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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:932–938, 1997
Vol. 283, No. 2
Printed in U.S.A.
Nor-binaltorphimine Precipitates Withdrawal and Excitatory
Amino Acid Release in the Locus Ceruleus of Butorphanol—but
Not Morphine-Dependent Rats1
YANGZHENG FENG, ROBIN W. ROCKHOLD and ING K. HO
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi
Accepted for publication July 11, 1997
Butorphanol (17-cyclobutylmethyl-3-14-dihydroxymorphinan) tartrate is available for clinical use as an unscheduled
opioid analgesic agent under the trade name of Stadol. A
member of the phenanthrene class of opioid analgesics, butorphanol is characterized as a mixed agonist-antagonist
that exerts an analgesic action with a potency seven times
greater than that of morphine (Dobkin et al., 1976). Indicated
for the relief of moderate to severe pain (Wilkinson, 1987),
butorphanol is considered to have a low abuse potential when
used according to therapeutic recommendations. Nevertheless, several clinical reports document the development of
dependence on butorphanol (Brown, 1985; Evans et al., 1985;
Jasinski et al., 1976), significant illegal diversion of the drug
from hospitals within the United States (Hoover and Williams, 1985) and frank abuse by teenagers (Smith and Davis,
1984). The recent release of a formulation for intranasal
application of butorphanol may increase the likelihood of
diversion and abuse of the agent, particularly among medical
personnel (Jasinski et al., 1988). The opioid receptor interaction profile of butorphanol differs significantly from that of
Received for publication April 11, 1997.
1
This work was supported by Grant DA-05828 from the National Institutes
on Drug Abuse. R.W.R. was supported by an award from the American Heart
Association, Mississippi Affiliate.
withdrawal were detected after nor-binaltorphimine only in butorphanol-dependent rats. Basal levels of glutamate and aspartate were not different between treatment groups. Nor-binaltorphimine in the butorphanol-dependent rats increased
glutamate to 227% and aspartate to 158% in the initial 15-min
sample (P , 0.01). Nor-binaltorphimine did not increase glutamate or aspartate concentrations in the morphine-dependent
or saline-treated groups. These results indicate a significantly
greater participation of kappa opioid receptors in the development of butorphanol, rather than morphine, dependence and
identify a differential neurochemical response to butorphanol
withdrawal within a defined brain region, the locus ceruleus.
the classic opioid analgesic morphine. Butorphanol has been
shown to interact with the kappa as well as the mu and delta
opioid receptors (Horan and Ho, 1989b), whereas morphine
binds as an agonist to primarily the mu and delta opioid
receptors (Abdelhamid et al., 1991; Gulya et al., 1988; Miyamoto et al., 1993). Although it is clear that the actions of
butorphanol at the mu and delta opioid receptors are important to the development of dependence (Jaw et al., 1993; Oh
et al., 1992), evidence indicates that interaction with the
kappa opioid receptor contributes significantly to the response (Feng et al., 1994a, 1994b; Jaw et al., 1993, 1994).
Additional data are needed to establish the relative degree of
the involvement of kappa opioid receptors in mediation of
specific aspects of butorphanol dependence, particularly with
respect to assessment of withdrawal from dependence.
Excitatory amino acid neurotransmitters, including glutamate and aspartate, participate in the opioid withdrawal
syndrome, an involvement that is evident particularly within
the pontine locus ceruleus (Rasmussen et al., 1990; Tanganelli et al., 1991). Recently, it has been recognized that naloxone-precipitated withdrawal from dependence on both
morphine and butorphanol is associated with increased extracellular fluid levels of glutamate and aspartate within the
locus ceruleus (Aghajanian et al., 1994; Feng et al., 1995,
ABBREVIATIONS: i.c.v., intracerebroventricular; i.p., intraperitoneal; CTOP, D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; NMDA, N-methyl-Daspartate; s.c., subcutaneous.
