Introduction

Top Abstract Introduction Methods Results Discussion References

Chronic administration of opioid agonists produces a physiological dependence revealed by the appearance of withdrawal signs when the maintenance dose of the agonist is withheld (abstinence-associated withdrawal) or when an opioid antagonist is administered (precipitated withdrawal). The signs of precipitated and abstinence-associated withdrawal are qualitatively similar and may be behavioral as well as physiological. In rodents, opioid dependence-related withdrawal has been characterized by use of behavioral measures, such as irritability and exploratory behaviors, as well as more objective physiological measures, such as defecation and hypothermia (Bläsig et al., 1973; Mucha et al., 1979). In monkeys, studies of opioid dependence-related withdrawal have relied primarily on subjective observations of behaviors such as drowsiness or restlessness (Seevers, 1936; Aceto et al., 1977, 1989). Physiological effects of morphine withdrawal in rhesus monkeys have received much less attention, although opioid withdrawal does produce hypothermia, hyperpnea and tachycardia in monkeys (Holtzman and Villarreal, 1969; Goldberg, 1976). More recently, naltrexone-precipitated withdrawal in monkeys receiving daily morphine also has been studied by drug discrimination procedures, in which either cessation of morphine injections or naltrexone administration occasions responding on a lever associated with naltrexone injection (France and Woods, 1989). Responding on the naltrexone-lever is attenuated by morphine and related agonists, which further suggests that the naltrexone discriminative stimulus is associated with opioid withdrawal and, therefore, dependence.

Many symptoms of withdrawal from morphine-like opioids appear to be opposite to the acute effects of agonist administration. For example, morphine withdrawal is characterized by mydriasis, hypothermia and reports of dysphoria, whereas acute morphine administration is associated with miosis, hyperthermia and reports of euphoria (Heishman et al., 1989; Preston et al., 1988). The ventilatory effects of acute morphine administration versus morphine withdrawal differ analogously; that is, ventilation is depressed after acute administration of morphine, whereas increased ventilation has been reported during opioid withdrawal (Martin et al., 1968). Both morphine abstinence and antagonist administration have been reported to produce hyperpnea in morphine-dependent subjects (Martin and Jasinski, 1969; Goldberg, 1976; Heishman et al., 1989). However, withdrawal-related changes in ventilation have not been fully characterized, and parametric relationships between abstinence and ventilation remain to be clarified.

The experiments presented here were designed to examine ventilatory effects of abstinence-associated and antagonist-precipitated withdrawal in rhesus monkeys receiving daily injections of morphine. In these experiments, the effects of the opioid antagonist, naltrexone, as well as the effects of withholding the maintenance dose of morphine for differing periods of time were assessed. These studies were completed in concert with other studies of tolerance to the ventilatory-depressant effects of mu opioid agonists (Paronis and Woods, 1997). The results of the present experiments demonstrate that ventilatory frequency increases during opioid withdrawal in a predictable manner that is consistent with the view that daily single injections of morphine produce opioid dependence. The orderliness of these effects suggests that similar procedures may be useful in future evaluations of opioid withdrawal in primates.

	Methods

Top Abstract Introduction Methods Results Discussion References

Subjects

Subjects were two adult male (909B and S67) and one adult female (F2) rhesus monkeys. The weights of the subjects remained relatively stable throughout the study and were 6.5 kg (F2), 7.5 kg (S67) and 9 kg (909B). Subjects 909B and F2 had been used previously in experiments involving opioids. Monkeys were housed singly in a temperature-controlled colony room with a 12-hr light/dark cycle (lights on at 6:00 A.M.). Water was available ad libitum, and the monkeys were fed approximately 30 biscuits (Purina Monkey Chow) daily, supplemented twice weekly with fresh fruit.

Apparatus

The apparatus used was similar to that described by Howell et al. (1988). Subjects were seated in a standard primate restraint chair enclosed within a sound-attenuating chamber. A Plexiglas helmet that was placed over the head of the subject served as a pressure-displacement plethysmograph. Customized Plexiglas plates and rubber dams were used to provide an air-tight seal around the monkey's neck. A continuous flow, 10 liters/min, of either air or a mixture of 1%, 3% or 5% CO₂ in air (hereafter referred to only by the CO₂ concentration) was introduced through a port at the front of the helmet, and was extracted at the same rate through a port in the back of the helmet. Changes in flow within the plethysmograph were measured by a pressure transducer connected to a polygraph (model 7E, Grass Instrument Co., Quincy, MA) and computer (IBM/PCjr). Pressure displacement was converted to a volume measure by a polygraph integrator (model 7P122E, Grass Instrument Co.). Minute volume (V_E) was determined by integration of changes in flow through the plethysmograph; frequency of ventilation (f) was directly determined; and tidal volume (V_T) was calculated as the quotient V_T = V_E/f.

