Abstract
Acute mu opioid agonist pretreatment (4 hr) dose-dependently sensitizes rats responding for food reinforcement to the rate-decreasing effects of naltrexone (NTX). In the present study, adult rats were trained to respond in an intracranial self-stimulation autotitration procedure in which responding resulted in electrical stimulation of the medial forebrain bundle that decreased in frequency until reset to the initial value. In an acute sensitization experiment, pretreatment (4 hr) doses of 3.0 and 10 mg/kg morphine reduced the ED25 value for the intracranial self-stimulation rate-decreasing effect of NTX from 28.2 mg/kg to 0.29 and 0.02 mg/kg, respectively. All mu-selective opioid agonists tested, fentanyl > levorphanol > methadone > morphine > meperidine (listed in order of decreasing potency), produced similar large increases in sensitivity to NTX. Acute sensitization was not induced by the kappa-selective opioid agonist spiradoline, the dextrorotary enantiomer of levorphanol, dextrorphan, or the nonopioid drugs d-amphetamine and pentobarbital. Pretreatment with morphine for 10 days by continuous subcutaneous infusion (15 mg/kg/day) reduced the ED25 value of NTX from 28.2 to 0.002 ± 1.48 mg/kg. The correlation of decreases in ED25 values for the rate-decreasing effect of NTX after both acute and chronic morphine administration is consistent with the theory that acute agonist-induced sensitization reflects receptor-mediated changes occurring early in the development of physical dependence.
Low or moderate (≤3.0 mg/kg) doses of the opioid antagonists naloxone and NTX have few observable effects in opioid-free subjects. However, subjects receiving morphine chronically show a substantial increase in sensitivity to both the physiological and behavioral effects of these antagonists (Eisenberg, 1982; Nakaki et al., 1981;Ramabadran, 1983). Most of the effects induced by antagonists after chronic morphine administration have been attributed to the induction of an opioid withdrawal syndrome (Kanof et al., 1992). Therefore, sensitization to opioid antagonists (precipitated withdrawal) has often been used as a quantitative indicant of opioid dependence (e.g., Wei et al., 1973).
The full opioid withdrawal syndrome seen in humans includes both (1) physiological signs, such as vomiting and diarrhea, and (2) motivational/subjective symptoms, such as dysphoria (Jaffe, 1990). The results of preclinical research have supported the theory that different neuroanatomical substrates may be involved in mediating the physiological or motivational manifestations of opioid withdrawal (Higgins and Sellers, 1994). For example, among other areas, the locus ceruleus and periaqueductal gray are implicated in the expression of physical withdrawal signs (Bozarth and Wise, 1984; Maldonado et al., 1992). Conversely, the nucleus accumbens and amygdala are critical sites for the expression of negative motivational signs of withdrawal (Stinus et al., 1990). Because theories of human drug abuse generally emphasize the motivational factors maintaining opioid self administration, preclinical methodologies that selectively assess the motivational changes related to opioid dependence and withdrawal receive much attention.
In addition to place and taste aversion conditioning, schedules of operant responding are sensitive to drug withdrawal-induced motivational changes. When morphine administration was abruptly discontinued or low doses of antagonists were administered during chronic morphine administration, rates of food reinforced operant responding were suppressed (Balster, 1985; Gellert and Sparber, 1977;Holtzman and Villarreal, 1973; Thompson and Schuster, 1964). Although sensitization to response-rate-decreasing effects of antagonists has been described most often in animals receiving chronic treatment, it can also occur after only a single dose of an opioid agonist. With food reinforced operant tasks, acute pretreatment of rats with morphine induced sensitization to antagonists (Meyer and Sparber, 1977) that was time (Young, 1986) and dose (Adams and Holtzman, 1990; Smits, 1975) dependent. In addition, the effect appeared to be centrally mediated (Adams and Holtzman, 1991). Sensitization was stereoselective among antagonists and relatively specific to mu opioid agonist pretreatment (Adams and Holtzman, 1990; White-Gbadebo and Holtzman, 1994a). The sensitization effect could be blocked by coadministration of naloxone with the morphine pretreatment (White-Gbadebo and Holtzman, 1994b). Taken together, these findings strongly suggest that the behavioral effects observed are mediated by central muopioid receptors. It is not clear that these behavioral effects are specific indicants of drug-induced motivational changes (dysphoria); nevertheless, the behavioral and physiological similarities in antagonist sensitization “syndromes” after acute or chronic morphine administration have given rise to the theory that acute sensitization reflects the initial receptor-mediated changes that result in physical dependence after chronic administration. In fact, the term “acute dependence” has been used to describe the acute sensitization effect in humans (Bickel et al., 1988).
In addition to food reinforced operant responding, operant responding for ICSS is sensitive to acute opioid administration (Wise and Bozarth, 1987). Both the current autotitration method (Schaefer and Michael, 1986) and discrete-trial current-threshold determination procedures (Schulteis et al., 1994) have been used to show that in rats, antagonist-precipitated withdrawal after a regimen of chronic morphine administration is associated with increased current thresholds for ICSS. Furthermore, these threshold increases were associated with a conditioned place aversion (Schulteis et al., 1994). Although reward thresholds were not elevated during unprecipitated withdrawal (Schaefer and Michael, 1986), the aforementioned data do support the hypothesis that altered performance in an ICSS autotitration task may be a sensitive preclinical indicant of the human-reported dysphoria associated with antagonist-precipitated opioid withdrawal. Conversely, both discrete-trial ICSS (Glick et al., 1982) and ICSS autotitration (Van Wolfswinkel et al., 1985; Van Wolfswinkel and Van Ree, 1985) procedures have been used to show that acute morphine (<5.0 mg/kg) lowers the threshold for ICSS responding, indicating a reward-enhancing effect.