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ABSTRACT
The relative involvement of kappa opioid receptors in the mediation of behavioral and neurochemical responses to withdrawal from chronic drug treatment with the opioid analgesic
butorphanol was studied using in vivo microdialysis to detail
extracellular fluid concentrations of glutamate and aspartate
within the locus ceruleus. Sprague-Dawley rats were rendered
opioid dependent after 3 days of intracerebroventricular (i.c.v.)
infusion of butorphanol (26 nmol/ml/hr) or morphine (26 nmol/
ml/hr) and after i.c.v. infusion of saline vehicle (1 ml/hr). Acute
withdrawal was precipitated by i.c.v. injection of the selective
kappa opioid receptor antagonist nor-binaltorphimine (48
nmol/5 ml) after the 3-day period of infusion. Behavioral signs of
1997
Butorphanol and Kappa Opioid Receptors
Methods
Surgical procedures. Male Sprague-Dawley rats (250–300 g;
Charles River, Wilmington, MA) were purchased and maintained
under conditions of 21 6 2°C with a 12 hr/12 hr light/dark cycle for
1 week before surgery. Each rat was anesthetized with Equithensin
(4.25 g of chloral hydrate, 2.23 g of MgSO4 z 7H2O, 0.972 g of
pentobarbital Na, 44.4 ml of propylene glycol, 10 ml of 95% ethanol
and distilled water to make a final volume of 100 ml; 0.3 ml/100 g of
body weight i.p.), and then placed in a stereotaxic apparatus. A
dorsal midline skin incision was made on the cranium, and soft
tissue was cleared from the skull by blunt dissection. The position of
the incisor bar was adjusted so that the skull sutures, bregma and
lambda, were at the same vertical position, leveling the dorsal skull
surface. A burr hole was made in the skull, the dura was incised and
a stainless steel cannula (26 gauge, 10 mm long) was implanted into
the right lateral cerebral ventricle to permit i.c.v. injection or infusion. A stylet (32-gauge sealed stainless steel tubing) was inserted
into the guide cannula to maintain cannula patency. The presence of
cerebrospinal fluid in the guide cannula was noted as verification of
proper ventricular placement. The coordinates for implantation were
(in mm relative to bregma) 0.5 posterior and 1.3 lateral (Paxinos and
Watson, 1986). The cannula tip was lowered 4.5 mm below the
surface of the dura. A CMA/11 microdialysis guide cannula (Bioanalytical Systems, West Lafayette, IN) was implanted with the tip
directed toward the locus ceruleus. The coordinates for implantation
were (in mm relative to bregma) 9.8 posterior and 1.1 lateral (Paxinos and Watson, 1986). The cannula tip was lowered 6.8 mm below
the surface of the dura. Five stainless steel screws were secured to
the skull, an aluminum protective cap was placed around each guide
cannula and dental acrylic (Lang Dental, Wheeling, IL) was applied
to anchor the assembly to the skull surface. Each rat received an s.c.
injection of 150,000 units of procaine penicillin G immediately after
surgical implantation of cannulae. One week was permitted for recovery from surgery before initiation of further experimental procedures. Each rat was killed after completion of an experimental protocol by injection of an overdose of Nembutal; the brain was removed
and post-fixed by immersion in 10% formalin. The i.c.v. cannula
track was verified visually by cutting vertically through the cannula
mark on the surface of the cortex. The remaining brain fragment was
frozen on dry ice, and 50-mm sections were cut through the pons
using a microtome-cryostat (Ames Lab-Tek, Westmont, IL). Animals
were included in statistical treatment of data only if probe placement
within the locus ceruleus was histologically verified (fig. 1).
Administration of drugs and induction of opioid dependence. Each animal was reanesthetized with ether and prepared for
implantation of an osmotic minipump (Alzet 2001; Alza, Palo Alto,
CA), filled with sterile solutions of butorphanol (26 nmol/ml), morphine (26 nmol/ml) or saline vehicle. Dependence was produced by
i.c.v. infusion (26 nmol/ml/hr for 3 days) of butorphanol or morphine.
Saline vehicle rats received a similar volume rate of 0.9% NaCl (1
ml/hr) over the 3-day infusion period. Both the infusion period and
dose paradigm have been used in studies from this laboratory (Feng
et al., 1996; Horan and Ho, 1991). Osmotic minipumps were implanted s.c. between the scapulae with the animals under ether
anesthesia. A 4-cm piece of Tygon tubing (0.38 mm inner diameter;
Cole-Palmer, Chicago, IL) was used to connect the outlet of the
minipump to a piece of L-shaped stainless steel injector tubing (32
gauge, 30 mm in length), which was placed into the i.c.v. guide
cannula. Butorphanol, morphine or saline vehicle solutions were
passed through a 0.2-mm sterile Acrodisk filter (Gelman Sciences,
Ann Arbor, MI) before they were introduced into the minipump
reservoirs. Minipumps were primed overnight at room temperature
in normal saline so the nominal flow rate (1 ml/hr) was attained
before implantation.
