The role of vasopressin on the effect of U-50,488 to block the development of morphine tolerance and physical dependence

U-50,488, a selective κ-opioid receptor agonist, has been reported to inhibit the development of antinociceptive tolerance to morphine in mice, rats and guinea pigs, but the mechanism involved in this action remains unknown. Since U-50,488 has been reported to supress the plasma vasopressin level, we investigated the role of vasopressin with U-50,488 in the male Sprague Dawley rat in this study. Animals (230–270 g) were chronically treated with morphine (10 mg/kg, i.p.) twice a day for 6 days in order to induce tolerance to antinociceptive effect measured by tail-flick test. Withdrawl symptoms were precipitated by naloxone (10 mg/kg, i.p.) on day 7. U-50,488 (i.p.) or AVP (i.p. or i.c.v.) or U-50,488 and AVP was (were) coadministered with chronic morphine to investigate their effects on morphine tolerance and dependence. We found that coadministration of 8 mg/kg U-50,488 (i.p.) with morphine almost completely block morphine tolerance and partially block withdrawal symptoms. In contrast, coadministration of AVP (0.3 μg/kg, i.p., or 0.01 μg, i.c.v.) with morphine and U-50,488, the effects of U-50,488 to block morphine tolerance and dependence were reversed. In addition, treatment of AVP antagonist (dPTyr(Me)AVP, 0.5 μg/kg, i.p. or 0.5 μg, i.c.v.) has the similar effect as U-50,488 to block morphine tolerance. In summary, the effect of U-50,488 to block morphine tolerance and dependence may relate to its inhibitory effect on AVP release. Received: 20 February 1996 / Accepted: 14 October 1996     

Development of physical dependence on morphine in respect to time and dosage and quantification of the precipitated withdrawal syndrome in rats

In rats implanted subcutaneously with morphine containing pellets different degrees of dependence were induced by varying the dosage, frequency of implantation and duration of exposure to morphine. Withdrawal was precipitated by intraperitoneal injection of morphine antagonists, mostly levallorphan. The absorption of morphine from the subcutaneous depots was estimated chemically.When withdrawal was precipitated with a constant dose of antagonist the frequency of occurrence of various counted signs and the presence of some checked signs were studied in respect to varying degrees of dependence. The results were compared to those obtained after administration of increasing doses of antagonist in groups of animals that had developed a constant degree of dependence.In both types of experiments the results were rather similar. Some signs became progressively more pronounced when dependence got stronger or the dose of the antagonist was increased. In contrast, other signs showed a maximal frequency at the lower degrees of dependence or after administration of the lower doses of antagonist and decreased or even disappeared when the degree of dependence was higher or the dose of antagonist further increased. Obviously, in withdrawal the intensity of recessive signs like writhing and wet dog shaking declines when dominant signs like jumping, flying (a vigorous kind of jumping) and teeth chattering increase. An inverse relationship between the occurrence of various signs could also be shown within the 30 min observation period. Changes in the integrative mechanisms controlling behaviour during withdrawal are supposed to be the reason for this shift of signs.In other experiments in which the interval between each morphine implantation was prolonged the frequency of some signs like jumping and teeth chattering tented to plateau. This finding seems to be correlated to some kind of steady state on resorption of morphine from the subcutaneous depots, as was found in chemical analysis.     

Effects of depletion of brain catecholamines during the development of morphine dependence on precipitated withdrawal in rats

Summary The significance of long term depletion of brain catecholamines (CAs) for the development of morphine dependence and for the expression of morphine withdrawal was studied in rats which were implanted with morphine pellets for 10 days. CAs were depleted by inhibition of tyrosine-hydroxylase with alpha-methyl-tyrosine (AMT) or by destruction of catecholaminergic nerve terminals with 6-hydroxydopamine (6-OHDA). In the acute experiments these drugs were applied within 24 hrs before precipitation of withdrawal; in the chronic experiments drug administration was started before the first implantation and in the case of AMT, continued repeatedly thereafter.With either method, acute depletion of brain CAs resulted in reduced intensity of withdrawal. When CAs were kept low through the whole time of morphine exposure and also at the time of withdrawal, the intensity of withdrawal was normal in the case of 6-OHDA administration and only slightly decreased in the case of AMT. When AMT administration was discontinued 40 hrs before precipitation of withdrawal the withdrawal pattern occurred with unchanged intensity.Our experimental data are compatible with the assumption that long lasting depletion of brain CAs is compensated for by induction of neuronal supersensitivity for noradrenaline (NA) and dopamine (DA). While both CAs play an important role in the full expression of the withdrawal syndrome their possible involvement in mechanisms leading to dependence seems to be unlikely although final statements cannot be made by the presented experiments.     