Although the aforementioned ICSS autotitration studies measured changes in current thresholds, work in this laboratory comparing responding (within-subjects) maintained by either current or frequency autotitration of ICSS indicated that both tasks (1) result in stable base-line responding, (2) provide a reward-selective measure (titration point) that is relatively independent of rate of responding but qualitatively different from a perceptual “threshold” (Easterling and Holtzman, 1997) and (3) show corresponding changes in response rate and titration point in response to systematic changes in stimulation parameters. Because frequency autotitration ICSS tasks generated higher rates of base-line responding, relative to corresponding current autotitration tasks, the present study examined changes in responding using this method.
Specifically, the present experiments were undertaken (1) to determine whether acute morphine-induced sensitization to the effects of naltrexone occurs in an ICSS frequency autotitration paradigm and (2) to compare acute (single-dose) sensitization with the degree of sensitization seen after prolonged (10 day) morphine administration. Furthermore, the pharmacological specificity of the acute sensitization effect was assessed by testing mu opioid receptor agonists in addition to morphine (i.e., fentanyl, levorphanol, methadone and meperidine); the kappa opioid agonist spiradoline; the nonopioid isomer of levorphanol, dextrorphan; and the nonopioid drugs pentobarbital and d-amphetamine.
Materials and Methods
Subjects and Surgical Procedures
Subjects.
Nine adult male Sprague-Dawley rats (Charles-River, Wilmington, MA) served as study animals. They were individually housed in standard polycarbonate cages with free access to food and water. The colony room was maintained under a 12-hr/12-hr light/dark cycle, with lights on at 6:00 a.m. Each rat weighed 250 to 350 g at the time of surgery.
ICSS electrode implantation.
The animals were anesthetized with a mixture of pentobarbital and chloral hydrate (Equithesin; 3.3 ml/kg intraperitoneal) and placed in the stereotaxic apparatus. A bipolar platinum electrode (tip diameter = 0.25 mm; Plastics One Inc., Roanoke, VA) was implanted in the medial forebrain bundle at the level of the lateral hypothalamus. The coordinates, using bregma as a reference point, were AP = 3.6 and D = −8.5. Lateral placement was balanced across subjects (i.e., L = 1.6 for half of the animals and L = −1.6 for the others). Three 2.4-mm jewelers’ screws were attached to the skull, forming a perimeter around the electrode site. Subsequently, dental acrylic was applied to the screws and electrode to form a pedestal firmly anchoring the electrode in place. After surgery and before training, animals were given a minimum 1-week recovery period.
Osmotic pump implantation.
At the time of osmotic pump implantation, rats weighed >500 g. Therefore, before the surgery, two model 2 ML2 Alzet osmotic pumps (Alza Corp., Palo Alto, CA) were prepared for each animal. Each pump was filled with a morphine solution of sufficient concentration to result in the delivery of 7.5 mg/kg/day morphine, for a total daily dose of 15 mg/kg. Immediately before the pump implantation surgery, animals were taken from the home cage, weighed and anesthetized with the inhalation anesthetic Metofane (2,2-dichloro-1,1-difluoro-ethyl methyl ether). A 1.5-cm-long sagittal midscapular incision was made, and the two minipumps were inserted subcutaneously into the incision in a rostrocaudal direction with their flow moderators entering first. The incision was closed with three or four stainless steel wound clips. After this 5-min procedure, animals emerged from anesthesia within 15 min.
Apparatus
For each animal, all training and testing sessions were conducted in one of three standard operant chambers (Coulbourn Instruments, Lehigh Valley, PA). Each chamber was housed within a larger ventilated, sound-attenuating enclosure. The chambers were equipped with two response levers, 4 cm apart, mounted on one wall 1 cm above the chamber floor. The right lever was designated the stimulation lever. The left lever was inactive. Speakers for tone presentation and white noise delivery, stimulus lights and a house light were mounted above the levers. An omnidirectional response lever, centered 1 cm from the wall opposite the response levers, hung from the ceiling of the chamber. This lever extended to within 0.5 cm of the chamber floor and was designated the “reset lever.”
Each operant chamber was interfaced to an IBM-compatible microcomputer and stimulator unit (Med Associates Inc., St. Albans, VT). The computer collected response data and controlled the presentation of stimuli. During training and testing sessions, the electrode of each animal was connected to the stimulator unit via a spring-covered lead, which was suspended above the chamber by a commutator.
ICSS Training
After recovery from the ICSS electrode implantation procedure, each animal was shaped to press the stimulation lever in two daily 30-min sessions. During initial sessions, CRF was available on the stimulation lever. Each response on the lever produced a 250-msec train of biphasic 0.5-msec-square waves at 100 Hz and at a constant current. The stimulation current intensity chosen for each animal was a “suprathreshold” value, ranging from 100 to 200 μA. For each animal, after the initial two training sessions, current intensity was decreased in 10-μA steps at 5-min intervals until response rate fell below 1.0 response/sec (criterion). Thereafter, the current that was 20 mA above that value was set as that animal’s stimulation current and it remained constant during all subsequent sessions. The mean current across all animals was 112 ± 13.6 μA. Selection of stimulation current intensity according to response rate data was designed to ensure that individual response rates were high (>1.0 response/sec) and that interanimal rate variability was reduced.