Measurement of behavioral signs during opioid withdrawal. Ten distinct behaviors (teeth-chattering, wet-dog shakes,
rearing, locomotion, stretching, scratching, ptosis, yawning, forepaw-tremor and penis-licking) were scored as behavioral signs of
withdrawal during the 30-min period after nor-binaltorphimine injection (i.c.v.). The reactions of each animal were evaluated by an
independent investigator who did not have knowledge of the nature
of the treatment received. The frequency of occurrence of each sign
during the 30-min observation period was used to compare responses
between the saline group and the nor-binaltorphimine-precipitated
withdrawal groups for statistical purposes.
Microdialysis sampling. The microdialysis probes (CMA/11, 2
mm tip) and guide cannulae were purchased from Bioanalytical
Systems (West Lafayette, IN) and used within 3 months. The dialysis membrane tip of the probe has an outer diameter of 240 mm and
an inner diameter of 210 mm, a dead volume of 1 ml and a molecular
weight cutoff of 20,000 Da. The in vitro recoveries of glutamate and
aspartate were determined by immersion of probes in Ringer’s solution containing 100 mM each of glutamate and aspartate at room
Fig. 1. Histological localization of microdialysis probe placement. The
photomicrograph depicts a cresyl violet-stained, 40-mm frozen section;
the track of the probe (arrow) can be seen to pass through the lateral
third of the locus ceruleus. The border of the track can be observed at
the level of the locus ceruleus. The probe was placed so as to extend
beyond the ventral border of the locus ceruleus because epoxy cement
occluded ;0.5 mm of the tip. Me5, mesencephalic trigeminal nucleus;
4V, fourth ventricle; MPB, medial parabrachial nucleus.
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1996; Zhang et al., 1994). Both behavioral signs of withdrawal and increased locus ceruleus concentrations of glutamate and aspartate can be elicited in butorphanol- and morphine-dependent rats after i.c.v. challenge injections of
selective antagonists at mu or delta opioid receptors; no differences in the magnitude of the responses can be distinguished between rats dependent on either opioid analgesic
(Feng et al., 1996). These similarities, and the documented
differences between butorphanol and morphine with respect
to kappa opioid receptor stimulation, suggest that it is of
interest to examine the relative role of the kappa opioid
receptor in mediation of the behavioral and neurochemical
responses to withdrawal from dependence on butorphanol in
comparison with withdrawal from morphine dependence. To
accomplish this objective, i.c.v. injections of the selective
kappa opioid receptor antagonist nor-binaltorphimine were
used to precipitate withdrawal in butorphanol- and morphine-dependent rats. Measurements of glutamate and aspartate concentrations from within the locus ceruleus were
performed using in vivo microdialysis in conscious rats and
correlated with the behavioral signs of withdrawal elicited
after nor-binaltorphimine challenge.
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Feng et al.
Vol. 283
Results
Basal levels of glutamate and aspartate did not differ (P .
0.05) among saline-treated (n 5 4; 10.75 6 1.22 and 8.16 6
0.66 mM) and butorphanol-dependent (n 5 6; 8.04 6 1.58 and
6.76 6 0.78 mM) or morphine-dependent (n 5 6; 9.62 6 1.76
and 8.29 6 1.64 mM) groups, respectively. Precipitation of
acute withdrawal after i.c.v. injection of nor-binaltorphimine
produced significant increases in the concentration of glutamate in butorphanol-dependent, but not in morphine-dependent or saline-treated rats, within the locus ceruleus (fig. 2).
