Psychopharmacology (1998) 140:5768

Springer-Verlag 1998

O R I G I N A L I N V E S T I G AT I O N

Mark A. Smith Mitchell J. Picker

Tolerance and cross-tolerance to the rate-suppressing effects

of opioids in butorphanol-treated rats: influence of maintenance dose
and relative efficacy at the mu receptor
Received: 25 November 1997 / Final version: 21 February 1998

Abstract The purpose of the present investigation was

to examine the development of tolerance to the rate-suppressing effects of mu and kappa opioids in rats administered either 3.0 (low) or 30 (high) mg/kg per day of
butorphanol, an opioid with low relative efficacy at the
mu receptor. The mu opioids butorphanol, buprenorphine, morphine, fentanyl and sufentanil, and the kappa
opioid U50,488 dose-dependently suppressed responding under all conditions examined. In rats administered
the low maintenance dose of butorphanol, tolerance developed to the effects of butorphanol, buprenorphine
and morphine, but not to fentanyl and sufentanil. In rats
administered the high maintenance dose, tolerance developed to all of the mu opioids examined. In both treatment groups, the degree to which tolerance developed
was greater for butorphanol and buprenorphine than for
morphine, fentanyl and sufentanil; and the degree to
which tolerance developed to these mu opioids was
greater in rats administered the high maintenance dose
of butorphanol. The tolerance that developed to morphine, fentanyl and sufentanil was not altered when tested at both 23 and 47 h following the previous maintenance dose of butorphanol, suggesting that these changes were not due to any acute pharmacological interactions between butorphanol and the test compound (i.e.,
antagonism). Tolerance was also conferred to the kappa
opioid U50,488 in both groups of rats, and in rats administered the high maintenance dose, this effect was
obtained when tested 23 and 47 h following the previous maintenance dose of butorphanol. Physical dependence developed in rats administered the high maintenance dose of butorphanol, as evidenced by the development of enhanced sensitivity to the rate-suppressing
effects of naloxone, and the finding that 30 mg/kg naloxone decreased body weight in a time-dependent manner. No physical dependence was apparent in rats administered the low maintenance dose of butorphanol.
M.A. Smith M.J. Picker ()
Department of Psychology,
University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-3270, USA

These data suggest that during chronic treatment with

butorphanol, (1) greater degrees of tolerance are conferred to drugs possessing low efficacy at the mu opioid
receptor, (2) tolerance is enhanced as the maintenance
dose of the toleragen is increased, and (3) mu-opioid
tolerance may be observed under conditions that do not
produce mu-opioid dependence.
Key words Butorphanol Cross tolerance Dependence
Relative efficacy Opioid Rat Tolerance

Introduction
Chronic exposure to the mu-opioid morphine can induce
tolerance to its effects within a given experimental preparation, a phenomenon believed to result from alterations
in the number and sensitivity of surface mu receptors
(see Puttfarcken et al. 1988; Puttfarcken and Cox 1989;
Yoburn et al. 1990, 1993). Tolerance can also be conferred to the effects of other mu opioids, with the magnitude of this effect varying across test drugs. In morphinetreated rats, for instance, a greater degree of tolerance is
conferred to the antinociceptive effects of buprenorphine
(Paronis and Holtzman 1992) and to the discriminativestimulus effects of nalbuphine (Young et al. 1991) than
to either morphine or etorphine. Also in morphine-treated rats, a greater degree of tolerance is conferred to the
rate-suppressing effects of butorphanol than to either
morphine or fentanyl (Picker et al. 1990; Hughes et al.
1996; Smith et al. 1997). In the studies cited above, it
was postulated that differences in the magnitude of tolerance was a consequence of differences in the relative efficacy of the test drugs at the mu opioid receptor, with
greater degrees of tolerance conferred to low-efficacy
mu opioids (butorphanol, buprenorphine, nalbuphine)
than to high-efficacy mu opioids (morphine, fentanyl, etorphine).
Although there is a wealth of data concerning the
development of tolerance in animals treated with highefficacy opioids such as morphine or fentanyl (e.g.,

58

Emmett-Oglesby et al. 1988; Paronis and Holtzman

1992, 1994; Yoburn et al. 1993), little is known about
the development of tolerance in animals chronically
treated with low-efficacy opioids. The data that do exist
with these drugs suggest there may be limitations as to
the role that relative efficacy plays in the development
of tolerance and cross-tolerance. For example, in pigeons treated chronically with the low-efficacy opioid
nalbuphine, tolerance failed to develop to its rate-suppressing effects even after 20 weeks of chronic treatment (Gerak and France 1996). Nevertheless, cross-tolerance was conferred to the effects morphine, fentanyl
and etonitazene, but the degree of tolerance was not
strongly related to their relative efficacy at the mu receptor.
One purpose of the present investigation was to
evaluate the behavioral effects of chronic administration of butorphanol, an opioid possessing lower efficacy at the mu receptor than morphine (Zimmerman et al.
1987; Butelman et al. 1995; Garner et al. 1997). In vivo, butorphanol produces mu-like discriminative stimulus effects (Shannon and Holtzman 1977; White and
Holtzman 1983; Picker and Dykstra 1989; Picker et al.
1993; Picker 1997), precipitates withdrawal in morphine-dependent animals (Horan and Ho 1989), and antagonizes the respiratory-depressive (Bowdle et al.
1987) and antinociceptive (Butelman et al. 1995; Garner et al. 1997) effects produced by high-efficacy mu
opioids. Despite observations that some of butorphanols effects are mediated by multiple opioid receptor
subtypes (Leander 1983a,b; Smith and Picker 1995), its
effects on schedule-controlled behavior appear to mediated by activity at the mu receptor. For example, in rats
responding under an FR20 schedule of food presentation, butorphanols rate-suppressing effects can be reversed by the mu-selective irreversible antagonist betafunaltrexamine (Pitts et al. 1996a), and pA2 values for
naloxone as an antagonist against butorphanols ratesuppressing effects are consistent with activity at mu,
but not kappa or delta opioid receptors (Pitts et al.
1996b).
A second purpose of the present investigation was to
examine the development of cross-tolerance to a range of
opioids possessing different degrees of efficacy at the
mu receptor. These compounds included several high-efficacy mu opioids (morphine, fentanyl and sufentanil)
and a low-efficacy mu opioid (buprenorphine). In order
to determine if the degree of tolerance was related to the
daily maintenance dose of butorphanol, groups of rats
were treated with either a low (3.0 mg/kg per day) or
high (30 mg/kg per day) maintenance dose of butorphanol. These maintenance doses were approximately 10and 100-fold larger than the dose required to decrease response rates to 50% of control values (i.e., ED50). Finally, in order to examine if the development of cross-tolerance was pharmacologically specific to drugs acting at
the mu opioid receptor, tests were conducted with the
kappa-opioid U50,488 and the non-opioid compound
pentobarbital.

