open symbols depict sites neither within the NRM or NGCp␣. Some sites overlapped
and are not visible. Numbers indicate the distance (mm) of the section caudal to the interaural line. The boundaries of the NRM and NGCp␣are
illustrated. Adapted from an atlas provided by Neurographics, Inc. (Kanata, Ontario, Canada).
Abbreviations used in the figures
dcn dorsal cochlear nucleus
icp inferior cerebellar peduncle
ngc nucleus reticularis gigantocellularis
nV spinal trigeminal nucleus
nVII facial motor nucleus
P pyramid
tb trapezoid body
7g genu of the seventh cranial nerve
7t tract of the seventh cranial nerve
R. W. Hurley et al. / Neuroscience 118 (2003) 789–796 791
gigantocellularis, did not significantly increase nocicep-
tive measures (data not shown). These sites were ex-
cluded from further analysis.
Antinociceptive effects of DPDPE, DELT and DAMGO
in vehicle-pretreated rats
Microinjection of 2.5 g DPDPE in the NRM or NGCp␣
significantly increased tail-flick latency in vehicle-pre-
treated rats with a peak effect occurring between 30 and
45 min (Fig. 2A and B). Microinjection of 0.94 g DELT in
the NRM or NGCp␣also significantly increased tail-flick
latency in vehicle-pretreated rats with a peak effect occur-
ring within 30 min (Fig. 3). Microinjection of 50 ng DAMGO
in the NRM or NGCp␣also increased tail-flick latency (Fig.
Fig. 2. Effects of 2.5 g DPDPE microinjected in the (A) NRM or (B)
NGCp␣of rats pretreated i.t. 10 min earlier with either saline (䊐), 30
g methysergide (Œ), 30 g yohimbine hydrochloride (}), 30
g CGP35348 (●), or 0.3 g bicuculine methiodide (■). Symbols
represent the mean⫾S.E.M. response latencies from five to 16 rats.
Asterisks indicate values that are significantly different from those in
the saline-treatment group at the corresponding time point (*P⬍0.05,
**P⬍0.01). The arrowhead indicates the time at which DPDPE was
microinjected in the ventromedial medulla.
Fig. 3. Effects of 0.94 g DELT microinjected in the (A) NRM or (B)
NGCp␣of rats pretreated i.t. 10 min earlier with either saline (䊐), 30
g methysergide (Œ), 30 g yohimbine hydrochloride (}), 30
g CGP35348 (●), or 0.3 g bicuculine methiodide (■). Symbols
represent the mean⫾S.E.M. response latencies from 11 to 20 rats.
Asterisks indicate values that are significantly different from those in
the saline-treatment group at the corresponding time point (*P⬍0.05,
**P⬍0.01). The arrowhead indicates the time at which DELT was
microinjected in the ventromedial medulla. Data for the effects of i.t.
yohimbine are taken from Grabow et al. (1999) and presented here for
the sake of completeness.
R. W. Hurley et al. / Neuroscience 118 (2003) 789–796792
4A, B). However, unlike DPDPE or DELT, the onset, mag-
nitude and duration of this increase was dependent on the
site of microinjection. Thus, DAMGO increased tail-flick
latency to peak values within 30 min of its injection in the
NRM, whereas the onset-to-peak effect was delayed to 45
min after microinjection in the NGCp␣.
At the doses used in this study, DPDPE and DAMGO
increased mean response latencies in the hotplate test to
15–20 s from baseline values of 8–10 s (data not shown).
Unfortunately, the magnitude of this increase was too
small to enable reliable detection of antagonism when it
occurred. Also, DELT was ineffective in the hotplate test at
the dose tested in this study (data not shown). For these
reasons, no attempt was made to quantify antagonism by
the i.t.-administered agents of the effects of these opioid
receptor agonists in the hotplate test.
Spinal pharmacology of DPDPE-mediated
antinociception
The increase in tail-flick latency produced by microinjection
of DPDPE in either NRM or NGCp␣was significantly an-
tagonized by pretreatment with either the serotonin recep-
tor antagonist methysergide or the ␣
2
adrenergic receptor
antagonist yohimbine (Fig. 2A, B, Table 1). The GABA
B
receptor antagonist CGP35348 antagonized the antinoci-
ceptive effect of DPDPE microinjected into the NRM, but
had no effect on the increase in tail-flick latency produced
by microinjection into the NGCp␣. In contrast, the GABA
A
receptor antagonist bicuculline partially antagonized the
effect produced by microinjection of DPDPE into the
NCGp␣, but not the NRM.