A maximal increase in glutamate concentration of 226.5%
above the basal level was noted in the initial 15-min sample
after nor-binaltorphimine challenge; the absolute value for
the glutamate concentration in that sample was 18.24 6 4.08
mM. The glutamate concentration remained elevated in the
second 15-min sample, as well, before returning to levels not
statistically different from the basal level in the third 15-min
sample. The glutamate levels in the initial samples from the
groups receiving morphine (10.65 6 1.23 mM) or saline
(10.23 6 1.04 mM) were not significantly different from basal
values in the respective group. Similarly, a significant increase in the locus ceruleus aspartate concentration was
noted in the initial 15-min sample after nor-binaltorphimine
challenge in the butorphanol-dependent group (11.42 6 1.17
mM; 158.3% of the basal value; fig. 3). The aspartate concentrations from the initial 15-min sample did not differ from
basal values in the groups receiving either morphine (7.47 6
1.62 mM) or saline (7.71 6 0.63 mM). Although aspartate
concentrations were not elevated to the same degree as those
of glutamate, aspartate levels were remained elevated for a
longer period. Significant increases above basal levels were
noted in aspartate in the first three 15-min samples after
nor-binaltorphimine challenge.
Behavioral evidence of withdrawal, as identified by the
incidence of 10 specific signs in the 30-min period after norbinaltorphimine challenge, was present only in butorphanoldependent rats (table 1). Significantly higher frequencies of 5
of the 10 signs (wet-dog shakes, teeth chattering, stretching,
scratching and fore-paw tremor) were noted in the butorphanol-dependent rats compared with morphine-dependent
Fig. 2. Increases in extracellular fluid levels of glutamate within the
locus ceruleus during nor-binaltorphimine-precipitated butorphanol
withdrawal. Animals received 3 days of i.c.v. infusion of butorphanol
(BUT; 26 nmol/ml/hr; n 5 6), morphine (MOR; 26 nmol/ml/hr; n 5 6) or
normal saline (SAL; 1 ml/hr; n 5 4). Nor-binaltorphimine (Nor-BNI; 48
nmol/5 ml) was given by i.c.v. injection over a 5-min period beginning at
the time indicated. Values are expressed as percentage changes
(mean 6 S.E.M.) from basal values. Basal values were calculated as a
single basal value that represented the average of the four individual
basal samples. * P , 0.01, ** P , 0.01 vs. control.
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temperature. Probes were perfused with Ringer’s solution at a rate of
2 ml/min, samples were collected and dialysate samples were subsequently analyzed by HPLC. The values from three or four consecutive samples were averaged to determine the average recovery values for each probe. The averaged recovery values of glutamate and
aspartate were 7.51 6 0.89% and 5.43 6 0.66%, respectively, of the
external concentrations of each amino acid for 16 probes. Due to the
variability of probe recovery, the extracellular levels of each amino
acid were calculated individually for each animal.
Analysis of amino acids. The method of Ellison et al. (1987) was
used, with minor modification, for measurement of amino acids.
Briefly, the measurements were performed on an HPLC (BAS 200;
Bioanalytical Systems) with electrochemical detection. A Rainin C18
column (150 3 4.6 mm I.D., 5 mm, 100 Å) was used. The mobile phase
consisted of 0.1 M sodium phosphate (mono and dibasic)/methanol
buffer (63:37, v/v; pH 5.2). The derivatizing agent consisted of
o-phthaldialdehyde (50 mg), 2-mercaptoethanol (40 ml), absolute ethanol (0.9 ml) and 0.1 M borate buffer to make a final volume of 10 ml.
The glutamate peak was verified by retention time, peak shape and
comparison of samples with a standard consisting of eight amino
acids (aspartate, glutamate, glutamine, histidine, arginine, glycine,
taurine, and alanine; each at a concentration of 5 mM).
General experimental procedures. One week after stereotaxic
surgery, each rat was anesthetized with ether, and an osmotic
minipump was implanted. An i.c.v. infusion of butorphanol, morphine or saline was initiated and maintained for 3 days as described
above. On the third day after initiation of i.c.v. infusion, each rat was
placed in an individual stainless steel wire-bottom cage, and a plastic
harness was secured loosely around the chest. This harness was
tethered to a microdialysis counterbalance arm, and a freshly calibrated microdialysis probe was placed into the locus ceruleus guide
cannula. The probe was perfused for the initial 18 hr at a low flow
rate (0.2 ml/min) using a CMA/100 microdialysis infusion pump.