Materials and methods

Animals and apparatus
Seventeen male Long-Evans rats were maintained at approximately 80% of their free-feeding body weights (295365 g) by restricting their daily intake of Purina Rat Chow. The rats were approximately 4 months old at the start of the experiment and were
housed individually in a colony room on a 12-h light/dark cycle.
Drinking water was available ad libitum in the home cage.
Six rat operant-conditioning chambers (Gerbrands model
G7322) were enclosed in ventilated, sound-attenuating cubicles.
Each chamber was equipped with two response levers, located 9.0
cm above the floor and 13.0 cm apart. Two stimulus lights were
mounted 7.0 cm above each lever, and a food receptacle into
which 45-mg Noyes pellets (P. J. Noyes Co., Lancaster, N.H.,
USA) were dispensed was centered 6.0 cm below the levers. Two
houselights were located on the ceiling above the rear wall. White
noise was present continuously within each chamber and throughout the experimental room. Experimental events were programmed and data were collected through the use of a microcomputer, using software and interfacing supplied by Med Associates,
Inc. (Georgia, Vt., USA).
Behavioral procedure
During the initial training sessions, rats were trained to lever press
on a multiple (10 min time-out) [5 min fixed ratio 1 (FR1)] schedule of food presentation. Training sessions consisted of five components of 15 min duration (75 min total), and started at precisely
1:00 p.m. each day. During training on the FR1 schedule, the
houselight was illuminated and a lever press on the left lever produced a food pellet. During the time-out period, the houselight
was extinguished and responding had no programmed consequences. Over the next 4 weeks, the FR requirement was gradually
increased until responding was maintained under an FR20 schedule of food presentation. Under this schedule, every 20th lever
press produced a food pellet. After an additional 4 weeks of training on the multiple schedule, dose-effect curves were determined
for various test compounds using a cumulative dosing procedure.
Under the cumulative dosing procedure, doses of a test compound
were administered at the beginning of each time-out period, such
that the dose increased the total amount of drug received in that
session by 0.25 or 0.5 log units. For example, the administration
of 1.0, 2.0, 2.6, 4.4 and 7.5 mg/kg of a test drug yielded cumulative doses of 1.0, 3.0, 5.6, 10.0 and 17.5 mg/kg. Otherwise, all
conditions were identical to those during training sessions. Test
sessions were conducted on Tuesdays and Fridays, training sessions were conducted on Mondays and Wednesdays, and saline
control sessions were conducted on Thursdays.
Induction and termination of tolerance
Groups I and II
Prior to chronic butorphanol treatment, group I (n=6) and group
II (n=6) were tested with butorphanol, morphine, fentanyl, buprenorphine, sufentanil, U50,488, pentobarbital and naloxone in that
order. Each dose-effect curve was determined twice before testing
the next compound. Following the determination of these prechronic dose-effect curves, rats from group I were injected daily
with 3.0 mg/kg butorphanol, whereas rats in group II were injected daily with 30 mg/kg butorphanol. In order to decrease the possibility that the maintenance dose of butorphanol would produce
acute, pharmacological interactions with doses of a test compound, each maintenance dose of butorphanol was administered
approximately 23 h prior to the next scheduled test session. Under these conditions, the maintenance dose of butorphanol was
administered daily at approximately 2:15 p.m. (immediately following each experimental session). After 6 weeks exposure to the

59
Table 1 Mean (SE) response
rates of rats in group I (n=6),
group II (n=6) and group III
(n=5). All values are expressed
in responses/s

Butorphanol
Pre-chronic
Chronic
Post-chronic
Buprenorphine
Pre-chronic
Chronic
Post-chronic
Morphine
Pre-chronic
Chronic
Post-chronic
Fentanyl
Pre-chronic
Chronic
Post-chronic
Sufentanil
Pre-chronic
Chronic
Post-chronic
U50,488
Pre-chronic
Chronic
Post-chronic
Pentobarbital
Pre-chronic
Chronic
Post-chronic
Naloxone
Pre-chronic
Chronic
Post-chronic

3.0 mg/kg per day (I)

30 mg/kg per day (II)

30 mg/kg per day (III)

1.014 (0.018)
0.798 (0.066)
0.864 (0.058)

1.466 (0.012)
1.390 (0.040)
1.395 (0.026)

0.998 (0.045)
0.885 (0.031)
0.878 (0.012)

0.896 (0.036)
0.893 (0.041)
0.822 (0.034)

1.342 (0.068)
1.249 (0.031)
1.288 (0.024)

0.844 (0.064)
0.779 (0.053)
0.902 (0.063)

1.301 (0.033)
1.358 (0.050)
1.405 (0.009)

0.916 (0.036)
0.920 (0.030)
0.807 (0.043)

0.964 (0.010)
0.781 (0.052)
0.884 (0.060)

1.392 (0.049)
1.358 (0.050)
1.369 (0.041)

1.098 (0.028)
0.967 (0.023)
0.846 (0.021)

1.015 (0.020)
0.916 (0.071)
0.849 (0.040)

1.425 (0.026)
1.258 (0.022)
1.321 (0.031)

0.923 (0.028)
0.897 (0.043)
0.872 (0.014)

0.994 (0.013)
0.943 (0.009)
0.860 (0.037)

1.437 (0.022)
1.356 (0.036)
1.353 (0.023)

1.199 (0.010)
0.987 (0.019)
0.934 (0.012)

0.898 (0.009)
0.961 (0.044)
0.864 (0.056)

1.422 (0.037)
1.475 (0.064)
1.388 (0.015)

0.984 (0.031)
0.900 (0.071)
0.803 (0.035)

1.497 (0.033)
1.309 (0.099)
1.310 (0.041)

chronic butorphanol regimen, during which time no test drugs

were administered but daily training sessions continued, dose-effect curves were redetermined for all drugs examined prior to butorphanol treatment in the order described above. During this
phase, test sessions were conducted on Fridays, training sessions
on Mondays, Tuesdays and Wednesdays, and saline control sessions on Thursdays. Following a single redetermination of the
dose-effect curves for each drug, which took approximately 8
weeks, the chronic butorphanol regimen was terminated and no
drugs were administered for the next 6 weeks. At the end of this
6-week period, dose-effect curves for all drugs were re-determined under a schedule identical to that employed during prechronic conditions.
Group III
Prior to chronic butorphanol treatment, group III (n=5) was tested
with butorphanol, morphine, fentanyl, sufentanil and U50,488. After each drug had been tested once, dose-effect curves for morphine,
fentanyl, sufentanil and U50,488 were redetermined following an
acute dose of 30 mg/kg butorphanol administered 23 h prior to the
test session. Following these tests, a chronic butorphanol regimen
was initiated in which rats received a daily injection of 30 mg/kg
butorphanol, with each injection administered at least 23 h before
the next scheduled experimental session. After 6 weeks of butorphanol treatment, the dose-effect curves for butorphanol, morphine,
fentanyl, sufentanil and U50,488 were redetermined as described
for groups I and II. The curves for morphine, fentanyl, sufentanil
and U50,488 were then redetermined a second time under chronic
conditions, with the exception that the previously scheduled maintenance dose of butorphanol was not administered. Under these con-

ditions, 47 h separated the administration of the previous maintenance dose of butorphanol and the following test session.
On Tuesday of week 10 of the chronic regimen, the regular
training session was suspended and measures of precipitated withdrawal were taken. Each rat was weighed, injected with 30 mg/kg
naloxone and returned to the home cage. Fifteen minutes following
the naloxone injection, each rat was removed from the home cage,
weighed a second time, and the presence or absence of diarrhea was
noted. This procedure was repeated again at 30 min and 60 min following naloxone injection. After the final weighing at 60 min, the
rats were injected with the daily maintenance dose of butorphanol
(30 mg/kg) before being returned to the home cage. This procedure
was repeated on Friday of week 10, with the exception that 1.0 ml/kg
saline was administered to each rat in lieu of naloxone.
When chronic testing was concluded, the butorphanol regimen
was terminated and no test drugs were administered for the next 6
weeks. The dose-effect curves for butorphanol, morphine, fentanyl,
sufentanil and U50,488 were then redetermined at both 6 and 10
weeks following treatment termination. One rat from group III exhibited adverse stereotypical behavioral during the chronic regimen
that consisted of chewing on the distal portion of the tail. This behavior ceased when chronic butorphanol treatment was terminated.
Data analysis
Rates of responding were derived by dividing the total number of
responses emitted during each FR component by the total time spent
within the component. During test sessions, rates of responding for
each FR component were expressed as a percentage of the response
rate for the relevant component from the most recent saline control
session. In most cases, dose-effect curves consisted of an initial