After this initial response rate screening, animals were introduced to a new CRF schedule in which every response on the stimulation lever resulted in the delivery of a constant-current electrical stimulus and every 15th response on that same lever resulted in a 5% (5 Hz) decrease (stepdown) in the frequency of the stimulating current (Easterling and Holtzman, 1997). Under this autotitration schedule, the reset lever was active. At any time during a 15-min session, one press of the reset lever resulted in the immediate and concurrent delivery of 1.5-sec tone and light presentations and reinstated the starting frequency of the stimulation that could be obtained by pressing the stimulation lever. The reset lever did not deliver stimulation. Therefore, the animal determined the lowest stimulation frequency at which responding during the session would be maintained: the titration point.
On each day (Monday through Friday) animals were trained on the CRF autotitration procedure in sessions consisting of five 15-min trials, each trial separated by a 15-min time-out period; animals remained in the chamber during the time-outs. The beginning of each trial was signaled by closing the door of the sound attenuating enclosure, illuminating the houselight and activating the white noise. The houselight and white noise then remained on for the duration of the trial. During each of the time-out periods, the door of the sound attenuating enclosure was opened and the houselight and white noise remained off. Subjects were trained on the CRF autotitration procedure until their mean titration point varied by <10% over five consecutive trials. Typically, training lasted 1 to 2 weeks. After responding on the training schedule reached stability, the number of trials in a training session was reduced to two. After the autotitration training sessions, animals were subjected to test sessions twice weekly. Test sessions were interspersed among ≥3 days of autotitration training sessions.
Testing Procedures
Behavioral baseline.
Before any drug testing, animals (n = 9) were given five consecutive test trials, with each preceded (15 min) by a saline injection. This was done to quantify shifts in base line during a session that were not due to drug administration. Because subsequent experiments involved initial treatment with opioid agonists (at 4 hr) followed by NTX, the effects of the prototypic opioid agonist morphine were initially determined at 4 hr. A single dose of morphine (10 mg/kg) was given 4 hr before a five-trial testing session during which repeated saline injections preceded each trial. Although an acute 10 mg/kg dose of morphine would be expected to eliminate responding soon after injection, at 4 hr response rates were predicted to have returned to predrug base-line values.
Morphine and NTX dose-effect curves.
Before determining the effects of NTX in animals receiving a morphine pretreatment, baseline morphine and base-line NTX dose-effect curves were generated. In each of these cumulative dosing test sessions, five 15-min autotitration trials were separated by 15-min time-out periods. Fifteen minutes before the first autotitration trial, animals were removed from their home cages, given an injection of saline and placed in the testing chamber. After that initial trial and each of the four subsequent trials, animals received, in half-log-unit increments, a series of cumulative morphine doses (e.g., 0.1, 0.2, 0.7 and 2.0 mg/kg for cumulative doses of 0.1, 0.3, 1.0 and 3.0) or a series of cumulative NTX doses (1.0–30 mg/kg), allowing a 30-min period between doses.
Acute sensitization.
At 4 hr before each test session, animals were weighed and saline (1 ml/kg subcutaneous) or one dose of a pretreatment drug was administered (see table 1 for pretreatment order). This morphine pretreatment interval has been reported to produce optimal sensitization to NTX in rats responding for food (Young, 1986). Within each drug pretreatment condition, the order of the doses was randomized. After pretreatment, animals were returned to their home cages for 4 hr. Each subsequent test session consisted of five 15-min autotitration trials separated by 15-min time-out periods. Fifteen minutes before the first autotitration trial, animals were removed from their home cages, given an injection of saline and placed in the testing chamber. After that initial trial and each of the four subsequent trials, animals received, in half-log-unit increments, a series of four cumulative NTX doses (0.001–30 mg/kg). Due to individual differences in sensitivity to NTX after each agonist pretreatment, not all animals received the same range of NTX doses. In fact, three dose ranges were used to determine a full range of effect for NTX in a given animal, and some pretreatment doses were tested more than once and followed by a greater range of NTX doses. After the completion of each test session, the animals were weighed.
Chronic morphine.
Table 2 provides a summary of the experimental protocol. Subjects received no drug treatment for a 1-month period after acute sensitization experiments. Because animals had received repeated opioid agonist and NTX combinations during the acute sensitization experiments, nondrug base-line values were redetermined, and acute naltrexone dose-effect curves were generated after both 4-hr saline and 4-hr morphine pretreatment (table 2, days −7 and −3). These data were then used for comparison with both the curves determined during the acute sensitization experiments and the NTX data collected during chronic morphine administration (table 2, day 10).
If changes in responsiveness to an antagonist occur with repeated NTX dosing, the effects of an agonist might differ as well. Therefore, an acute morphine dose-effect curve was determined. These “prepump” data, when compared with the morphine dose-response data gathered on the seventh day of osmotic pump operation (table 2, day 7), may indicate the development of tolerance to the response rate-decreasing effects of morphine.