Nicotine physical dependence and tolerance in the mouse following chronic oral administration

Rationale Although nicotine dependence and tolerance develop in rats, few studies have examined these processes in the mouse. Establishing such mouse models would eventually allow for an examination of the role of specific nicotinic receptor subtypes in mediating these processes (i.e. through the use of receptor knockouts).Objectives The goals of the present study were to establish mouse models of nicotine dependence and tolerance.Methods Mice were chronically exposed to nicotine (0–200 g/ml) in their drinking solution and assayed for plasma nicotine and cotinine levels, withdrawal signs following nicotine cessation (spontaneous withdrawal) or nicotinic antagonist administration (precipitated withdrawal), or nicotine tolerance. Dependence assays included somatic sign observations (paw tremors, backing and head shakes), tail-flick, plantar stimulation, elevated plus-maze and spontaneous activity. Tolerance was assayed using tail-flick, hot-plate and body temperature tests.Results Plasma nicotine and cotinine levels were elevated during oral nicotine exposure (15.85 ng/ml and 538.00 ng/ml, respectively) and quickly declined following nicotine cessation (<1 ng/ml and <2 ng/ml, respectively), providing evidence that the oral route was pharmacologically relevant. Nicotine withdrawal increased numbers of somatic signs (spontaneous and mecamylamine-precipitated withdrawal) and/or hyperalgesia (spontaneous withdrawal only). Chronic nicotine exposure also produced tolerance, as indicated by reduced responsivity to acute nicotine in assays of analgesia and hypothermia.Conclusions These results indicate that chronic oral nicotine produces dependence and tolerance in the mouse. Further, nicotine dependence may be mediated by multiple nicotinic receptor subtypes, since specific nicotinic receptor antagonists failed to precipitate withdrawal.     

Effect of agmatine on the development of morphine dependence in rats: potential role of cAMP system

Agmatine is an endogenous amine derived from arginine that potentiates morphine analgesia and blocks symptoms of naloxone-precipitated morphine withdrawal in rats. In this study, we sought to determine whether treatment with agmatine during the development of morphine dependence inhibits the withdrawal symptoms and that the effect is mediated by cAMP system. Exposure of rats to morphine for 7 days resulted in marked naloxone-induced withdrawal symptoms and agmatine treatment along with morphine significantly decreasing the withdrawal symptoms. The levels of cAMP were markedly increased in morphine-treated rat brain slices when incubated with naloxone and this increase was significantly reduced in rats treated with morphine and agmatine. The induction of tyrosine hydroxylase after morphine exposure was also reduced in locus coeruleus when agmatine was administered along with morphine. We conclude that agmatine reduces the development of dependence to morphine and that this effect is probably mediated by the inhibition of cAMP signaling pathway during chronic morphine exposure.     

Preferences for morphine in rats: Validation of an experimental model of dependence

Summary Rats were induced to administer morphine to themselves by drinking solutions of it in preference to water; this behaviour was found to be a valid model of morphine dependence. Previous passive medication with morphine was not necessary; initial aversions for the bitter morphine solutions were converted into preferences after the rats were repeatedly given only morphine solutions to drink in order to relieve thirst. The consumption of solutions of quinine which were initially equally aversive did not increase, suggesting that the repeated pairing of a bitter taste with relief of thirst did not account for the preferences for the morphine solutions. It appeared that the post-ingestional effects of morphine provided primary reinforcement for the rats; they were able to regulate their daily intake of the drug after being injected with varying doses of it and they lost weight abruptly during enforced abstinence. There was also evidence that the bitter taste of morphine had become a secondary reinforcer for rats with established preferences.The work was supported by a grant from the Medical Research Council and by grant MH-03313 from the U.S. Public Health Service.One of us (R. K.) held a Beit Memorial Research Fellowship.     

Hyperalgesia during withdrawal as a means of measuring the degree of dependence in morphine dependent rats

A modified flinch-jump procedure was used to detect changes in sensitivity to electric footshock in rats. In preliminary studies, dose-related increases in reaction thresholds (analgesia) were observed following intraperitoneal administration of 3, 6, or 9 mg/kg of morphine with the peak effect 60 min after injection. Analgesia and development of tolerance to the analgesic effects of morphine were detected 2–6 h and 12–24 h, respectively, after the subcutaneous (s.c.) implantation of a morphine pellet (75 mg base). In studies of withdrawal, rats made dependent by the s.c. implantation of morphine pellets showed significant decreases in reaction thresholds (hyperalgesia) and correlated decreases in body weight following removal of the pellet. The greatest changes during withdrawal occurred 12–36 h after pellet removal and the return of the reaction threshold to preremoval levels was associated with the return of normal diurnal fluctuation of body weight. Rats made dependent by s.c. administration of varying doses of morphine every 8 h for 11 days showed similar decreases in body weight and reaction threshold following abrupt cessation of drug injections. Animals receiving larger doses of morphine showed greater changes in the two measures, as well as a more rapid onset and more prolonged duration of effect. Peak effects were observed 48–60 h after the last injection of morphine. In another experiment, rats were made dependent to morphine by the pellet implantation procedure. Following i.p. administration of varying doses of naloxone, a morphine antagonist, there were dose-related decreases in reaction threshold and in body weight with the greatest decrease occurring 60–120 min after the injection. These investigations indicate that the hyperalgesia during withdrawal is a useful index of the degree of physical dependence in rats.Supported in part by Grant NS-10323 from NINDS.     