60
dose of the test compound that had little or no effect on rates of responding and proceeded up to a dose that decreased markedly or
eliminated responding. For each dose-effect curve, ED50 values
were computed for each rat mathematically (least squares method)
by log-linear interpolation using at least three points on the descending portion of the curve. Using data from each rat, relative potency
estimates (95% confidence limits) for pre-chronic versus chronic
and pre-chronic versus post-chronic ED50 values were computed using standard statistical software. Relative potency estimates were
considered statistically significant when the 95% confidence limits
did not overlap 1.0. One-way ANOVAs were also performed on individual potency ratios for selected test compounds within each
group of rats. In some cases, these ANOVAs were followed by ttests corrected for multiple pairwise comparisons to determine significant differences among particular compounds. Where appropriate, two-way ANOVAs were also performed for the factors maintenance dose versus test compound. In all cases, P values of less than
0.05 were considered statistically significant.

of rats, rates of responding remained relatively stable

throughout the course of the experiment, although slight
differences were observed across groups. For example,
rats from group I had the lowest response rates, averaging 0.95, 0.87 and 0.86 responses/s under pre-chronic,
chronic and post-chronic conditions, respectively. Rats
from group III had slightly higher responses rates, averaging 1.03, 0.93 and 0.87 responses/s, whereas rats from
groups II had the highest response rates, averaging 1.41,
1.34 and 1.35 responses/s. Under all conditions, these
values were generally similar to those observed during
baseline training sessions in which no injection was administered. Rates of responding within a given session
were also consistent across components, and this was
true in both saline control and baseline training sessions.

Drugs

Butorphanol and buprenorphine

Buprenorphine hydrochloride, morphine sulfate (both supplied by
the National Institute on Drug Abuse, Rockville, Md., USA), butorphanol tartrate (generously supplied by Bristol-Myers, Wallingford, Conn., USA), fentanyl citrate (generously supplied by
McNeil Laboratories Inc., Fort Washington, Pa., USA), pentobarbital hydrochloride, naloxone hydrochloride (Sigma Chemical
Co., St Louis, Mo., USA), U50,488 methanesulfonate (Research
Biochemical Int., Natick, Mass., USA) and sufentanil (donated by
J. Woods, University of Michigan) were dissolved in saline and injected SC in a volume of 1.0 ml/kg during test sessions. High doses (>10.0 mg/kg) of butorphanol produced skin lesions at the site
of injection when administered SC, thus necessitating that the daily maintenance dose be administered IP in a volume of 1.0 ml/kg.

Results
Control data
Data obtained during saline control sessions are depicted
in Table 1 for all three groups of rats. Within each group
Fig. 1 Effects of butorphanol
(upper panels) and buprenorphine (lower panels) in rats responding under an FR20 schedule of food presentation. Left
panels depict data collected in
rats from group I (n=6), whereas right panels depict data collected in rats from group II
(n=6). Ordinates reflect rates of
responding and are expressed
as a percentage of saline control values. Abscissae reflect
doses in mg/kg. Circles represent data obtained prior to butorphanol treatment (pre), squares
represent data obtained during
treatment when tested at 23 h
following the previous maintenance dose of butorphanol
(chronic), and triangles represent data obtained 6 weeks following treatment (post). Vertical lines on data points represent the SE; when not indicated,
the SE fell within the data point

Figure 1 (top panels) depicts the effects of butorphanol

on rates of responding in rats administered 3.0 mg/kg per
day (group I) and 30 mg/kg per day (group II) butorphanol. Under all conditions and in all rats, butorphanol produced dose-dependent decreases in rates of responding.
The potency of butorphanol to suppress response rates
was decreased when tested under chronic conditions,
with the dose-effect curves shifting 15-fold and 55-fold
to the right in rats administered the low and high maintenance dose, respectively. When examined 6 weeks following termination of chronic butorphanol treatment, the
potency of butorphanol was increased relative to that obtained under pre-chronic conditions (Fig. 1 and Table 2).
The rate-suppressing effects of butorphanol were also
examined in group III, which was treated with 30 mg/kg
per day butorphanol. As seen in Table 3, the butorphanol
dose-effect curve was shifted 137-fold to the right under

61
Table 2 Relative potency estimates (95% confidence limits) of
test compounds in rats from group I (n=6) and group II (n=6).
Comparisons are made for pre-chronic (pre) versus chronic and
for pre-chronic versus post-chronic (post)

Butorphanol
Pre vs chronic
Pre vs post
Buprenorphine
Pre vs chronic
Pre vs post
Morphine
Pre vs chronic
Pre vs post
Fentanyl
Pre vs chronic
Pre vs post
Sufentanil
Pre vs chronic
Pre vs post
U50,488
Pre vs chronic
Pre vs post
Pentobarbital
Pre vs chronic
Pre vs post
a

3.0 mg/kg per day (I)

30 mg/kg per day (II)

14.88 (3.7070.15)a
0.37 (0.140.97)a

54.58 (20.24139.59)a
0.41 (0.180.99)a

5.17 (2.4610.45)a
0.39 (0.170.80)a

11.72 (5.5025.69)a
0.27 (0.140.54)a

2.26 (1.563.32)a
0.72 (0.261.54)

2.31 (1.403.79)a
0.32 (0.150.61)a

1.74 (0.893.98)
0.48 (0.280.90)a

2.51 (1.244.84)a
0.29 (0.150.54)a

1.06 (0.412.20)
0.29 (0.120.68)a

2.90 (1.316.29)a
0.37 (0.180.78)a

5.18 (2.4313.40)a
1.34 (0.762.53)

5.54 (3.987.55)a
1.23 (0.791.94)

1.05 (0.681.62)
1.01 (0.631.58)

1.76 (0.903.16)
1.90 (0.983.36)

Statistically significant (P<0.05)

chronic conditions in this group of rats. In contrast to the

effects observed in groups I and II, the potency of butorphanol was not increased when re-examined 6 weeks following termination of treatment, a finding also observed
when butorphanol was re-examined at 10 weeks.
The effects of buprenorphine on rates of responding
are shown in the lower panels of Fig. 1. Like butorphanol, buprenorphine produced dose-dependent decreases in
response rates across all conditions. During chronic butorphanol treatment, the dose-effect curves for buprenorphine were shifted approximately 5- and 12-fold to the
right in rats administered the low and high maintenance
dose, respectively (Table 2). When examined 6 weeks
following termination of the butorphanol regimen, the buprenorphine dose-effect curve was shifted approximately
3-fold to the left of that obtained under pre-chronic conditions, an effect obtained in both groups of rats.
Morphine, fentanyl and sufentanil
As shown in Fig. 2, morphine, fentanyl and sufentanil
dose-dependently decreased rates of responding in rats
administered the low (group I) and high (group II) maintenance dose of butorphanol. Analysis of relative potency
estimates revealed that butorphanol treatment shifted the
morphine dose-effect curve approximately 2-fold to the
right in both groups of rats (Table 2). In rats administered
the high maintenance dose of butorphanol, the dose-effect
curves for fentanyl and sufentanil were shifted between
2- and 3-fold to the right. In rats administered the low