On the first two mornings (9:00 a.m. to 12:00 noon) of the after week, nondrug base-line data were collected in two-trial ICSS sessions (table2, days −1, 0). On the morning of the third day, base-line data were collected (9:00 to 12:00 noon). However, immediately after this session, osmotic pumps delivering 15 mg/kg/day morphine solution over the course of 2 weeks were implanted in the animals. To assess the development of tolerance during this period, on each of the following 14 mornings, ICSS data were collected (9:00 to 12:00 noon). In addition, on day 7, cumulative morphine (1.0–30 mg/kg) was administered during ICSS testing trials. On day 10, cumulative NTX (0.001–0.03 mg/kg) doses were administered during ICSS testing, and behavioral withdrawal signs were noted. Pumps were removed on day 14, and ICSS testing continued for an additional 3 days.
Drugs
Morphine sulfate (Penick Co., Nutley, NJ); naltrexone hydrochloride, sodium pentobarbital (Sigma Chemical Co., St. Louis MO); levorphanol tartrate, dextrorphan tartrate (Roche Laboratories, Nutley, NJ); fentanyl citrate (McNeil Laboratories, Fort Washington, PA); spiradoline methane sulfonate (The Upjohn Company, Kalamazoo, MI), methadone hydrochloride (Mallinkrodt, St. Louis, MO) and meperidine hydrochloride (Penick Co., Nutley, NJ) were dissolved in 0.9% saline. All drug doses are reported as the free base and were administered subcutaneous in a volume of 1 ml/kg.
Data Analyses
The data collected during each autotitration trial included number of responses per 1 sec of trial time and the mean frequency of stimulation at which a animal pressed the reset lever during a trial (titration point). To permit the assessment of pretreatment drug effects, these raw data were transformed to a percentage-of-base-line value based on the mean of the data obtained during the preceding 7 training days. For example, group titration point and response rate base-line values averaged over the course of acute sensitization testing were 64.69 ± 2.1 Hz and 1.88 ± 0.17 responses/sec, respectively. Likewise, in the chronic morphine experiment group, base-line values were 63.6 ± 2.1 Hz and 1.85 ±.18 responses/sec, respectively. Comparisons among data obtained at equivalent drug doses were made by analysis of variance (ANOVA) and post hoccomparisons (Tukey’s test) appropriate for repeated measures.
After saline, when a cumulative dose of 30 mg/kg NTX was administered acutely (15 min) during the course of an ICSS testing session, a ∼25% decrease in response rate was seen (ED25 = 28.2 ± 1.45). Furthermore, pilot work indicated that in some animals, response rates were reduced <50% even at ≥100 mg/kg NTX. Based on these NTX data and the desire to minimize withdrawal-related distress in later testing, it was decided that sensitization to NTX could best be assessed in subsequent experiments by quantifying shifts in the ED25 of the response rate measure. In addition, this measure alleviated the need to test doses of NTX that completely suppressed responding. ED25 calculations were omitted for any dose of a pretreatment drug that significantly attenuated response rate before NTX administration (e.g., 0.1 mg/kg fentanyl, 56 mg/kg meperidine and 1.0 and 3.0 mg/kg spiradoline). Although all titration point data were analyzed, these data are generally presented only if they differed from base line.
The response rate percent-of-base-line data generated by each animal during cumulative NTX dosing were also used to generate an ED25 value for the rate-decreasing effect of NTX. Under each drug pretreatment condition, individual ED25 values were interpolated by linear regression. Subsequently, a mean ED25 value (n = 7–9) and a S.E.M. value were calculated for each pretreatment condition. In addition, these ED25 values were subjected to one-way ANOVA appropriate for repeated measures. Post hoc comparisons were made using Tukey’s test. The α level set for all comparisons was P ≤ .05.
During the chronic morphine experiment, animals were weighed before and after each test session. The resulting difference scores were compared among experimental conditions (i.e., acute vs. chronic morphine) using Student’s t tests. In addition, ED25 data for before and during osmotic pump operation were calculated for the rate-decreasing effects of morphine. Two of the animals died during the chronic administration experiment. The deaths were due to apparent morphine overdose (30 mg/kg) during the morphine challenge on day 7. In addition, one of the animals stopped responding during the chronic phase of the experiment. Postmortem histology indicated that in this animal a cancerous growth had shifted the tip of the stimulation electrode. Therefore, data from these three animals were discarded in the analyses of data from the chronic treatment experiment.
Results
Behavioral Base Line
After an initial 1-week training period, responding (titration point or response rate) during ICSS autotitration sessions exhibited little (5–10%) variability across days. Group titration point and response rate base-line values averaged over the course of acute sensitization testing were 64.69 ± 2.1 and 1.88 ± 0.17, respectively. Before drug testing, animals (n = 9) were given five consecutive trials, with each preceded (15 min) by a saline injection. When the group data from these consecutive trials were analyzed, mean titration point did not change [F(4,32) = 0.67] from its initial (trial 1) mean value of 62.6 ± 1.56 Hz across the five trials of the session. Although there were no significant differences in response rate among the subsequent four trials (1.56 ± 0.15 responses/sec), post hoccomparisons indicated that response rates did decrease slightly [F(4,32) = 5.29, P < .05] after the first trial (i.e., 10.1 ± 5.2%). However, they remained stable across subsequent trials.