Each experimental sequence began with disconnection of the tubing
between the osmotic minipump and the i.c.v. inlet cannula. The
infusion rate through the probe was increased to 2 ml/min for an
equilibration period of 2 to 3 hr. Thereafter, a series of four or five
sequential 15-min samples were collected for determination of basal
concentrations of glutamate and aspartate. Nor-binaltorphimine was
injected i.c.v. (48 nmol/5 ml) over a 5-min period using a hand-held
microliter syringe. Samples were collected during the hour immediately after nor-binaltorphimine injection. This dose of nor-binaltorphimine was chosen based on the results of prior studies (Jaw et al.,
1993, 1994). In particular, the dose was determined from the study of
Jaw et al. (1994), who compared the effects of a range of nor-binaltorphimine doses (12–100 nmol i.c.v.) with those of naloxone in the
elicitation of behavioral signs of acute withdrawal in butorphanoldependent rats.
Statistics. The Student’s t test was used to test for differences
between the basal level of an amino acid and the maximal value
obtained after nor-binaltorphimine challenge. One-way analysis of
variance and the Newman-Keul test were used to test among the
amino acid concentrations in the butorphanol, morphine and saline
groups. Calculated values of P , 0.05 were considered statistically
significant. Values for amino acid concentrations that are expressed
as percentage change from basal values were calculated from a
single basal value that represented the average of the four individual
basal samples taken immediately before nor-binaltorphimine challenge. The frequencies of occurrence of each behavioral sign were
analyzed by one-way analysis of variance, and the Bonferroni test
was used to differentiate among the mean values of these three
groups. Mean 6 S.E.M. values are reported.
1997
Butorphanol and Kappa Opioid Receptors
TABLE 1
Frequencies of withdrawal signs precipitated by i.c.v. injection of
the kappa opioid receptor antagonist nor-binaltorphimine in
butorphanol- or morphine-dependent rats
Rats were treated with i.c.v. infusion of butorphanol (26 nmol/ml/hr), morphine (26
nmol/ml/hr) or saline (1 ml/hr) for 3 days and then challenged with i.c.v. norbinaltorphimine (48 nmol/5 ml).
Wet-dog shakes
Teeth chattering
Rearing
Locomotion
Stretching
Penis-licking
Scratching
Ptosis
Yawning
Forepaw-tremor
Butorphanol
(n 5 6)
Morphine (n 5 6)
Saline (n 5 4)
10.67 6 3.00
9.50 6 2.14
1.16 6 0.40
2.67 6 1.20
5.83 6 1.57
1.50 6 0.80
1.50 6 0.50
1.00 6 0.44
3.00 6 0.08
3.17 6 1.08
1.33 6 0.80b
0.17 6 0.17b
0
0.17 6 0.17
0b
0.17 6 0.17
0.17 6 0.17a
0.17 6 0.17
0.16 6 0.16
0b
1.50 6 0.86a
0b,c
0.50 6 0.50
0.25 6 0.25
0a
0
0
0
0
0
a
P , 0.05, b P , 0.01 (analysis of variance test), withdrawal score of butorphanol group vs. morphine or saline group.
c
0, no positive signs detected.
and/or saline-treated animals. The withdrawal scores in the
morphine-dependent group did not differ from those of the
saline-treated animals.
Discussion
The relative involvement of opioid receptor subtypes in the
development of dependence on, and withdrawal from, butorphanol differs from that observed with the prototype opioid
analgesic morphine. The results of the present study extend
the body of evidence that supports this conclusion by documenting that the kappa opioid receptor plays a role in neurochemical and behavioral indices of withdrawal in butorphanol-, but not morphine, dependent rats. This is
demonstrated by the finding that acute precipitation of withdrawal from butorphanol, but not morphine, dependence can
be elicited by i.c.v. injection of the selective kappa opioid
receptor antagonist nor-binaltorphimine. The results indicate further that excitatory amino acid neurotransmission
within the pontine locus ceruleus is altered during withdrawal from dependence on butorphanol.
Morphine has been demonstrated to activate primarily the
mu opioid receptor (Gulya et al., 1988), with possible agonistic actions also at the delta opioid receptor subtype (Abdelhamid et al., 1991; Martin et al., 1976; Miyamoto et al., 1993).