Fig. 2 Effects of morphine (upper panels), fentanyl (middle panels) and sufentanil (lower panels) in rats responding under an
FR20 schedule of food presentation. Left panels depict data collected in rats from group I (n=6), whereas right panels depict data
collected in rats from group II (n=6). Ordinates reflect rates of responding and are expressed as a percentage of saline control values. Abscissae reflect doses in mg/kg. Circles represent data obtained prior to butorphanol treatment (pre), squares represent data
obtained during treatment when tested at 23 h following the previous maintenance dose of butorphanol (chronic), and triangles represent data obtained 6 weeks following treatment (post). Vertical
lines on data points represent the SE; when not indicated, the SE
fell within the data point

maintenance dose, however, these curves were not significantly altered. When re-examined 6 weeks following termination of butorphanol treatment, the potency of each of
these opioids was increased relative to that observed prior
to treatment, with the exception of morphine in rats administered the low maintenance dose of butorphanol (Table 2).
The interactions between butorphanol and morphine,
fentanyl, and sufentanil were examined further in rats
from group III, which received 30 mg/kg per day butorphanol. Figure 3 shows that in this group of rats these
opioids produced dose-dependent decreases in rates of
responding, effects that were observed under all conditions. Under pre-chronic conditions, these opioids were
administered alone and in combination with 30 mg/kg
butorphanol administered 23 h prior to testing. Pretreatment with butorphanol in this manner enhanced the ratesuppressing effects of low doses of all three opioids, pro-

62
Table 3 Relative potency estimates (95% confidence limits) of
test compounds in rats from group III (n=5). Comparisons are
made for pre-chronic (pre) versus chronic and for pre-chronic versus post-chronic (post)
30 mg/kg per day (III)
Butorphanol
Prea vs chronicb
Prea vs postd
Prea vs poste
Morphine
Prea vs chronicb
Prea vs chronicc
Prea vs postd
Prea vs postc
Fentanyl
Prea vs chronicb
Prea vs chronicc
Prea vs postd
Prea vs poste
Sufentanil
Prea vs chronicb
Prea vs chronicc
Prea vs postd
Prea vs poste
U50,488
Prea vs chronicb
Prea vs chronicc
Prea vs postd
Prea vs poste
a
b
c
d
e
f

137.28 (58.06392.03)f
0.75 (0.401.55)
1.01 (0.561.82)
3.56 (1.956.00)f
3.90 (1.728.00)f
1.02 (0.462.31)
0.89 (0.481.58)
4.85 (3.088.03)f
4.80 (2.768.99)f
1.67 (0.972.83)
1.46 (0.942.38)
4.83 (3.397.09)f
5.14 (3.3810.87)f
1.22 (0.811.85)
1.04 (0.661.64)
6.46 (3.8110.57)f
6.66 (3.7411.42)f
0.98 (0.551.74)
0.82 (0.421.55)

Determined when drug was administered alone

Determined 23 h following maintenance dose
Determined 47 h following maintenance dose
Determined 6 weeks following treatment termination
Determined 10 weeks following treatment termination
Statistically significant (P<0.05)

ducing a leftward and downward shift in their dose-effect curves. Under chronic conditions, the effects of
these mu opioids were examined at both 23 and 47 h following administration of the previous maintenance dose
of butorphanol. At both time points, the dose-effect
curves for morphine, fentanyl and sufentanil were shifted
to the right of those observed under pre-chronic conditions, with each drug generating similar relative potency
estimates at the 23- and 47-h time points (Table 3). In
contrast to that observed in groups I and II, the potencies
of these opioids were not increased when re-examined 6
weeks following termination of butorphanol treatment.
Furthermore, follow-up tests conducted 10 weeks following treatment termination still failed to show an increase in the potency of morphine, fentanyl and sufentanil in this group of rats (Table 3, Fig. 3).
Mu opioids and butorphanol maintenance dose
Figure 4 depicts potency ratios (expressed in log units)
for butorphanol, buprenorphine, morphine, fentanyl and
sufentanil in rats administered the low (group I) and high
(group II) maintenance dose of butorphanol. All values
reflect the degree to which a particular dose-effect was

Fig. 3 Effects of morphine (upper panel), fentanyl (middle panel)

and sufentanil (lower panel) in rats from group III (n=5) responding under an FR20 schedule of food presentation. Ordinates reflect rates of responding and are expressed as a percentage of saline control values. Abscissae reflect doses in mg/kg. Filled circles
represent data obtained prior to butorphanol treatment when administered alone [pre (alone)], open circles represent data obtained prior to butorphanol treatment when 30 mg/kg butorphanol
was administered 23 h before testing [pre (w/ butor)], filled squares represent data obtained during treatment when tested at 23 h
following the previous maintenance dose of butorphanol [chronic
(23 h)], open squares represent data obtained during treatment
when tested at 47 h following the previous maintenance dose of
butorphanol [chronic (47 h)], filled triangles represent data obtained 6 weeks following treatment [post (6 wks)], and open triangles represent data obtained 10 weeks following treatment [post
(10 wks)]. Vertical lines on data points represent the SE; when not
indicated, the SE fell within the data point

shifted rightward under chronic conditions, which often

differed substantially between drugs and across groups.
In rats administered the low maintenance dose of butorphanol, pairwise comparisons revealed significant differences (P<0.05) between the shifts for butorphanol and
fentanyl, butorphanol and sufentanil, and buprenorphine
and sufentanil. In rats administered the high maintenance
dose of butorphanol, significant differences were observed between butorphanol and sufentanil, butorphanol
and fentanyl, butorphanol and morphine, buprenorphine
and fentanyl, and buprenorphine and morphine. In addition, a two-way ANOVA revealed a main effect for
maintenance dose, with the rightward shifts being greater

63

Fig. 4 Potency ratios of butorphanol (BUT), buprenorphine (BUP),

morphine (MOR), fentanyl (FEN) and sufentanil (SUF) in rats responding under an FR20 schedule of food presentation. Left panels depict data collected in rats from group I (n=6), whereas right
panels depict data collected in rats from group II (n=6). Striped
bars reflect the ratio of the pre-chronic ED 50 versus the chronic
ED50 as expressed in log units. Horizontal lines above bars indicate groups that were significantly different from one another
(P<0.05)

in rats administered the high maintenance dose of butorphanol (F4,25=5.87, P<0.05). No significant differences
were observed between drugs or across groups under
post-chronic conditions.
U50,488 and pentobarbital
The effects of U50,488 on rates of responding in groups
I, II and III are depicted in Fig. 5. Under all conditions
and in all three groups of rats, U50,488 decreased rates
of responding. The potency of U50,488 to suppress response rates was decreased under chronic conditions,
with its dose-effect curves shifting 5-, 5- and 6-fold to
the right in groups I, II and III, respectively (Tables 2
and 3). In rats from group III, the U50,488 dose-effect
Fig. 5 Effects of U50,488 (upper panels) and pentobarbital
(lower panels) in rats responding under an FR20 schedule of
food presentation. Left panels
depict data collected in rats
from group I (n=6), center panels depict data collected in rats
from group II (n=6), and right
panels depict data collected
in group III (n=5). Ordinates
reflect rates of responding and
are expressed as a percentage
of saline control values. Abscissae reflect doses in mg/kg. Asterisk (*) indicates a single data
point that was 169% of saline
control values. Other details are
as described in Fig. 3

curve was determined twice under chronic conditions, at

23 and 47 h following the previous maintenance dose of
butorphanol. Figure 5 and Table 3 show that altering the
time interval between butorphanol administration and
testing in this manner did not alter the potency of
U50,488 when tested under chronic conditions. When
U50,488 was re-examined at 6 (groups I, II, III) and 10
(groups II, III) weeks following treatment termination,
its potency was almost identical to that obtained prior to
butorphanol treatment.
Figure 5 also depicts the effects of pentobarbital in
rats administered the low (group I) and high (group II)
maintenance dose of butorphanol. Pentobarbital decreased rates of responding under all conditions in a
dose-dependent manner. In contrast to that observed with
the other drugs tested, chronic butorphanol treatment had
little effect on the potency of pentobarbital to suppress
response rates (see also Table 2).
Naloxone
The effects of naloxone on response rates are shown in
Fig. 6. In rats administered the low-maintenance dose of
butorphanol (group I), doses as high as 30 mg/kg nalox-