Experimental sessions generally consisted of four to six consecutive 30-min cycles, each comprising a 23-min exposure to air followed by a 7-min exposure to 5% CO₂. Data from the first air/CO₂ cycle were used for session-control values. Cumulative dosing procedures similar to those described by Howell et al. (1988) were used. At the start of each test cycle, graded doses of drug were administered i.m. so that the total dose increased by 0.25 or 0.5 log unit increments throughout the session. When ventilatory responses to different concentrations of CO₂ were measured, a single dosing procedure was used. In these instances, experimental sessions consisted of two 63-min cycles in which 7-min exposures to increasing concentrations of CO₂ alternated with 14-min exposures to air. Animals received an i.m. injection of drug after the end of the first cycle. Data obtained from the first cycle served as session-control values.

Experimental Design

Drug tests were conducted during four phases, baseline, single-dosing, double-dosing and abstinence, during which the subjects were repeatedly exposed to morphine and other mu opioid agonists, the opioid antagonist, naltrexone, and several nonopioid drugs. Experimental sessions were conducted at least twice a week to ensure stable patterns of ventilation, and at least 2 days intervened between drug tests. Five to seven days intervened between tests when the daily dosing schedule was interrupted to assess abstinence-associated withdrawal effects, or when the monkeys received large doses of drugs. The order of the drug tests was randomized between subjects within each phase of the study to minimize the influence of duration of exposure to the maintenance dose of morphine on the responses to the test drug.

Baseline phase. During the baseline phase, the ventilatory effects of morphine and naltrexone were determined in all monkeys. This phase of the study lasted 14 weeks in S67 and 16 weeks in 909B and F2. Data obtained during this phase are referred to throughout this paper as baseline values, and should be distinguished from the session-control values obtained at the start of individual test sessions.

Single-dosing phase. During the single-dosing phase, the monkeys received injections of 3.2 mg/kg morphine every morning, either in the test chamber or, on days when they were not tested, in the home cage. Except where noted, tests occurred 24 hr after injection of the maintenance dose of morphine. Only the ventilatory effects of morphine were studied during the first 4 weeks of the single-dosing phase. Subsequently, the ventilatory effects of a range of opioid drugs, including naltrexone, morphine, nalbuphine, butorphanol, ethylketazocine and fentanyl, and several nonopioid drugs (midazolam, flumazenil and -carboline-3-carboxylic acid; reported in Paronis et al., 1994) were examined. The order in which drugs were studied was randomized among subjects. When naltrexone or a nonopioid drug was studied, the daily morphine dose was administered immediately after the test session. The effects of abstinence-associated withdrawal were assessed by withholding the maintenance dose of morphine for either 48 or 72 hr. This phase of the study lasted 40 weeks in S67 and 57 weeks in 909B and F2.

Double-dosing phase. During the double-dosing phase, monkeys received two injections of 3.2 mg/kg of morphine daily. The injections were spaced by 12 ± 2 hr, and usually were given at 9:00 A.M. and 9:00 P.M. This phase of the study lasted 12 weeks in S67 and 17 weeks in 909B and F2. During this time the ventilatory effects of naltrexone and morphine were examined in all monkeys; except where noted, tests occurred 24 hr after morphine.

Abstinence phase. During the abstinence phase, daily morphine injections were discontinued and the monkeys were drug-free for 4 weeks. Experiments continued throughout this time to assess any effects of abstinence-associated withdrawal. After at least 28 days, the ventilatory effects of morphine and naltrexone were redetermined in all monkeys.

Data Analysis

Data from the last 3 min of each exposure to air or CO₂ were used for analysis of drug effects on ventilation. The ventilatory stimulant effects of 3% and 5% CO₂ were calculated as a ventilatory ratio (V_ECO₂/V_EAir). Effects of naltrexone and abstinence-associated withdrawal on ventilation were subjected to repeated measures one-way ANOVA, followed by Dunnett's multiple comparison test. Significance was set at P < .05.