Acute Drug Pretreatment
When a single dose of morphine (10 mg/kg) was given 4 hr before a five-trial testing session during which repeated saline injections preceded each trial, no changes in titration point [F(4,41) = 1.22] were observed among trials within the 2.5-hr session. However, a small progressive [F(4,41) = 8.05, P < .05] decrease in response rate was noted over the course of the five trials, resulting in a response rate of 89 ± 4.3% of predrug base line by trial five. ANOVA indicated that these response rate data were not different from those obtained in the previous repeated saline-only condition. Therefore, in the absence of NTX injections, 4-hr pretreatment with morphine has no effect on response rate over a series of five trials.
When cumulative doses of NTX (1–30 mg/kg) were administered acutely (15 min) during the course of an ICSS testing session, the slight increases in titration point data were not dose dependent [F(4,36) = .87] (fig. 1, top). However, significant [F(4,36) = 4.38] decreases were noted in response rate (fig. 1). Post hoc comparisons indicated that the significant (25%) response rate reduction was accounted for by the highest dose (30 mg/kg) of NTX tested, and an ED25 of 28.2 ± 1.45 mg/kg was calculated for this rate decrease (table3).
As indicated in table 1, a second saline pretreatment determination was made approximately midway through the agonist testing sequence. The data from this determination did not differ [F(1,42) = .71) from the first saline pretreatment condition data. Therefore, both these data sets were combined for the saline values in figures1, 2, 3, 4.
Figure 1 and table 3 show that in contrast to saline pretreatment, acute pretreatment doses of 3.0 and 10 mg/kg morphine significantly reduced the ED25 value for the ICSS rate-decreasing effect of NTX from 28.2 mg/kg to 0.29 ± 5.5 and 0.02 ± 2.63 mg/kg, respectively. In addition, the 10 mg/kg morphine pretreatment caused a decrease in titration point (fig. 1). This decrease in titration point was either transient or was attenuated in a dose-related manner after increasing doses of NTX. Figure 2 shows that levorphanol pretreatment (1.0 mg/kg) also resulted in a titration point decrease (17%) at 4 hr. Again, this effect appeared to be attenuated by naltrexone administration. These titration point data show that higher doses of these two agonists may affect titration point up to 4 hr after administration.
Based on response rate ED25 data, all mu opioid receptor-selective agonists tested produced large increases in sensitivity to NTX (table 3, figs. 1, 2, 3), with a potency order of fentanyl > levorphanol > methadone > morphine > meperidine. This sensitization effect occurred at agonist pretreatment doses that did not cause changes in response rates 4 hr after administration (figs. 1, 2, 3). Table 3 lists ED25 values for all drug pretreatment conditions.
Although the two highest doses of the kappa-selective opioid agonist spiradoline disrupted responding before NTX testing, a half-log lower dose (0.3 mg/kg) did not induce sensitization to NTX (table 3, fig. 4). Neither dextrorphan, the dextrorotary enantiomer of levorphanol, nor the nonopioid drugs d-amphetamine and pentobarbital induced significant sensitization to NTX (table 3, fig.2). For comparison, the doses of levorphanol, at only 0.1 and 0.33 times the dose of dextrorphan used, induced 40- and 400-fold sensitization to NTX. Furthermore, the doses of pentobarbital andd-amphetamine used were sufficient to result in observable changes in activity during the pretreatment period: sedation and increased activity, respectively.
Chronic administration
Baselines.
After a month-long drug-free period, animals began the chronic administration experiment. Within the first week of resumed training, baseline titration point and response rate data stabilized at mean levels no different from those seen before the acute administration experiments (63.6 ± 2.1 and 1.85 ±.18 responses/sec, respectively). After saline pretreatment, the NTX dose-response curve (fig. 5, bottom) was not significantly different from the corresponding curve determined during the acute agonist pretreatment experiment (fig. 1). These data indicate that minimal tolerance or sensitization occurs to the response rate-decreasing effect of NTX after repeated exposure to NTX or to opioid agonists. However, in contrast to those initial titration point determinations (fig. 1), figure 5 (top) shows that when a dose of 30 mg/kg NTX was given without morphine pretreatment, titration point was significantly elevated (15%). This suggests that a small sensitization to the titration-point-altering effects of a high dose of NTX may have developed over the course of the repeated NTX administration.
A pretreatment dose of 10 mg/kg morphine reduced the ED25value for the ICSS rate-decreasing effect of NTX, from 29.51 ± 2.04 after saline to 0.001 ± 1.51 after morphine. Furthermore, based on these ED25 data, the degree of sensitization to the rate-decreasing effects of NTX did not differ from that seen during the initial determinations (acute sensitization experiment). After morphine pretreatment, there was a significant titration point increase (10%) on NTX (0.01 mg/kg) administration (fig. 5) as seen with saline pretreatment and 30 mg/kg NTX. In contrast to the data collected during the acute sensitization experiment (fig. 1, top), there was no titration point decrease 4 hr after a dose of 10 mg/kg morphine (fig.5, top, Pre-pump MOR).
Figure 6 shows that after an extended drug-free period, cumulative morphine administration caused significant decreases in ICSS response rate at 3.0 and 5.6 mg/kg (40% and 75%, respectively). However, a significant titration point decrease was noted only at the larger dose (5.6 mg/kg).