64

Fig. 6 Effects of naloxone in rats responding under an FR20

schedule of food presentation. Left panels depict data collected in
rats from group I (n=6), whereas right panels depict data collected
in rats from group II (n=6). Ordinates reflect rates of responding
and are expressed as a percentage of saline control values. Abscissae reflect doses in mg/kg. Circles represent data obtained prior to
butorphanol treatment (pre), squares represent data obtained during treatment when tested at 23 h following the previous maintenance dose of butorphanol (chronic), and triangles represent data
obtained 6 weeks following treatment (post). Vertical lines on data
points represent the SE; when not indicated, the SE fell within the
data point

tested with naloxone and saline 3 days apart, beginning

approximately 10 weeks after the initiation of the chronic regimen. When 30 mg/kg naloxone was administered,
three out of five rats lost at least 10 g (approximately
3.5% of total body weight) over a 60-min observation
period beginning immediately following the injection.
Severe diarrhea accompanied this weight loss at all time
intervals measured. In contrast, when 1.0 ml/kg saline
was administered, no rat lost more than 6 g (approximately 2% of total body weight) over the 60-min observation
period. Under these conditions, diarrhea was not observed at any time point examined. A two-way ANOVA
revealed main effects for both injection (F2,12=5.09,
P<0.05) and time (F2,12=4.70, P<0.05) on loss of body
weight.

Discussion

Fig. 7 Effects of saline (filled bars) and naloxone (striped bars)

administration in rats from group III (n=5). Ordinate reflects loss
of body weight expressed as a percentage of pre-injection body
weight. Abscissa reflects post-injection time intervals. Vertical
lines above bars represent the SE

one failed to alter rates of responding when tested under

pre-chronic, chronic and post-chronic conditions. In rats
administered the high maintenance dose of butorphanol
(group II), naloxone failed to suppress response rates
when examined under pre-chronic conditions, but produced a dose-dependent decrease in response rates when
examined under chronic conditions. Under these conditions, the 30 mg/kg dose of naloxone completely eliminated responding in four out of six rats and substantially
reduced rates of responding in the other two. When reexamined following termination of the chronic butorphanol regimen, naloxone again failed to suppress rates of
responding up to a dose of 30 mg/kg.
Figure 7 depicts the effects of naloxone and saline
on body weight when administered to rats administered
30 mg/kg per day butorphanol (group III). Rats were

In the present investigation, chronic treatment with 3.0

(low) and 30 (high) mg/kg per day butorphanol produced
tolerance to the rate-decreasing effects of butorphanol,
buprenorphine, morphine and U50,488. Only at the high
maintenance dose of butorphanol, however, did tolerance
develop to the effects of fentanyl and sufentanil. Crosstolerance was not conferred to pentobarbital in rats administered either maintenance dose of butorphanol, suggesting that the development of cross-tolerance was
pharmacologically specific to compounds acting at mu
and kappa opioid receptors. Several pieces of data suggest that the effects observed during the chronic regimen
were not due to acute pharmacological interactions between the daily maintenance dose of butorphanol and the
test compounds (i.e., antagonism). First, 23-h pretreatment with 30 mg/kg butorphanol failed to antagonize the
rate-suppressing effects of morphine, fentanyl, sufentanil
and U50,488 when administered under pre-chronic conditions. Examination of these data reveals that butorphanol pretreatment shifted the curves for morphine, fentanyl and sufentanil downward and slightly to the left, an
effect that may be attributed to residual traces of butorphanol present at the time of behavioral testing. Second,
when these opioids were examined during chronic butorphanol treatment, the potency of each opioid was the
same when examined at both 23 and 47 h following administration of the previous maintenance dose. If residu-

65

al traces of butorphanol were producing antagonist activity 23 h following administration of the maintenance
dose, then the rightward shifts observed for these opioids
would be substantially reduced when butorphanol was
given an additional 24 h of clearance. Under no circumstances were these effects observed. Finally, if the rightward shifts observed under chronic conditions had indeed been a consequence of butorphanol antagonism,
then the magnitude of these shifts would have been comparable across the mu opioids examined and reflective of
a competitive interaction at a single receptor site (see
Bertalmio and Woods 1987; Dykstra et al. 1987; Walker
et al. 1994). As depicted in Fig. 4, large and significant
differences were noted in the relative potency estimates
for the five mu opioids examined.
Differential degrees of tolerance development between opioids are often attributed to differences in their
relative efficacy at a given receptor site, with greater degrees of tolerance conferred to low-efficacy than highefficacy opioids (Stevens and Yaksh 1989; Sosnowski
and Yaksh 1990; Paronis and Holtzman 1994). This explanation is consistent with a growing body of knowledge concerning drug-receptor interactions. For example, numerous studies indicate that functional alterations
in cellular physiology occur during a regimen of chronic
drug administration such as changes in the size of the receptor pool (Law et al. 1983; Rogers and el-Fakahany
1986; Diaz et al. 1995; Malatynska et al. 1996; Chen et
al. 1997) and changes in receptor-effector coupling
mechanisms (Su et al. 1980; Law et al. 1985; Puttfarcken
et al. 1988; Chakrabarti et al. 1995; Pei et al. 1995). Because drugs with low efficacy require a greater fractional
receptor occupancy to produce a given effect, any such
alterations in receptor number or function should produce greater alterations in the effects produced by lowefficacy than by high-efficacy opioids. In the present investigation, this would be evidenced as greater degrees
of tolerance conferred to drugs possessing low efficacy
at the mu receptor.
Data obtained in the present experiment provide limited support for the role of relative efficacy in the development of tolerance to mu opioids. In rats administered
both the low and high maintenance dose of butorphanol,
greater tolerance was conferred to the effects of the lowefficacy mu opioids butorphanol and buprenorphine than
to the high-efficacy mu opioids morphine, fentanyl and
sufentanil (for estimates of relative efficacy see Zimmerman et al. 1987; Adams et al. 1990; Mjanger and Yaksh
1991; Morgan and Picker, 1998). These findings are similar to those reported in morphine-treated animals (Negus et al. 1989; Picker et al. 1990; Paronis and Holtzman
1994; Smith et al. 1997) and are what would be predicted if the receptor reserve was functionally reduced. Of
particular interest was the finding that the degree to
which tolerance developed to the rate-suppressing effects
of butorphanol and morphine were similar to that reported in morphine-treated rats. For example, in rats chronically treated with 4.0 mg/kg per day (SC), 30 mg/kg per
day (SC) or approximately 50 mg/kg per day (PO) mor-