Drugs

Morphine sulfate (Mallinckrodt, Inc., St. Louis, MO) and naltrexone HCl (Endo Laboratories, Inc., Garden City, NY) were dissolved in sterile water and injected i.m. in a volume of 0.1 to1.0 ml. Drug doses are expressed as the weight of the salt.

	Results

Top Abstract Introduction Methods Results Discussion References

Baseline. Ventilatory responses to increasing concentrations of CO₂ were obtained before and after vehicle injections on 3 consecutive days. Vehicle injections did not systematically alter ventilation, and data collected before and after the vehicle injections were combined to obtain baseline response values given in table 1. Exposure to CO₂ increased all measures of ventilation in a concentration-dependent manner in each monkey, and increases in ventilatory rate, tidal volume and minute volume achieved statistical significance during exposure to 3% and 5% CO₂ (table 1). The responses to CO₂ were similar for individual monkeys; the mean ventilatory ratio of 3% CO₂ compared with air was 1.82 ± 0.07 and the mean ventilatory ratio in the presence 5% CO₂ compared with air was 3.13 ± 0.04.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Naltrexone effects on ventilation in the presence of either normal air () or 5% CO2 () under baseline conditions. Points above "ctrl" represent mean session-control values obtained before the first injection of naltrexone. Abscissa: cumulative naltrexone dose. Ordinates: respiration frequency in breaths per minute (top), tidal volume in milliliters per breath (middle), minute volume in liters per min (bottom). Each point represents the group mean, based on one or more observations in three subjects, vertical lines are ± 1 S.E. (* P < .05 versus session-control value.)

Precipitated withdrawal. Naltrexone, administered 24 hr after morphine during the single-dosing phase, increased breathing frequency in an orderly manner. The effects of naltrexone during the single-dosing phase were markedly different in both potency and magnitude compared with its effects during the baseline phase (fig. 2). Ventilatory frequency was significantly increased by doses of naltrexone as low as 0.0032 to 0.01 mg/kg, reflecting an increased potency of approximately 4 orders of magnitude. In addition to increased potency, the magnitude of the responses to naltrexone was greater during the single-dosing phase than during the baseline phase. For example, under baseline conditions, 32 mg/kg naltrexone increased respiratory rate in air 44%, from 25 ± 1 to 36 ± 1 breaths/min; in monkeys receiving a single daily injection of 3.2 mg/kg morphine, 0.01 mg/kg naltrexone increased ventilatory frequency in air 62%, from 32 ± 4 to 52 ± 4 breaths/min (fig. 2). Naltrexone had no effect on tidal volumes, thus increases in minute volumes were consequential to the increases in ventilatory rate.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Naltrexone effects on ventilation in the presence of normal air during the baseline phase (; data are redrawn from fig. 1), single-dosing phase (), double-dosing phase () and abstinence phase (). Other details are as in figure 1. (* P < .05 versus session-control value.)

View larger version (K): [in this window] [in a new window]   Fig. 3.   Effects of naltrexone administered as single doses on ventilatory frequency in the presence of air (), 3% () and 5% () CO2 during the single-dosing phase. Points above "ctrl" represent mean control values obtained during the baseline phase. Other details are as in figure 1. (* P < .05 versus baseline control value.)

View larger version (K): [in this window] [in a new window]   Fig. 4.   Naltrexone effects on ventilatory frequency in the presence of normal air during the single- and double-dosing phase at 24 hr (; data are redrawn from fig. 2), 3 hr () and 12 hr () after the last previous morphine injection. The horizontal lines represent the mean breathing frequency during the baseline phase; dotted lines are ± 95% confidence limit. Note that the range of the ordinate increases from A to B. Other details are as in figure 1. (* P < .05 versus baseline control value.)

Abstinence-associated withdrawal. The effects of abstinence-associated withdrawal were assessed during the single-dosing phase by withholding the maintenance dose of morphine for up to 72 hr. Later time points were not tested to avoid further disruption of the chronic dosing regimen. During the double-dosing phase, the maintenance dose of morphine was withheld for up to 48 hr; and, in addition, ventilation was measured during a 4-week period after cessation of the daily morphine treatment. Similar to the effects of naltrexone-precipitated withdrawal, abstinence-associated withdrawal was accompanied by an increase in ventilatory frequency, whereas measures of minute volume and tidal volume were not affected (fig. 5). The ventilatory effects of abstinence-associated withdrawal were relatively mild compared with the effects of naltrexone-precipitated withdrawal and were statistically significant only during the double-dosing phase. A peak effect, 47 ± 7 breaths/min, was seen at 48 hr after the last morphine injection. Termination of daily morphine injections did not result in significant changes to the ventilatory responses to 3% CO₂ and 5% CO₂; this is reflected in the fairly stable ventilatory ratios obtained during the abstinence phase (fig. 6). Although ventilatory measures were obtained for 28 days, recovery appeared complete in all monkeys by 5 to 7 days.