Spinal pharmacology of DELT-mediated
antinociception
The increase in tail-flick latency produced by microinjec-
tion of DELT in the NRM was not antagonized by pre-
treatment with either bicuculline or methysergide. Al-
though pretreatment with yohimbine or CGP35348 di-
minished the effects of DELT, this modest antagonism
did not achieve statistical significance (Fig. 3A, Table 1).
The increase in tail-flick latency produced by microinjec-
tion of DELT in the NGCp␣was also not antagonized by
either bicuculline or methysergide. However, in contrast
to the NRM, it was nearly completely antagonized by
pretreatment with either CGP35348 or yohimbine (Fig.
3B, Table 1).
Spinal pharmacology of DAMGO-mediated
antinociception
Pretreatment with methysergide completely prevented the
increase in tail-flick latency produced by microinjection of
DAMGO in the NRM (Fig. 4A, Table 1). The antinocicep-
tive effect of DAMGO was also partially antagonized by
pretreatment with CGP35348, and unaffected by either
bicuculline or yohimbine. In contrast, the delayed antino-
ciceptive effect of DAMGO microinjected into the NGCp␣
was consistently attenuated only by methysergide (Fig.
4B).
DISCUSSION
Activation of ␦
1
,␦
2
or opioid receptors in the RVM
produces antinociception
The NRM and NGCp␣are often considered to be a func-
tional unit because electrical or chemical activation of neu-
rons in either region can produce similar antinociceptive
effects. However, substantial pharmacological and ana-
Fig. 4. Effects of 50 ng DAMGO microinjected in the (A) NRM or (B)
NGCp␣of rats pretreated i.t. 10 min earlier with either saline (䊐), 30
g methysergide (Œ), 30 g yohimbine hydrochloride (}), 30
g CGP35348 (●), or 0.3 g bicuculine methiodide (■). Symbols repre-
sent the mean⫾S.E.M. response latencies from five to 12 rats. Asterisks
indicate values that are significantly different from those in the saline-
treatment group at the corresponding time point (*P⬍0.05, **P⬍0.01).
The arrowhead indicates the time at which DAMGO was microinjected in
the ventromedial medulla.
R. W. Hurley et al. / Neuroscience 118 (2003) 789–796 793
tomical evidence indicates that the afferent and efferent
connections of these two nuclei and their neurotransmitter
content are not identical. For example, the antinociception
produced by electrical stimulation or microinjection of L-
glutamate in the NRM or NGCp␣can be differentially
antagonized by i.t.-administered serotonergic (Satoh et al.,
1983; McGowan and Hammond, 1993b), noradrenergic
(Satoh et al., 1983), opioidergic (Hammond et al., 1997)
and GABAergic (McGowan and Hammond, 1993a,b) re-
ceptor antagonists. Also, enkephalinergic neurons in the
NRM and NGCp␣differentially innervate catecholaminer-
gic and non-catecholaminergic neurons in the A7 nucleus
of the dorsolateral pontine tegmentum (Holden and Proud-
fit, 1998). Finally, afferent inputs to the NRM and NGCp␣
are thought to arise from separate populations of neurons
in the periaqueductal gray (Beitz et al., 1983; Cameron et
al., 1995). This study investigated the spinal pharmacology
of antinociception produced by microinjection of prototypic
␦
1
,␦
2
or opioid receptor agonists in the NRM or the
NGCp␣. Our ability to differentiate drug effects in these two
nuclei is supported by results of previous studies that used
a larger injection volume of 0.5 l (McGowan and Ham-
mond, 1993a,b; Hammond et al., 1997), and by the finding
that 90% of radiolabeled -endorphin or morphine injected
in 0.5 l in the medulla diffused no farther than 0.4 mm
from the intended target by 30 min (Tseng and Collins,
1991).