phine, the dose-effect curves for butorphanol were shifted at least 10-fold to the right, far greater than the shifts
obtained with morphine (Picker et al. 1990; Oliveto et al.
1991; Hughes et al. 1996; Smith et al. 1997). Such findings indicate that the degree to which tolerance develops
to the effects of mu opioids depends more on the relative
efficacy of the test drug, and less on the relative efficacy
of the toleragen.
Despite differences in their relative efficacy at the mu
receptor (see Adams et al. 1990; Mjanger and Yaksh
1991), there were no consistent differences in the extent
to which tolerance was conferred to morphine, fentanyl
and sufentanil in either group of rats. This finding is consistent with previous studies reporting that the extent to
which tolerance is conferred to the effects of mu opioids
on scheduled-controlled behavior cannot always be predicted on the basis of their relative efficacy (e.g., Picker
and Yarbrough 1991; Hughes et al. 1996; Smith et al.
1997). Such failures to find a clear relationship between
relative efficacy and the magnitude of tolerance development may be a consequence of the small degree of tolerance typically conferred to high-efficacy opioids. In animals administered the high maintenance dose of butorphanol, the dose-effect curves for buprenorphine and butorphanol were shifted as much as 12- and 137-fold to
the right, respectively, whereas the dose-effects curves
for morphine, fentanyl and sufentanil were shifted by no
greater than 5-fold. It is possible that differences between morphine, fentanyl and sufentanil were too small
to quantify accurately; had greater shifts been observed
between these compounds, differences in the degree to
which tolerance developed may have been more readily
apparent.
When data were collapsed across the five mu opioids
examined, greater tolerance was observed in rats administered 30 mg/kg per day (groups II and III) than 3.0
mg/kg per day butorphanol (group I), a finding that can
be attributed to differences between groups for butorphanol, buprenorphine, fentanyl and sufentanil. No significant differences were observed across groups for morphine, a finding that is likely attributable to the small degree of tolerance that was conferred to its rate-suppressing effects (see discussion above). Previous studies have
reported that high maintenance doses of both morphine
(Mucha et al. 1979; Adams and Holtzman 1990; Young
et al. 1990) and etorphine (Yoburn et al. 1993) induce
greater degrees of tolerance than do low maintenance
doses. Supporting these findings are studies conducted in
vitro reporting that increasing the maintenance dose of
an opioid during chronic treatment leads to greater alterations in the number and sensitivity of opioid receptors
(Baumhaker et al. 1994; Belcheva et al. 1994; Law and
Bergbaken 1995). As mentioned above, cellular adaptations may serve as one mechanism by which the nervous
system maintains equilibrium during chronic agonist
treatment. If such is the case, then increasing the maintenance dose of the agonist should result in greater functional reductions in receptor reserve. Such adaptations
would account for the large degrees of tolerance that

66

were generally observed in rats from group II and III,

which were treated with the high maintenance dose of
butorphanol.
An interesting albeit unexpected finding was the observation that enhanced sensitivity developed to the ratesuppressing effects of several opioids when tested under
post-chronic conditions. When re-determined 6 weeks
following termination of butorphanol treatment, the
dose-effect curves for butorphanol, buprenorphine, morphine, fentanyl and sufentanil were shifted leftward relative to their pre-chronic positions in groups I and II. The
loss of opioidergic tone following cessation of butorphanol treatment may have resulted in compensatory increases in the number of surface mu receptors, thereby
rendering mu agonists more potent at suppressing response rate. A previous study reported an increase in mu
receptor density 1435 days following treatment with the
mu-alkylating agent clocinnamox (Zernig et al. 1994),
which was correlated with an increase in the potency of
morphine to produced effects in an antinociceptive assay.
Similarly, an increase in receptor density would account
for the enhanced sensitivity observed in groups I and II
in the present investigation.
Extending an earlier report (Feng et al. 1994), butorphanol conferred tolerance to the effects of U50,488 in
all groups of rats, although the magnitude of these effects was not a function of the butorphanol maintenance
dose. Previous studies have concluded that the rate-decreasing effects of U50,488 are mediated by its activity
at the kappa opioid receptor, because these effects are
antagonized by the kappa-selective antagonist norbinaltorphimine (Picker et al. 1996) but not by the mu-selective antagonist beta-funaltrexamine (Pitts et al. 1996a).
The ability of butorphanol to confer tolerance to a kappa
opioid is somewhat intriguing because mu-selective opioids such as morphine do not share this ability (Craft et
al. 1989; Doty et al. 1989; Craft and Dykstra 1990; Picker
et al. 1991); as a consequence, the tolerance conferred to
U50,488 in the present investigation cannot be attributed
to butorphanols activity at mu receptors. Although previous studies have concluded that butorphanols ratesuppressing effects are mu-mediated (see introduction),
it is possible that butorphanol possesses kappa-mediated
activity in this procedure, but only at doses higher than
those that could be tested under pre-chronic conditions.
Supporting this possibility is the finding that the relative
order of agonist potency differed across conditions (e.g.,
butorphanol was more potent than morphine under prechronic conditions but less potent than morphine under
chronic conditions), which may be an indication that the
mechanism of action for butorphanol changed qualitatively. In addition, the rightward shifts obtained with butorphanol in rats administered the high maintenance dose
of butorphanol (groups II and III) were non-parallel,
which may also reflect qualitative changes in butorphanols mechanism of rate-suppression. It is likely that the
development of tolerance to butorphanols mu-agonist
activity unmasked butorphanols activity at other receptor sites, including its activity at kappa receptors.

Once this activity was unmasked, concomitant changes

within the kappa-opioid system could have developed in
concert with those within the mu-opioid system. The implications of such findings are intriguing, in that compounds with affinity for multiple receptor sites may confer tolerance across pharmacological classes, regardless
of which receptor population mediates their effects under
any one set of conditions.
In rats administered the high maintenance dose of butorphanol, naloxone induced time-dependent decreases
in body weight that was accompanied by diarrhea, suggesting that 30 mg/kg per day butorphanol was sufficient
to induce a state of physical dependence. Naloxone also
dose-dependently suppressed response rates in rats administered this maintenance dose, a finding consistent
with a large body of literature reporting that opioid antagonists disrupt operant behavior in opioid-dependent
animals (e.g., Leander et al. 1975; Brady and Holtzman
1980; Picker and Yarbrough 1991; Picker et al. 1992;
Smith et al. 1997). Maximal levels of rate suppression
were not observed, however, until cumulative doses of
naloxone reached 30 mg/kg, an amount approximately
300-fold greater than that required to eliminate responding in previous studies. Such data suggest that the development of physical dependence in these rats was not mediated entirely by butorphanols activity at either mu or
kappa receptors. Indeed, butorphanol withdrawal can be
precipitated by mu-, kappa- and delta-selective antagonists (Jaw et al. 1993a,b,c), and it is likely that activity at
multiple receptor subtypes mediated the development of
physical dependence observed in this group of rats.
In rats administered the low maintenance dose of butorphanol, naloxone failed to suppress response rates
when tested under chronic conditions, suggesting that
3.0 mg/kg per day was not sufficient to induce a mu-like
physical dependence. Despite an absence of physical dependence, tolerance was conferred to several mu opioids
and a kappa opioid in this group of rats. Previous studies
conducted in vivo have noted a disassociation between
opioid-induced tolerance and opioid-induced dependence, but these studies typically report the development
of dependence in the absence of tolerance (Johnson and
Duggan 1984; Rahman et al. 1994). The findings obtained in the present investigation may be a consequence
of butorphanols low efficacy at the mu receptor. Indeed,
previous studies have reported that low-efficacy opioids
induce greater degrees of tolerance (Stevens and Yaksh
1989) but produce less physical dependence (Cowan et
al. 1977) than do high-efficacy opioids.
In conclusion, tolerance was conferred to the ratesuppressing effects of several mu opioids in butorphanol-treated rats. The magnitude of tolerance and crosstolerance was influenced by both the maintenance dose
of butorphanol and the relative efficacy of the test compound. Such findings can be attributed to changes in the
number and sensitivity of mu opioid receptors, but data
collected with U50,488 suggest that alterations in kappa
receptors may have occurred as well. The tolerance that
developed to butorphanol and other mu opioids were ob-

67

served under conditions that did not produce mu-opioid

dependence, suggesting that the cellular adaptations responsible for the development of tolerance are not completely identical to those responsible for the development
of physical dependence.
Acknowledgements This study was supported by US Public Service Grant DA10277 from the National Institute on Drug Abuse.
M. A. S. was supported by Predoctoral Fellowship DA05713 from
the National Institute on Drug Abuse. Animals used in this study
were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of North
Carolina, and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy Press 1996). The authors would especially like to thank Christopher Mathewson for expert technical assistance.