View larger version (K): [in this window] [in a new window]   Fig. 5.   Abstinence-associated withdrawal effects on ventilation in the presence of normal air during the single-dosing phase (left column) or after the double-dosing phase (right column). Abscissae: time since the last morphine injection in days. Ordinates: ventilation frequency in breaths per minute (top), tidal volume in milliliters per breath (middle), minute volume in liters per min (bottom). Each point represents the group mean, based on one or more observations in three subjects, vertical lines are ± 1 S.E. Solid horizontal lines represent the mean values during the baseline phase; dotted lines are ± 95% confidence limit. (* P < .05 versus baseline control value.)

View larger version (K): [in this window] [in a new window]   Fig. 6.   Ventilatory effects of 3% CO2 () and 5% CO2 () during the abstinence phase. Abscissa: time since the last morphine injection in days. Ordinate: ratio of minute volume in CO2 to minute volume in air. The dotted line represents the mean ventilatory ratio at 3% CO2 during the baseline phase; the dashed line represents the mean ventilatory ratio at 5% CO2 during the baseline phase. Each point represents the group mean, based on one observation in three subjects, vertical lines are ± 1 S.E.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Before the onset of the daily morphine dosing regimen, doses of naltrexone up to 32 mg/kg had only modest effects on ventilatory rate in rhesus monkeys. In morphine-maintained rhesus monkeys, however, ventilatory rate was markedly increased by low doses, 0.0032 to 0.01 mg/kg, of naltrexone (precipitated withdrawal) or by discontinuation of morphine treatment (abstinence-associated withdrawal). The ventilatory effects of both naltrexone and morphine abstinence were enhanced when the daily maintenance dose of morphine was increased. Moreover, after cessation of all daily dosing with morphine, baseline ventilatory values and the original effects of naltrexone were recovered. The general similarity in the effects of naltrexone injections and discontinuation of morphine maintenance in the present experiments are in accord with other comparisons of antagonist-precipitated and spontaneous abstinence (Wikler et al., 1953). For example, the administration of nalorphine to human subjects receiving 60 to 240 mg/day morphine produced the same constellation of effects, e.g., tachypnea, nausea and yawning, as were reported after abrupt cessation of the morphine treatment. These findings are consistent with the view that during the chronic dosing phases of the present study, the monkeys were dependent on morphine, and the naltrexone- and abstinence-induced increases in ventilatory rate reflect states of opioid withdrawal.

Previous studies that have measured ventilation during antagonist-precipitated opioid withdrawal have produced mixed results. For example, patients enrolled in methadone treatment programs, and presumably opioid dependent, showed no changes in ventilation after injections of 0.2 mg/kg naloxone (Preston et al., 1988), whereas an earlier study reported that nalorphine increased ventilatory rate in humans dependent on morphine, methadone or heroin (Wikler et al., 1953). Similarly, in an acute opioid-dependence procedure in humans, naloxone administered 6 hr after morphine dose-dependently increased ventilatory rate, but only when specific time and dose conditions were satisfied (Heishman et al., 1989, 1990; Kirby et al., 1990). Results from animal studies have been more consistent, and opioid antagonist-induced increases in respiration have been reported after either continuous or repeated agonist administration in rats, dogs and monkeys (Baraban et al., 1993; Gilbert and Martin, 1976; Goldberg, 1976). For example, injections of 0.2 mg/kg nalorphine in rhesus monkeys maximally dependent on morphine (8-12 mg/kg/day) increased respiration to 40 to 60 breaths/min (Goldberg, 1976). The results of the present experiments are in agreement with and extend previous findings, demonstrating that naltrexone will increase ventilation in morphine-dependent monkeys in a manner that is dependent on both the acutely administered naltrexone dose and the maintenance dose of morphine.