Spinal pharmacology of opioid receptor agonist-
mediated antinociception is site dependent
Microinjection of DAMGO, DPDPE or DELT in the RVM of
the rat produced a sustained antinociception (Tseng and
Collins, 1991; Rossi et al., 1994; Ossipov et al., 1995;
Thorat and Hammond, 1997; Tershner et al., 2000). The
magnitude, onset and duration of the increase in tail-flick
latency produced by DELT were similar after injection in
either the NRM or the NGCp␣. However, the antinocicep-
tion produced by microinjection of DELT in the NGCp␣was
much more effectively antagonized by either CGP35348 or
by yohimbine, than that evoked from sites in the NRM.
Thus, the antinociception evoked by activation of ␦
2
opioid
receptors in the NGCp␣, but not the NRM, is mediated by
spinal ␣
2
and GABA
B
receptors.
The spinal pharmacology of DPDPE-mediated antino-
ciception evoked from the NRM or the NGCp␣also dif-
fered. Intrathecal pretreatment with methysergide or yo-
himbine antagonized the antinociception produced by mi-
croinjection of DPDPE in either the NRM or the NGCp␣.
However, the antinociception evoked from the NRM was
also antagonized by the GABA
B
receptor antagonist
CGP35348, but not by the GABA
A
receptor antagonist
bicuculline, whereas that evoked from the NGCp␣was
antagonized by the bicuculline, but not by CGP35348.
Thus, although DPDPE produces comparable antinocicep-
tion in either the NRM or the NGCp␣, it does so through
different neural pathways. The antinociception produced
by microinjection of L-glutamate in the NRM or NGCp␣
was also preferentially antagonized by i.t. administration of
CGP35348 or bicuculline, respectively (McGowan and
Hammond, 1993a,b) This similarity suggests that ␦
1
opioid
receptor agonists and excitatory amino acid receptor ago-
nists may share a common neuronal substrate for the
production of antinociception.
The onset of DAMGO’s effects at sites in the NGCp␣
was considerably delayed compared with its onset at sites
in the NRM, suggesting that the antinociception results
from diffusion of DAMGO to active sites within the NRM.
The observation that methysergide, which antagonized the
increase in tail-flick latency produced by microinjection of
DAMGO in the NRM, also antagonized its effect 45 min
after microinjection in the NGCp␣supports this proposal.
Although one would also expect CGP35348 to have simi-
larly antagonized the antinociceptive effects of DAMGO in
NGCp␣, this effect did not achieve statistical significance.
Opioid receptor agonists produce antinociception
that is mediated by different spinal cord receptors
The spinal pharmacology of the antinociception produced
by microinjection of DELT or DAMGO in the RVM was
distinct. In the NRM, DAMGO produced antinociception
that was mediated by spinal serotonin
1/2
and GABA
B
re-
ceptors, whereas the effects of DELT were not significantly
attenuated by any antagonist. In the NGCp␣, DELT pro-
duced antinociception that was mediated by spinal ␣
2
ad-
renergic and GABA
B
receptors, whereas the effects of
DAMGO were mediated by spinal serotonin
1/2
receptors.
This differential profile suggests that, at the doses admin-
istered in this study, DELT and DAMGO retain their re-
spective selectivity for ␦
2
and opioid receptors. It is also
consistent with the inability of opioid receptor antago-
nists to antagonize the antinociceptive effect of DELT mi-
Table 1. Effects of intrathecal pretreatment with receptor antagonists on the increase in tail-flick latency produced by microinjection of DPDPE, DELT
or DAMGO in the rostral ventromedial medulla of the rat
Intrathecal antagonist Opioid receptor agonists
DPDPE DELT DAMGO
NRM NGCp␣NRM NGCp␣NRM NGCp␣
Methysergide 30 g Yes Yes No No Yes Yes
Yohimbine 30 g Yes Yes No Yes No No
Bicuculline 0.3 g No Yes No No No No
CGP35348 30 g Yes No No Yes Yes No
a
DPDPE, [D-Pen
2,5
]enkephalin; DELT, [D-Ala
3
, Glu
4
]deltorphin; DAMGO, [D-Ala
2
, NMePhe
4
, Gly-ol
5
]enkephalin.
R. W. Hurley et al. / Neuroscience 118 (2003) 789–796794
croinjected in the RVM or administered intracerebroven-
tricularly (Fraser et al., 2000; Hurley and Hammond, 2001).