References
Adams JU, Holtzman SG (1990) Tolerance and dependence after
continuous morphine infusion from osmotic pumps measured
by operant responding in rats. Psychopharmacology 100:
451458
Adams JU, Paronis CA, Holtzman SG (1990) Assessment of relative intrinsic activity of mu-opioid analgesics in vivo by using
beta-funaltrexamine. J Pharmacol Exp Ther 255:10271032
Baumhaker Y, Ben-Dor T, Bar-Hamburger R, Sarne (1994) Characterization of a triple opioid system in the human neurobastoma NMB cell line. Brain Res 665:94100
Belcheva MM, Barg J, McHale RJ, Dawn S, Ho MT, Ignatova E,
Coscia CJ (1994) Differential down- and up-regulation of rat
brain opioid receptor types and subtypes by buprenorphine.
Dev Brain Res 80:159162
Bertalmio AJ, Woods JH (1987) Differentiation between mu and
kappa receptor-mediated effects in opioid drug discrimination:apparent pA2 analysis. J Pharmacol Exp Ther 243:
591597
Bowdle TA, Greichen SL, Bjustrom RL, Schoene RB (1987) Butorphanol improves CO2 response and ventilation after fentanyl anesthesia. Anesth Analg 66:517522
Brady LS, Holtzman SG (1980) Schedule-controlled behavior in
the morphine-dependent and post-dependent rat. Psychopharmacology 70:1118
Butelman ER, Winger G, Zernig G, Woods JH (1995) Butorphanol: characterization of agonist and antagonist effects in rhesus monkeys. J Pharmacol Exp Ther 272:845843
Chakrabarti S, Law PY, Loh HH (1995) Neuroblastoma Neuro2 A
cells stably expressing a cloned mu-opioid receptor: a specific
cellular model to study acute and chronic effects of morphine.
Brain Res Mol Brain Res 30:269278
Chen JJ, Dymshitz J, Vasko MR (1997) Regulation of opioid receptors in rat sensory neurons in culture. Mol Pharmacol
51:666673
Craft RM, Dykstra LA (1990) Differential cross-tolerance to opioids in squirrel monkeys responding under a shock titration
schedule. J Pharmacol Exp Ther 252:945952
Craft RM, Picker MJ, Dykstra LA (1989) Differential cross-tolerance to opioid agonists in morphine-tolerant pigeons responding under a schedule of food presentation. J Pharmacol Exp
Ther 249:386393
Cowan A, Lewis JW, McFarlane IR (1977) Agonist and antagonist
properties of buprenorphine, a new antinociceptive agent. Br J
Pharmacol 60:537545
Diaz A, Ruiz F, Florez J, Hurle MA, Pazos A (1995) Mu-opioid
receptor regulation during opioid tolerance and supersensitivity in rat central nervous system. J Pharmacol Exp Ther 243:
15451551
Doty P, Picker MJ, Dykstra LA (1989) Differential cross-tolerance
to opioid agonists in morphine-tolerant squirrel monkeys re-

sponding under a schedule of food presentation. Eur J Pharmacol 174:171180

Dykstra LA, Gmerek DE, Winger G, Woods JH (1987) Kappa opioids in rhesus monkeys. II. Analysis of the antagonist actions
of quadazocine and beta-funaltrexamine. J Pharmacol Exp Ther
242:421427
Emmett-Oglesby MW, Shippenberg TS and Herz A (1988) Tolerance and cross-tolerance to the discriminative stimulus properties of fentanyl and morphine. J Pharmacol Exp Ther 245:1723
Garner HR, Burke TF, Lawhorn CD, Stoner JM, Wessinger WD
(1997) Butorphanol-mediated antinocieption in mice: partial
agonist effects and mu receptor involvement. J Pharmacol Exp
Ther 282:12531261
Gerak LR, France CP (1996) changes in sensitivity to the rate-decreasing effects of opioids in pigeons treated acutely or chronically with nalbuphine. Behav Pharmacol 7:437447
Feng YZ, Narita M, Tseng YT, Hoskins B, Ho IK (1994) Crosstolerance between butorphanol and morphine in rats. Pharmacol
Biochem Behav 49:657661
Horan P, Ho IK (1989) Butorphanol precipitates abstinence in
morphine dependent rats. Eur J Pharmacol 170:265268
Hughes CE, Dykstra LA, Picker MJ (1996) Behavioral tolerance
and cross-tolerance to the response rat-decreasing effects of
mu opioids in rats. Behav Pharmacol 7:228236
Jaw SP, Hoskins B, Ho IK (1993a) Opioid antagonists and butorphanol dependence. Pharmacol Biochem Behav 44:497500
Jaw SP, Makimura M, Hoskins, Ho IK (1993b) Effects of nor-binaltorphimine on butorphanol dependence. Eur J Pharmacol
239:133140
Jaw SP, Hoskins B, Ho IK (1993c) Involvement of delta-opioid receptors in physical dependence on butorphanol. Eur J Pharmacol 240:6772
Johnson SM, Duggan AW (1984) Dependence in the absence of
tolerance to morphine. Eur J Pharmacol 97:507508
Law PY, Bergabaken C (1995) Properties of delta opioid receptors
in neuroblastoma NS20Y:receptor activation and neuroblastoma proliferation. J Pharmacol Exp Ther 272:322332
Law PY, Hom DS, Loh HH (1983) Opiate receptor down regulation and densensitization in neuroblastoma glioma NG10815 hybrid cells are two separate cellular adaption processes.
Mol Pharmacol 25:413424
Law PY, Hom DS, Loh HH (1985) Multiple affinity states of opiate
receptor in neuroblastoma glioma NG105-15 hybrid cells. J
Biol Chem 260:35613569
Leander JD (1983a) A kappa opioid effect: increased urination in
the rat. J Pharmacol Exp Ther 224:8994
Leander JD (1983b) Evidence that nalorphine, butorphanol and
oxilorphan are partial agonists at a kappa opioid receptor. Eur
J Pharmacol 86:467470
Leander JD, McMillan DE, Harris LS (1975) Effects of narcotic
agonist and antagonists on schedule-induced water and morphine ingestion. J Pharmacol Exp Ther 195:271278
Malatynska E, Wang Y, Knapp RJ, Waite S, Calderon S, Rice K,
Hruby VJ, Yamamura HI, Roeske WR (1996) Human delta
opioid receptor: functional studies on stably transfected Chinese hamster ovary cells after acute and chronic treatment
with the selective nonpeptidic agonist SNC-80. J Pharmacol
Exp Ther 248:10831089
Mjanger E, Yaksh TL (1991) Characteristics of dose-dependent
antagonism by beta-funaltrexamine of the antinociceptive effects of intrathecal mu agonists. J Pharmacol Exp Ther 258:
544550
Morgan D, Picker MJ (1998) The opioid irreversible antagonist
betafunaltrexamine differentiates the discriminative stimulus
effects of opioids with high and low efficacy at the opioid
receptor. Psychopharmacology 140:2028
Mucha RF, Kalant H, Linseman MA (1979) Quantitative relationships among measures of morphine tolerance and physical dependence in the rat. Pharmacol Biochem Behav 10:397405
Negus SS, Picker MJ, Dykstra LA (1989) Kappa antagonist properties of buprenorphine in non-tolerant and morphine-tolerant
rats. Psychopharmacology 98:141143