The enhanced potency of naltrexone in increasing ventilatory rate in morphine-maintained monkeys is in general agreement with reports of other behavioral effects of antagonists in opioid-dependent subjects, compared with effects in nondependent subjects. For example, in rats responding under a schedule of food presentation, the dose-response curve of naltrexone was displaced 5000-fold to the left after implantation of morphine-filled osmotic minipumps (Adams and Holtzman, 1990). Similarly, both naloxone and nalorphine were approximately 100-fold more potent in disrupting fixed-ratio performance under a schedule of food presentation in etonitazene-dependent rhesus monkeys than in nondependent monkeys (Tang, 1981). The naltrexone-induced increases in ventilation in the present study demonstrate that the potency with which an opioid antagonist exerts unconditioned physiological effects is also enhanced during a period of morphine maintenance.

Previous investigations have reported that exposure to low doses of opioid antagonists during periods of morphine maintenance may sensitize monkeys to behavioral and autonomic effects of the antagonist in the absence of agonist treatment (Goldberg and Schuster, 1970; Goldberg, 1976). Moreover, Warren and Morse (1989) demonstrated that an enduring sensitization to the effects of naltrexone on food-maintained behavior can develop in nondependent monkeys simply as a result of repeated exposure to the antagonist. In the latter experiments, the enhanced potency of naltrexone was retained for several months, dissipating only when the naltrexone was administered under conditions in which behavior was not maintained by food delivery. It is likely that such sensitization to naltrexone represents, in part, a conditioned response to its initial effects rather than pharmacodynamic changes in the effects of naltrexone (Bergman and Schuster, 1985; Warren and Morse, 1989; Schindler et al., 1990). In the present experiment, exposure to low doses of naltrexone also might have elicited interoceptive cues that modified the responses to subsequent naltrexone injections. However, similar responses to naltrexone were obtained using either cumulative or single-dosing procedures. Moreover, the ventilatory effects of naltrexone were apparent only during chronic morphine treatment, which suggests that the enhanced potency of naltrexone primarily reflected alterations in its pharmacodynamic actions rather than conditioned effects of repeated exposure.

Ventilatory measures were transiently increased after cessation of the daily morphine administration, but recovered to predependent baseline values within 1 week of terminating the daily dosing regimen. The duration of morphine withdrawal as measured by changes in ventilation agree with the results of previous studies of spontaneous opioid withdrawal in rhesus monkeys. In previous investigations in monkeys, chronic morphine regimens that lasted for months were followed by abstinence syndromes that persisted for only 4 to 8 days (Holtzman and Villarreal, 1971, 1973; Bergman and Schuster, 1985). Similarly, a study of opioid withdrawal in humans suggested that signs of abstinence first emerge 6 to 8 hours after cessation of the agonist treatment, peak within 72 hr and then gradually subside over 7 to 10 days (Kolb and Himmelsbach, 1938). In other studies, however, the withdrawal syndrome in humans has been reported to last for several months (Martin et al., 1968; Martin and Jasinski, 1969). Furthermore, in the latter study, the most pronounced respiratory effect of the withdrawal syndrome was an immediate, dramatic, hypersensitivity to CO₂ that lasted for several weeks and was then followed by a prolonged hyposensitivity to CO₂ which persisted for an additional 23 weeks. These findings are in marked contrast to the present results, showing no change in sensitivity to CO₂ during either abstinence-associated or antagonist-precipitated withdrawal in monkeys. In addition to possible species differences, it is possible that the contrasting results of the studies by Martin and his colleagues and the present experiments are related to different morphine dosing schedules or the different methods used to assess ventilatory responses.

Overall, the ventilatory effects of naltrexone appear to provide sensitive, quantitative measures of precipitated opioid withdrawal in morphine-maintained monkeys. The magnitude of the ventilatory effects of precipitated withdrawal vary dose-dependently with the dose of the antagonist as well as the maintenance dose of the agonist. Hence, ventilatory measures may be useful in future studies addressing questions of opioid withdrawal in a primate species.

The appearance of withdrawal signs reveals a physiological dependence on the chronically administered drug, and the development of dependence on a drug is frequently accompanied by tolerance to the acute effects of the same drug. As described in a companion paper (Paronis and Woods, 1997) the morphine-dosing regimen used in this study produced tolerance to the antinociceptive effects of morphine; however, no changes were seen in the ventilatory depressant effects of morphine. Taken together, the results presented here and in the companion paper suggest that opioid tolerance and dependence may be dissociable effects of chronic morphine exposure in rhesus monkeys.