Pump implant.
Figure 7 shows daily changes in titration point and response rate 2 days before, during and 3 days after the implantation of morphine-filled (15 mg/kg/day) osmotic pumps. There were significant decreases in both titration point and response rate 24 hr after the pump was implanted (day 1). Tolerance to the titration point decrease developed the next day (day 2). However, only partial tolerance to the response rate-decreasing effects of morphine developed at that time, and response rate did not return to prepump base-line levels until day 3.
Morphine challenge.
Figure 6 (bottom) shows that 7 days after animals were implanted with osmotic pumps, the administration (fig. 7, b) of 2-fold-higher doses (ED50 = 6.31 ± 1.34) of morphine was necessary to suppress response rates by 50% compared with the corresponding prepump curve (ED50 = 3.18 ± 1.47). In addition, morphine caused a significant increase in titration point (fig. 6, top), a change opposite that seen during prepump testing. Therefore, tolerance to the acute titration-point-decreasing effects of morphine occurred and was not surmountable at the doses tested. Due to the behavioral depressant effects of morphine, it was not possible to test higher doses. Titration point remained significantly elevated ≥24 hr after the testing of cumulative morphine doses (fig. 7, day 8). Taken together, these data show a clear differential effect of morphine challenge in tolerant animals compared with nontolerant animals.
Naltrexone challenge.
Figure 5 shows the effects of NTX after saline, one 4- hr (10 mg/kg) morphine injection or 10 days of chronic morphine infusion via osmotic pump. During chronic morphine infusion, the ED25 value for the rate-decreasing effect of NTX was reduced from 29.5 ± 2.04 mg/kg after saline to 0.002 ± 1.48 mg/kg (fig. 5, bottom). However, the ED25 value of the Pump-day 10 group was not significantly different from the ED25 value obtained after 4-hr morphine challenge. Table 3shows ED25 data for all three conditions.
Although the rate-decreasing effect of NTX was affected comparably by morphine administered as a single 10 mg/kg dose or continuouslyvia osmotic pumps, titration points were differentially affected by these two treatments (fig. 5, top). The 4-hr pretreatment resulted in a maximum 10% elevation of titration point at 0.01 mg/kg NTX. In contrast, during continuous infusion of morphine, titration point was increased 17% by as little as 0.003 mg/kg NTX, and 0.1 mg/kg NTX produced a peak increase of 30%. Analysis revealed that the difference between the two pretreatment conditions was significant [F(1,32) = 4.38]. Furthermore, during the morphine infusion, NTX challenge resulted in signs of physical withdrawal not seen after 4-hr morphine challenge. These signs included “wet-dog” shakes, gnawing movements, salivation, defecation and aggressive postures, irritability and vocalization on handling. Also, there was a significantly greater [t(1,11) = 4.35] mean weight loss observed between the beginning and the end of the testing session (15.8 ± 3.6 g) compared with the mean 9 ± 2.2 g loss observed during the acute pretreatment condition.
Pump removal.
Figure 7 shows that there was response rate disruption on day 11, the day after the NTX challenge (fig. 7, c). However, response rates returned to base-line levels within 2 days. Both response rate and titration point were decreased significantly the day after pump removal (fig. 7, d) but returned toward prepump base-line values 1 day later. Physical signs associated with withdrawal were noted the day after the pump was removed; these included gnawing movements, defecation and irritability and vocalization on handling. However, symptom severity appeared to be attenuated compared with the intensity noted during the NTX challenge session, and weight loss did not exceed control levels.
Discussion
The present report provides further support for the observation that morphine-induced sensitization to naltrexone is a dose-relatedmu opioid receptor-mediated effect. A significant and dose-related sensitization to the ICSS response rate-decreasing effect of NTX was observed after a 4-hr pretreatment with each of the fivemu opioid agonists tested. Sensitization to NTX differed among the opioid agonists tested and seemed to correspond to the selectivity of the agonist for the mu-receptor subtype. However, sensitization to NTX was not seen after the acute (4 hr) administration of a kappa opioid agonist or nonopioid drugs. The present findings are consistent with those of previous studies from this laboratory (Adams and Holtzman, 1990; White-Gbadebo and Holtzman, 1994a, 1994b) and extend the generality of the acute NTX sensitization phenomenon to a non-food-maintained baseline.
In these acute (4 hr) pretreatment tests, enhanced sensitivity to NTX was not accompanied by tolerance to the effects of morphine. Sensitization to the ICSS rate-decreasing effect of NTX also occurred after 10 days of morphine infusion via osmotic pump: a chronic dosing regimen resulting in significant tolerance to the initial effects of morphine. Response rate ED25 data indicated that the degrees of sensitization to NTX after either acute (10 mg/kg, 4 hr) or chronic (15 mg/kg/day, 10 day) morphine pretreatment were approximately equivalent (fig. 5). This dissociation between the NTX-sensitizing and tolerance-inducing effects of morphine adds further support for the theory that opioid dependence and opioid tolerance are not exclusively interdependent phenomena.