68
Oliveto AH, Picker MJ, Dykstra LA (1991) Acute and chronic
morphine administration: effects of mixed action opioids in
rats and squirrel monkeys responding under a schedule of food
presentation. J Pharmacol Exp Ther 257:818
Paronis CA, Holtzman SG (1992) Development of tolerance to the
analgesic activity of mu agonists after continuous infusion of
morphine, meperidine or fentanyl in rats. J Pharmacol Exp Ther
262:19
Paronis CA, Holtzman SG (1994) Sensitization and tolerance to
the discriminative stimulus effects of mu-opioid agonists. Psychopharmacology 114:601610
Pei G, Kieffer BL, Lefkowitz RJ, Freedman NJ (1995) Agonistdependent phosphorylation of the mouse delta-opioid receptor:
involvement of G protein-coupled receptor kinases but not
protein kinase C. Mol Pharmacol 48:173177
Picker MJ (1997) Discriminitive stimulus effects of the mixedopioid agonist/antagonist dezocine: cross-substitution by mu
and delta opioid agonists. J Pharmacol Exp Ther 283:19
Picker MJ, Dykstra LA (1989) Discriminative stimulus effects of
mu and kappa opioids in the pigeon: analysis of the effects of
full and partial mu and kappa agonists. J Pharmacol Exp Ther
249:557566
Picker MJ, Yarbrough J (1991) Cross tolerance and enhanced sensitivity to the response rate-decreasing effects of opioids with
varying degrees of efficacy at the mu receptor. Psychopharmacology 105:459466
Picker MJ, Negus SS, Craft RM (1990) Butorphanols efficacy at
mu and kappa opioid receptors: inferences based on the schedule-controlled behavior of nontolerant and morphine-tolerant
rats and on the responding of rats under a drug discrimination
procedure. Pharmacol Biochem Behav 36:563568
Picker MJ, Negus SS, Powell KR (1991) Differential cross-tolerance to mu and kappa opioid agonists in morphine-tolerance
rats responding under a schedule of food presentation. Psychopharmacology 103:129135
Picker MJ, Craft RM, Negus, Powell KR, Mattox SR, Jones SR,
Hargrove BK, Dykstra LA (1992) Intermediate efficacy mu
agonists: examination of their morphine-like stimulus effects
and response rate decreasing effects in morphine-tolerant rats.
J Pharmacol Exp Ther 263:668681
Picker MJ, Yarbrough J, Hughes CE, Smith MA, Morgan D,
Dykstra LA (1993) Agonist and antagonist effects of mixed
action opioids in the pigeon drug discrimination procedure: influence of training dose, intrinsic efficacy and inter-animal
differences. J Pharmacol Exp Ther 266:756767
Picker MJ, Mathewson C, Allen RM (1996) Opioids and rate of
positively reinforced behavior: III. Antagonism by the longlasting kappa antagonist norbinaltrophimine. Behav Pharmacol
6:495504
Pitts RC, West JP, Hapke DM, Morgan D, Dykstra LA, Picker MJ
(1996a) Opioids and rate of positively reinforced behavior: II.
Antagonism by beta-funaltrexamine. Exp Clin Psychopharmacol 4:389395
Pitts RC, West JP, Morgan D, Dykstra LA, Picker MJ (1996b)
Opioids and rate of positively reinforced behavior: differential
antagonism by naltrexone. Behav Pharmacol 7:205215
Puttfarcken PS, Cox BM (1989) Morphine-induced desensitization
and down-regulation at mu-receptors in 7315c pituitary cells.
Mol Pharmacol 45:19371942

Puttfarcken PS, Werling LL, Cox BM (1988) Effects of chronic

morphine exposure on opioid inhibition of adenylyl cyclase in
7315c cell membranes: a useful model for the study of tolerance at mu receptors. Mol Pharmacol 33:520527
Rahman AF, Takahashi M, Kaneto H (1994) Morphine dependence with or without tolerance in formalin-treated mice: further evidence for disassociation. Jpn J Pharmacol 66:277280
Rogers NF, el-Fakahany E (1986) Morphine-induced opioid receptor down-regulation detected in intact adult rat brain cells. Eur
J Pharmacol 124:221230
Shannon HE, Holtzman SG (1977) Further evaluation of the discriminative stimulus effects of morphine in the rat. J Pharmacol Exp Ther 201:5566
Smith MA, Picker MJ (1995) Examination of the kappa agonist
and antagonist properties of opioids in the rat drug discrimination procedure: influence of training dose and intrinsic efficacy. Behav Pharmacol 6:703717
Smith MA, Pitts RA, Picker MJ (1997) Tolerance and cross tolerance to the accuracy- and rate-decreasing effects of mu opioids
in rats responding under a fixed-consecutive-number schedule.
Drug Alcohol Depend 46:1930
Sosnowski M, Yaksh TL (1990) Differential cross tolerance between intrathecal morphine and sufentanil in the rat. Anesthesiology 73:11411147
Stevens CW, Yaksh TL (1989) Potency of infused spinal antinociceptive agents is inversely related to magnitude of tolerance
after continuous infusion. J Pharmacol Exp Ther 250:18
Su YF, Harden TK, Perkins JP (1980) Cathecholamine-specific
densensitization of adenylate cyclase: evidence for a multistep process. J Biol Chem 255:74107419
Walker EA, Makhay MM, House JD, Young AM (1994) In vivo
apparent pA2 analysis for naltrexone antagonism of discriminative stimulus and analgesic effects of opiate agonist in rats. J
Pharmacol Exp Ther 271:959968
White JM, Holtzman SG (1983) Further characterization of the
three-choice morphine, cyclazocine and saline discrimination
paradigm: opioids with agonist and antagonist properties. J
Pharmacol Exp Ther 224:9599
Yoburn BC, Sierra V, Lutfy (1990) Simultaneous development of
opioid tolerance and opioid antagonist-induced receptor upregulation. Brain Res 529:143148
Yoburn BC, Billings B, Duttaroy A (1993) Opioid receptor regulation in mice. J Pharmacol Exp Ther 265:314320
Young AM, Sannerud CA, Steigerwald ES, Doty MD, Lipinski WJ
Tetrick LE (1990) Tolerance to morphine stimulus control: role
of morphine maintenance dose. Psychopharmacology 102:5967
Young AM, Kapitsopoulos G, Makhay MM (1991) Tolerance to
morphine-like stimulus effects of mu opioid agonists. J Pharmacol Exp Ther 257:795805
Zernig G, Butelman ER, Lewis JW, Walker EA, Woods JH (1994)
In vivo determination of mu opioid receptor turnover in rhesus
monkeys after irreversible blockade with clocinnamox. J Pharmacol Exp Ther 269:5765
Zimmerman DM, Leander JD, Reel JK, Hynes MD (1987) Use of
beta-funaltrexamine to determine mu opioid receptor involvement in the analgesic activity of various opioid ligands. J
Pharmacol Exp Ther 241:374378