Induction of tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space.

Rats implanted with chronic catheters in the spinal subarachnoid space were given twice daily injections for 7 days of morphine sulfate, either intrathecally into the lumbar subarachnoid space (15 or 50 microng) or i.p. (20 mg/kg). The development of tolerance, as manifested in a reduction of the analgetic efficacy of these injections on the hot plate and tail flick, occurred in a dose dependent fashion over a period of 7 days. At this time, injections of i.p. morphine into animals which had received spinal morphine and vice versa revealed the existence of a two way cross tolerance between spinal and systemically administered morphine. Injection of naloxone into the spinal cord of animals exposed to i.p. morphine or conversely, i.p. naloxone in animals tolerant to intrathecal morphine, yielded a hyperreflexia and extreme sensitivity to handling. Other signs commonly observed in percipitated withdrawal, however, such as wet shakes and weight loss, were not observed.     

Development and loss of tolerance to morphine in the rat

The development of a differential tolerance to morphine was investigated with respect to the mean effective dose, the threshold dose of tolerance, the degree of tolerance after a fixed dose, and the speed of tolerance loss. The mean effective doses, the threshold doses of tolerance, and the degree of tolerance differed considerably from effect to effect, whereas in all tests tolerance loss remained the same. The mean effective doses were not correlated to threshold doses of tolerance, degree of tolerance, or to the loss of tolerance, but a strong correlation exists between threshold doses of tolerance and degree of tolerance to all effects measured. Consequences of these results upon current theories of tolerance are discussed.     

The effects of p-chlorophenylalanine on morphine analgesia,tolerance and dependence development in two strains of rats

The effects of p-chlorophenylalanine (p-CPA; 100 mg/kg/day for 3 days) on morphine analgesia and the development of tolerance and physical dependence were investigated in Sprague-Dawley (SD) and Fisher (F) strains of albino rats. Using a modified flinch-jump method to detect changes in reactivity to electric footshock, F strain rats were more reactive to the footshock than SD rats, but showed less relative increase in threshold (analgesia) than SD rats following various doses of morphine. Pretreatment with p-CPA attenuated significantly morphine analgesia in SD, but not F rats. In animals implanted subcutaneously with morphine pellets, p-CPA appeared to delay the development of tolerance to morphine in both strains of rats. Hyperalgesia and loss of body weight resulted from administration of naloxone to pellet-implanted rats and p-CPA pretreatment lessened these withdrawal effects significantly in SD rats only. These results emphasize the importance of strain differences in the study of morphine analgesia and development of tolerance and dependence. Assuming differences in the function of the serotonergic inhibitory system in the two strains of rats, these data provide general support for the involvement of brain 5-HT mechanisms in modulating, if not mediating the effects of morphine.     

Intermittent naloxone attenuates the development of physical dependence on methadone in rhesus monkeys

Rhesus monkeys that received 15 daily injections of methadone (2 mg/kg i.m.) exhibited a characteristic opiate withdrawal syndrome after injection of naloxone (0.5 mg/kg i.m) on the 16th day. In comparison, injection of naloxone (0.5 mg/kg i.m.) once every 2 days during a similar 15 day methadone treatment period in these same monkeys significantly attenuated the severity of the opiate withdrawal syndrome exhibited after naloxone injection on the 16th day. Each naloxone administration during the 15 day methadone treatment period elicited an opiate withdrawal syndrome that did not significantly differ on each of the 7 days it was given and was less severe than the syndrome precipitated by naloxone following 15 days of methadone without intermittent naloxone. The lack of increments in the withdrawal response to the seven naloxone injections during the 15 days of methadone treatment and the attenuation of the withdrawal response to naloxone on day 16 after intermittent naloxone administration during the 15 day methadone treatment period support the hypothesis that naloxone modifies opiate receptor mechanisms so that they revert to an agonist-naive state following antagonist exposure. These findings suggest that various agonist and antagonist drugs opiate combinations or mixed agonist-antagonist drug could be clinically useful in the management of situations where physical dependence on opiates is a problem.     