When morphine is administered acutely to drug-free animals, moderate doses produce an initial decrease in the rate of lever pressing, which is sometimes followed by a rebound increase (Lorens and Mitchell, 1973;Schaefer and Holtzman, 1977). Furthermore, both discrete-trial (Glicket al., 1982; Marcus and Kornetsky, 1974) and autotitration (Van Wolfswinkel et al., 1985; Van Wolfswinkel and Van Ree, 1985) ICSS procedures have been used to show that morphine (≤5.0 mg/kg) lowers the threshold for ICSS responding, interpreted as a reward-enhancing effect. The present study is consistent with these earlier data in that before chronic morphine administration, significant decreases in titration point and response rate were seen on acute (15-min) morphine (3.0 and 5.6 mg/kg) administration. Titration point decreases were also seen 4 hr after administration at the highest doses of morphine and levorphanol tested in the acute sensitization experiment. Therefore, it might be concluded that opioid agonists other than morphine lower titration point. However, because this effect was not noted after all of the mu opioid-selective agonists tested or on a second 4-hr morphine determination (chronic morphine experiment), these titration point changes might have reflected inherent variability in the titration point measure or they may have been dependent on the presence of the agonist at the receptor, making them dependent on the pharmacokinetics of the particular compound in question. Pharmacokinetic factors also may account for the differential degrees of sensitization to the rate-decreasing effects of NTX seen after the mu opioid agonists tested. Alternatively, failure to note changes in titration point after all mu opioid agonists may indicate that tolerance to the titration-point-decreasing effects of agonists develops over the course of repeated testing.
Previous studies (Esposito and Kornetsky, 1977) have shown that the magnitude of the ICSS threshold decrease seen on acute (10-min) morphine administration does not change over the course of repeated once daily morphine injections (6–12 mg/kg, ≤27 days), indicating a lack of tolerance to the rewarding effects of morphine. In contrast to the results obtained with daily dosing, the present study showed the development of tolerance to both the rate and titration-point-altering effects of morphine. During a continuous infusion of morphine deliveredvia osmotic pumps, the morphine dose-response curve for response rate was shifted to the right 2-fold after 7 days, and the direction of the effect before and after pump implant was the same: a decrease. These data indicate the development of tolerance to the response rate-decreasing effects of morphine. Also, the present titration point data were consistent with the corresponding response rate data. After 7 days of continuous morphine infusion, doses of morphine approaching a dose that results in almost complete response rate suppression (10 mg/kg) no longer result in a titration point decrease. The different morphine dosing regimens used in the Esposito and Kornetsky study (1977) and the present study most likely account for the differential development of tolerance in the two studies.
The fact that rats were tolerant to the titration-point-lowering effects of morphine after infusion of the drug is consistent with human clinical data indicating tolerance to the euphorigenic effects of morphine in addicts (Jaffe, 1990). Due to response rate suppression at the higher doses of morphine (>10 mg/kg), in the present study it was not possible to determine whether tolerance to the titration-point-lowering effect of morphine was surmountable. Further work, perhaps using a reward-selective methodology less sensitive to response suppression, may answer this question. Regardless of the effects of chronic morphine administration, an acute 4-hr pretreatment with morphine, should not have induced tolerance to the effects of morphine itself on ICSS behavior (Lorens and Mitchell, 1973). Therefore, in the acute sensitization experiments, tolerance to morphine did not appear to be a prerequisite for the development of any sensitization to NTX induced by morphine (Takemori et al., 1973; Tulunay and Takemori, 1974a, 1974b).
In opioid naive animals, the effects of lower doses of NTX are small. However, it is possible that repeated exposure to an antagonist can result in sensitization to that antagonist. Consistent with previous reports (Adams and Holtzman, 1990; Young, 1986), a negligible change in sensitivity to the rate-decreasing effects of NTX alone occurred in rats over the course of this study (figs. 1 and 5). Sensitization to opioid antagonists after repeated antagonist administration appears to be a species-specific effect (Adams and Holtzman, 1990). Monkeys exhibit such sensitization (Dykstra, 1983), where pigeons do not (Goldberg et al., 1981). After repeated administration of the drug, a small degree of sensitization to NTX occurs in rats (Schindler et al., 1993; present study). In the present study, animals showed little enhanced sensitivity to the response rate-decreasing effects of NTX over the course of repeated testing. However, they did exhibit a significantly enhanced titration point elevation (figs. 1 and. 5). These titration point data may indicate that sensitization to the motivational effects of NTX develops independent of response rate changes over the course of repeated intermittent dosing with NTX or opioid agonists.
The autotitration method has been used to show that antagonist-precipitated withdrawal after a regimen of chronic morphine administration is associated with increased current titration points for ICSS (Schaefer and Michael, 1986). In that study, current titration points were not elevated during unprecipitated withdrawal. Likewise, we found frequency titration point elevations during precipitated withdrawal but not unprecipitated withdrawal. In fact, a frequency titration point decrease was seen after pump removal. As it has been shown that reductions in operant behavior occurring during spontaneous withdrawal are generally less severe than those occurring during antagonist-precipitated withdrawal, the present findings, along with those of Schaefer and Michael (1986), may indicate that the autotitration procedure is simply insensitive to spontaneous withdrawal-induced motivational changes.