In contrast, butorphanol is an agonist not only at the mu and
delta opioid receptors but also at the kappa opioid receptor
(Horan and Ho, 1989b), with binding affinity ratios to mu,
delta and kappa opioid receptors of 1:4:25 (Chang et al., 1983;
Lahti et al., 1985). The characteristics of action of butorphanol at the mu opioid receptor are such that it is capable of
precipitating withdrawal in rats made dependent on morphine (Horan and Ho, 1989a; Pircio et al., 1976) and can
substitute for agonists at the mu opioid receptor in drug
discrimination paradigms (Picker and Dykstra, 1989). However, butorphanol has been shown to be self-administered to
a lesser degree than was the mu opioid receptor agonist
codeine and capable of producing antagonism of the analgesic
effects of high-efficacy mu opioid receptor agonists (Woods
and Gmerek, 1985). Butorphanol exerts a kappa opioid receptor agonist-like dependence profile (Woods and Gmerek,
1985) and has been demonstrated to cause increases in urinary output, although not to the extent as that produced by
bremazocine and other high-efficacy kappa opioid receptor
agonists. In addition, butorphanol has been reported to blunt
the urinary output (Leander, 1983a, 1983b) and analgesic
responses (Pircio et al., 1976) to administration of high-efficacy kappa opioid receptor agonists. These observations have
led to the classification of butorphanol as a mu and kappa
opioid receptor agonist with intermediate efficacy (Leander,
1983b; Picker and Dykstra, 1989).
An earlier report from this laboratory found that morphine
and butorphanol were equipotent in the induction of dependence, as demonstrated by the behavioral signs elicited after
acute precipitation of withdrawal by challenge with the nonselective opioid antagonist naloxone (Horan and Ho, 1991). It
is also of note that the general pattern for the behavioral
response to precipitated withdrawal of morphine and butorphanol is similar (Jaw et al., 1994). The development of
tolerance has been demonstrated in both the tail-flick and
acetic acid writhing tests of analgesia after 1 to 3 days of the
i.c.v. infusion of butorphanol (Feng et al., 1994b). In addition,
cross-tolerance between butorphanol and morphine could be
produced through i.c.v. infusion of the two agents (Feng et al.,
1994a). In the latter study, chronic i.c.v. administration of
butorphanol produced similar rightward shifts of the analgesic response to morphine in both the tail-flick and acetic acid
writhing tests (Feng et al., 1994a). A recent investigation
from this laboratory, using a protocol similar to that used in
the present study, examined the ability of selective antagonists at mu and delta opioid receptors to precipitate withdrawal in rats rendered dependent on butorphanol or morphine. The i.c.v. injections of the selective mu opioid receptor
antagonist CTOP and the delta opioid receptor antagonist
naltrindole elicited equivalent behavioral signs of withdrawal and increases in locus ceruleus concentrations of glutamate and aspartate in butorphanol- and morphine-dependent rats (Feng et al., 1994c, 1996). The results have been
interpreted to indicate that dependence on either butorpha-
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Fig. 3. Increases in extracellular fluid levels of aspartate within the locus
ceruleus during nor-binaltorphimine-precipitated butorphanol withdrawal. Animals received 3 days of i.c.v. injection of butorphanol (BUT;
26 nmol/ml/hr; n 5 6), morphine (MOR; 26 nmol/ml/hr; n 5 6) or saline
(SAL; 1 ml/hr, n 5 4). Nor-binaltorphimine (Nor-BNI; 48 nmol/5 ml) was
given by i.c.v. injection over a 5-min period beginning at the time
indicated. Values are expressed as percentage changes (mean 6
S.E.M.) from basal values. Basal values were calculated as a single
basal value that represented the average of the four individual basal
samples. * P , 0.05, ** P , 0.01 vs. control.
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TABLE 2
Comparison between concentrations of glutamate in extracellular fluid dialysate samples taken from the locus ceruleus of conscious
rats in the present study with those from Feng et al. (1996)
Values presented are from 15-min samples taken before (Basal) and from the initial 15 min samples taken immediately after i.c.v. injections of nor-binaltorphimine (48
nmol/5 ml) in rats rendered dependent on butorphanol or morphine and in rats treated with saline. The data are compared with previously published data (Feng et al.,
1996) in which acute withdrawal was precipitated by i.c.v. injection of either CTOP (48 nmol/5 ml) or naltrindole (100 nmol/5 ml). Mean 6 S.E.M. values are given. The
numbers in parentheses denote the number of animals tested in each group.