Two previous studies, which used antagonists of the ␦
1
and ␦
2
opioid receptor, concluded that DPDPE and DELT
act at different subtypes of the ␦opioid receptor in the RVM
to produce antinociception (Ossipov et al., 1995; Thorat
and Hammond, 1997). The present finding that the spinal
pharmacology of these two agonists is quite different fur-
ther supports this contention. However, the situation is not
as clear with respect to differentiation of ␦
1
and opioid
receptors. Recently, the antinociceptive effects of DPDPE
were proposed to be additionally mediated by a direct
action at opioid receptors for which it has low affinity, or
a synergistic or additive interaction with opioid receptors
that are constitutively active (He and Lee, 1998; Fraser et
al., 2000; Hosohata et al., 2000). Although the design of
the present studies did not provide for a direct test of this
hypothesis, it is notable that the spinal pharmacology of
DPDPE-mediated antinociception was similar to that of
DAMGO for sites in the NRM. Thus, i.t. administration of
methysergide and of CGP35348, but not bicuculline, atten-
uated the increase in tail-flick latency produced by either
agonist. However, whereas i.t. administration of yohimbine
antagonized the antinociceptive effect of DPDPE, it did not
antagonize that produced by DAMGO (this study, but see
Tseng and Collins, 1991) or by morphine (Jensen and
Yaksh, 1986a). Furthermore, the spinal pharmacology of
DPDPE and of DAMGO microinjected in the NGCp␣dif-
fered. Were the antinociceptive effects of DPDPE in the
RVM dependent on activation of a opioid receptor, a
stronger concordance in the spinal pharmacology of DP-
DPE- and DAMGO-mediated antinociception would have
been expected.
The antagonism of DPDPE-, DAMGO- or DELT-medi-
ated antinociception by the GABA receptor antagonists
CGP35348 or bicuculline was always accompanied by an
ability of either yohimbine or methysergide to produce a
comparable level of antagonism. It is therefore plausible to
propose that GABA released in the spinal cord originates
predominantly from interneurons that are situated postsyn-
aptic to either serotonergic or noradrenergic bulbospinal
neurons. Several observations support this idea. The an-
tinociception produced by i.t. administration of serotonin or
serotonin
3
receptor agonists or the inhibition of dorsal horn
neuronal responses to noxious stimulation produced by
iontophoretic application of serotonin
3
receptor agonists is
antagonized by GABA
A
or GABA
B
receptor antagonists
(Alhaider et al., 1991; Nadeson et al., 1996). Also, the
increased release of GABA in the spinal cord produced
by i.c.v. administration of morphine is antagonized by i.t.
administration of a serotonin
3
receptor antagonist
(Kawamata et al., 2002). Finally, norepinephrine or ␣
1
adrenergic receptor agonists dramatically increase the fre-
quency of GABAergic miniature inhibitory post-synaptic
currents recorded in lamina II neurons, suggesting that
norepinephrine increases the release of GABA in the dor-
sal horn (Baba et al., 2000a,b). Thus, it is likely that the
antinociception produced by microinjection of opioid recep-
tor agonists in the RVM is not only mediated by a release
of serotonin or norepinephrine acting at serotonin
1/2
and ␣
2
adrenergic receptors situated on spinothalamic tract neu-
rons, but also by an action at serotonin
3
and ␣
1
adrenergic
receptors situated on GABAergic interneurons that in turn
release GABA to act at either GABA
A
or GABA
B
receptors.
That said, a contribution of GABA released from the ter-
minals of a small population of bulbospinal GABAergic
neurons cannot be excluded (Reichling and Basbaum,
1990; Jones et al., 1991).
In summary, the spinal pharmacology of opioid-medi-
ated antinociception is highly dependent on the type of
receptor and the site of microinjection in the RVM. Thus,
different neural substrates mediate the antinociceptive ef-
fects of ␦
1
,␦
2
and opioid receptor agonists in the RVM.
Acknowledgements—This study was supported by U.S. Public
Health Service grants DA06736 (D.L.H.) and DA05784 (R.W.H.)
from the National Institute on Drug Abuse. We thank Sanjay N.
Thorat, Kenneth Park, and Laura Skrocki for their assistance with
aspects of this work.
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(Accepted 25 November 2002)
R. W. Hurley et al. / Neuroscience 118 (2003) 789–796796