Under both the 4-hr and the 10-day morphine treatment conditions, titration point increases and response rate decreases occurred on NTX challenge (fig. 5). However, the magnitude of the titration point increase under the two conditions was significantly different at equivalent low (0.003 mg/kg) doses of NTX. Furthermore, rats in the chronic (10 day) morphine condition exhibited a 20% higher maximum titration point that was associated with more observable physical withdrawal signs, such as weight loss. These titration point data may indicate qualitatively greater motivational changes on NTX challenge in “dependent” animals. Due to response rate disruption, higher (≥1 mg/kg) antagonist doses were not tested in this study. Therefore, many of the somatic signs of morphine withdrawal (e.g., wet dog shakes, jumping, teeth chattering and ptosis) that generally emerge only with antagonist challenge doses that are larger than those having significant effects on schedule controlled behavior (Schulteis et al., 1994) may have been absent in either the acute or chronic morphine studies, thereby precluding the detection of a differential effect of the duration of morphine pretreatment (4 hr vs. 10 day). A longer duration of morphine administration may be functionally equivalent, in altering ICSS responding, to an increased NTX challenge dose and might account for the 4-hr vs. 10-day condition titration point differences during NTX challenge. Alternatively, prolonged (10 day) morphine administration may result in a true qualitative difference in maximal responsiveness to the effects of NTX on ICSS responding that may not be overcome by increasing the NTX dose. As increasing NTX doses do not appear to increase the maximal titration point in the 4-hr pretreatment group, this latter explanation appears most likely.
After morphine pretreatment, progressive increases in sensitization to NTX occurred over multiple testing sessions in rats responding for food reinforcement (Adams and Holtzman, 1990). In the present study, rats responding for ICSS after morphine pretreatment (4 hr) did not show a progressive increase in sensitization to the rate-decreasing effects of NTX over repeated determinations (figs. 1 and 5). It has been suggested (Adams and Holtzman, 1990) that after morphine pretreatment, very low doses of NTX given early in the session may serve as discriminative stimuli for the unconditioned rate-decreasing effects of the larger doses received later, resulting in a conditioned rate suppression at lower doses. Very low doses of NTX have been shown to serve as discriminative stimuli in animals made dependent on morphine (Gellert and Holtzman, 1979). In the present study, however, a lack of progressively increasing sensitization to NTX after repeatedmu-agonist administration provided no evidence that behavioral conditioning played a role in the development of sensitization. This long-term consistency of drug effects may be one of the advantages of a behavioral baseline maintained by a reinforcer other than food (Adams and Holtzman, 1990).
The fact that large shifts in sensitivity to NTX were observed with themu opioid selective agonists both before and after testing with the nonopioid compounds indicates that any lack of effect seen after the nonopioid compounds was not due to an order effect. As observed with food reinforced responding (Adams and Holtzman, 1990), the kappa-selective agonist spiradoline did not induce significant sensitization, even at doses (3.0 mg/kg) that substantially suppressed responding 4 hr after administration. Although the challenge doses of NTX that were used in the present study were larger than those that would be expected to be receptor subtype selective, the fact that naltrexone is slightly less potent as an antagonist at kappaopioid receptors than at mu opioid receptors might partially account for this finding (Von Voightlander et al., 1983). Behaviorally active doses of d-amphetamine and pentobarbital and an adequate dose of dextrorphan (Herling et al., 1983), relative to levorphanol, did not induce sensitization to NTX. These results concur with those seen in a food reinforced paradigm (Adams and Holtzman, 1990) and demonstrate further the pharmacological specificity of acute agonist-induced sensitization to opioid antagonists.
In humans, physical dependence is often assessed by the emergence of a withdrawal syndrome on removal of morphine or by administration of an opioid antagonist. In animal models, operant responding has long been used as a baseline against which to study the development of tolerance occurring with chronic administration (Holtzman and Villarreal, 1973). Furthermore, in animals, a disruption of responding (rate decrease) seen after removal of the drug is often used as a gauge of dependence (Balster, 1985). However, animal models are often criticized for failing to provide a reward-specific measure of the motivational changes occurring during withdrawal that have been documented in humans. In the present study, changes in titration point and response rate were noted when animals were undergoing precipitated withdrawal. Therefore, the present animal model may have utility for assessing the neuronal mechanisms that underlie motivational states associated with acute and chronic opioid administration and opioid withdrawal. Furthermore, the correspondence in ED25 values for the rate-decreasing effect of NTX after both acute and chronic morphine administration is consistent with the theory that acute agonist-induced sensitization reflects receptor-mediated changes occurring early in the development of physical dependence.
Acknowledgments
The authors gratefully acknowledge The Upjohn Company, Penick Company, Roche Laboratories and McNeil Laboratories for generously providing drugs used in the study.
Footnotes
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Send reprint requests to: Dr. Keith W. Easterling, Emory University School of Medicine, Department of Pharmacology, Atlanta, GA 30322.
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↵1 This investigation was supported by National Institutes of Health Grant DA00541 and by Research Scientist Award K05/DA00008 to S.G.H.
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↵2 Portions of this work were presented at the at the 57th annual meeting of the College on Problems of Drug Dependence (1995, Scottsdale, AR) and the 25th annual meeting of the Society for Neuroscience (1995, San Diego, CA).
- Abbreviations:
- ANOVA
- analysis of variance
- CRF
- continuous reinforcement
- ICSS
- intracranial self-stimulation
- NTX
- naltrexone hydrochloride
- Received May 8, 1996.
- Accepted December 24, 1996.
- The American Society for Pharmacology and Experimental Therapeutics