Glutamate concentration
Basal
Antagonist
Butorphanol
Nor-binaltorphimine
(48 nmol/5 ml)
CTOP†
(48 nmol/5 ml)
Naltrindole†
(100 nmol/5 ml)
8.04 6 1.58
(6)
10.17 6 0.75
(5)
11.66 6 1.19
(5)
Morphine
9.62 6 1.76
(6)
10.03 6 1.12
(5)
12.68 6 0.91
(5)
Initial 15-min sample
Saline
mM
10.75 6 1.22
(4)
9.99 6 1.27
(6)
9.59 6 1.27
(4)
Butorphanol
Morphine
Saline
18.24 6 4.08b
(6)
25.73 6 1.19b
(5)
17.47 6 1.89a
(5)
10.65 6 1.23
(6)
31.17 6 5.61b
(5)
21.56 6 3.07a
(5)
10.23 6 1.05
(4)
12.63 6 1.59
(6)
12.77 6 1.60
(4)
Asterisks denote a statistical difference between an indicated and basal value for each group (a P , 0.05, b P , 0.01). Data from Feng et al. (1996) are reproduced
by permission (†).
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mu opioid receptor agonist morphine in the mouse writhing
test for analgesic efficacy (Takemori et al., 1988). A 160-fold
selectivity for kappa compared with mu and delta opioid
receptors has been reported for nor-binaltorphimine in radioligand binding assays (Takemori et al., 1988). Naloxone differs from nor-binaltorphimine in that it can exert relatively
nonselective antagonistic effects at mu and delta, as well as
kappa, opioid receptors (Patterson et al., 1984). Both naloxone and nor-binaltorphimine challenge can precipitate behavioral signs of acute withdrawal in butorphanol-dependent
rats (Jaw et al., 1994). However, protection of kappa opioid
receptors by pretreatment with nor-binaltorphimine minimizes behavioral symptoms of acute, naloxone-precipitated
withdrawal in butorphanol-dependent rats (Jaw et al., 1993).
It has also been shown that continuous i.c.v. infusion of
butorphanol for 3 days will induce cross-tolerance to the
analgesic action of the selective kappa opioid receptor agonist
U-50,488 as well as to morphine (Feng et al., 1994a). The
results of the present study detail a kappa opioid receptor
action to the actions of butorphanol, as demonstrated by the
ability of nor-binaltorphimine to precipitate increases in locus ceruleus concentrations of glutamate and aspartate, as
well as behavioral symptoms of opioid withdrawal in butorphanol- but not in morphine-dependent rats.
The contention that the locus ceruleus participates in morphine withdrawal is supported by an abundance of electrophysiological data (Rasmussen et al., 1990, 1991a; Rasmussen and Aghajanian, 1989). In this regard, the hallmark of
withdrawal from morphine dependence is hyperactivity of
noradrenergic neurons within the locus ceruleus (Aghajanian, 1978), a response that has been correlated with behavioral symptoms of opioid withdrawal (Gold et al., 1980; Redmond and Krystal, 1984). Indeed, the locus ceruleus has been
identified as a principal site within the brain from which the
behavioral signs of withdrawal from morphine dependence
can be elicited by local tissue injections of the naloxone derivative methyl naloxonium (Maldonado et al., 1992). The
issue addressed by the present study stems from the aforementioned body of data and is 2-fold. First, does a neurotransmitter system other than the opioid system or the alpha
adrenoceptor system (Aghajanian, 1978) also contribute to
the mediation of opioid withdrawal? Second, does withdrawal
nol or morphine involves roughly equivalent actions at mu
and delta opioid receptors.
In contrast, nor-binaltorphimine challenge precipitates
withdrawal selectively in butorphanol-dependent rats, which
supports the hypothesis of kappa opioid receptor involvement
in the development of dependence on butorphanol. Specifically, 48 nmol of nor-binaltorphimine was found to elevate
locus ceruleus concentrations of both glutamate and aspartate in butorphanol- but not in morphine-dependent rats. For
example, the degree of elevation in glutamate, above basal
levels, was 227% in butorphanol-dependent rats. This compares with maximal elevations in glutamate of 253% and
150%, respectively, after precipitation of withdrawal from
butorphanol dependence by i.c.v. injections of the selective
mu and delta opioid receptor antagonists CTOP and naltrindole (Feng et al., 1996). Table 2 is provided to permit comparison of basal and maximal elevations in glutamate levels
between this and our previous study (Feng et al., 1996).
Although nor-binaltorphimine injection increased locus ceruleus glutamate levels only in butorphanol-dependent rats,
CTOP and naltrindole elicited increases in glutamate in both
morphine- and butorphanol-dependent rats (Feng et al.,
1996). Finally, elicitation of the behavioral signs of withdrawal by nor-binaltorphimine was evident in butorphanolbut not in morphine-dependent rats. This latter result is
meaningful because in a previous study, i.c.v. infusion of the
agents CTOP and naltrindole elicited significant behavioral
evidence of withdrawal in both morphine- and butorphanoldependent animals (Feng et al., 1996). Although direct comparison of results from the present study with those of Feng
et al. (1996) is not possible due to slight differences in the
behavioral scoring protocol, the data support a significantly
greater involvement of kappa opioid receptors in withdrawal
from butorphanol dependence. Nor-binaltorphimine is a
bivalent pharmacophore consisting of two molecules of naltrexone bound by a pyrrole ring, which possesses a 20-fold
selectivity for kappa over mu opioid receptors in the guinea
pig ileum and exerts a long-lasting antagonistic action (Portoghese et al., 1987). Moreover, after i.c.v. administration,
nor-binaltorphimine exerts a 100-fold selectivity for kappa
over mu opioid receptors when tested against the selective
kappa opioid receptor agonist U-50,488 and the prototypical
1997
937
gantocellularis have been shown to attenuate the hyperactivity of locus ceruleus neurons associated with opioid withdrawal (Rasmussen and Aghajanian, 1989), a response that
is believed to be mediated by an excitatory amino acid pathway from the nucleus paragigantocellularis to the locus ceruleus. These data have led investigators to suggest that the
source of the increased glutamate originates from activation
of glutamatergic nerve projections from the nucleus paragigantocellularis to the locus ceruleus (Ennis et al., 1992;
Zhang et al., 1994). No direct evidence is available concerning
the source of withdrawal-associated glutamate overflow from
the locus ceruleus. However, behavioral signs that mimic
those evoked during precipitated withdrawal from dependence on butorphanol can be elicited after electrical stimulation of the nucleus paragigantocellularis in conscious rats
(Liu et al., 1997). This strengthens an argument for involvement of the nucleus paragigantocellularis in excitatory
events related to opioid withdrawal. Additional studies to
examine glutamate efflux from the locus ceruleus after electrical stimulation of the nucleus paragigantocellularis are in
progress.
McFadzean et al. (1987) noted that administration of the
selective kappa opioid receptor agonist U 50488 to rat brain
slice preparations could depress excitatory synaptic transmission to locus ceruleus neurons. This raises the possibility
that the increases noted in glutamate in the present study
were the result of blockade, by nor-binaltorphimine, of this
previously identified, presynaptic mechanism. However, the
i.c.v. administration of butorphanol to naive rats does not
alter locus ceruleus levels of glutamate (Feng et al., 1995;
Hoshi et al., 1996), as would be expected if the presynaptic
pathway were the principal mechanism underlying a selective kappa opioid receptor mediation of the observed phenomena. One potential explanation for this apparent discrepancy
lies in the observation of McFadzean et al. (1987) that only a
modest percentage (38%) of the excitatory synaptic potential
within the locus ceruleus could be depressed by U-50488.
They suggested that kappa opioid receptors might inhibit
presynaptic glutamate release only on specific neuronal afferents to the locus ceruleus. In the naive rat, the depression
produced by butorphanol may not sufficiently reduce extracellular concentrations of glutamate to be detected by in vivo
microdialysis techniques.
The results of the present study are significant in that they
demonstrate that excitatory amino acid levels within the
locus ceruleus can be increased and behavioral symptoms of
withdrawal can be elicited selectively by nor-binaltorphimine
in butorphanol-dependent rats. Thus, the physical dependence produced by chronic administration of butorphanol can
be attributed to interaction with the kappa opioid receptor, a
phenomenon that does not occur when dependence is produced by similar administration regimens of morphine. Additional investigations will need to be conducted to determine
the site of the opioid receptors on which butorphanol acts to
mediate withdrawal.
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