Some relations between tolerance and physical dependence to morphine in mice

Tolerance to morphine analgesia and precipatated physical dependence were studied in mice under different conditions. There was a gradual loss of tolerance during the continous absorption of morphine from a pellet. Tolerance was decreased by nalorphine during morphine absorption. An attenuated physical dependence was observed two or four days after a single dose of morphine. In animals previously treated with pellets of morphine, single doses of morphine induced less tolerance than in mice that had never been implanted with pellets; in both cases cycloheximide prevented development of tolerance. Tolerance persisted for more than 20 days after absorption of the morphine pellet. These results reinforce the hypothesis that tolerance and physical dependence are produced by a similar mechanism and that an inhibitory process of tolerance exists.     

The production of morphine tolerance and physical dependence by the oral route in the rat

A method for the chronical administration of morphine by the oral route is discussed and compared with the production of physical dependence to morphine by injection. The method recommends the administration of Morphine HCl dissolved in a 45% sucrose syrup and given orally for 4 weeks. The initial concentration of morphine in the syrup was 1 mg/ml and was increased weekly up to 4 mg/ml at the end of the experiment. This procedure rendered the animals physically dependent on morphine as observed by drug withdrawal, when abstinence symptoms were easily identified.     

NKP608, an NK1 receptor antagonist, has an anxiolytic action in the social interaction test in rats

RATIONALE: Evidence is starting to accumulate that NK1 receptor antagonists might have anxiolytic effects in animal tests and in patients. OBJECTIVE: To examine the effects of NKP608, a substance P antagonist acting at NK1 receptors, in various conditions of the social interaction test of anxiety and to determine its effects after 3 and 6 weeks of treatment. METHODS: Rats were tested after vehicle, 0.01 or 0.1 mg/kg PO in three conditions of the social interaction test that varied in the level of anxiety generated. Thus pairs of rats were tested in an arena with which they were unfamiliar that was lit by high (HU) or low (LU) light and in the condition that generated the lowest level of anxiety, i.e. an arena with which they were familiar, lit by low light (LF). They were also tested after 3 and 6 weeks of treatment with 0.03 mg/kg and after 24 h withdrawal from these chronic treatments. RESULTS: NKP608 had significant anxiolytic effects at 0.01, 0.03 and 0.1 mg/kg PO in the HU and LU test conditions, but was without effect in the LF condition, except for an increased incidence of bite attacks at 0.1 mg/kg. The anxiolytic effect of 0.03 mg/kg remained after 3 weeks of chronic treatment and there was no anxiogenic effect after 24 h of drug withdrawal. Following 6 weeks of chronic treatment (0.03 mg/kg per day), tolerance had developed, but no anxiogenic withdrawal effect was seen 24 h after the last dose. CONCLUSIONS: These results provide further evidence that substance P may play a role in mediating states of anxiety and suggest that the selective NK1 receptor antagonist NKP608 may prove a useful anxiolytic compound.     

Acute morphine tolerance in new born and young rats

The development of tolerance after two consecutive doses of morphine administered at 4 h intervals was studied in 16, 25 and 38 day-old Sprague-Dawley rats. Tolerance was measured by the diminution of the rats' response to morphine on a hot plate (56 C). The ages selected correspond to clear stages of the brain barrier permeability to morphine. A loss of sensitivity to morphine was observed according to age. Acute tolerance developed at all ages tested, except when higher doses were employed in 25 and 38 day-old rats. When acute tolerance had developed it persisted for a long time. It is concluded that the development of acute tolerance to morphine analgesia does not depend on the stage of development of the rat.Partially supported by grants from the Gildemeister Foundation, Santiago, Chile, and from the Fondo de Investigaciones Científicas, Universidad Católica de Chile, Santiago, Chile     

The antagonizing effect of aspartic acid on the brain levels of monoamines and free amino acids during the development of tolerance to and physical dependence on morphine

Since it has been shown in a previous study that aspartic acid prevents the development of physical dependence on and tolerance to morphine and antagonizes the abstinence syndrom signs, the biochemical bases of that prevention were investigated in the present study. The brain contents of serotonin, DA, NA, and free amino acids of the rats given aspartic acid and morphine separately and in combination were determined. It has been observed that most of the morphine-induced changes in the brain were normalized in the group given aspartic acid and morphine together. The relative ineffectiveness of aspartic acid in normalizing some amino acid levels decreased by morphine was discussed and some logical explanations were found.     

Quantitative relationships among measures of morphine tolerance and physical dependence in the rat

The influence of treatment dose on a number of characteristics of opiate tolerance was studied in male Sprague-Dawley rats treated with daily intraperitoneal (IP) injections of morphine sulfate. Zero, 7.5, 15, 25 or 45 mg/kg/day was given for 34 consecutive days and the degree of morphine effect on four different tests was periodically assessed. Dose-related effects on tailflick latency (tail immersion test, 1.5 hr post injection), swimming test (2 hr post injection), and body weight gain revealed the development of tolerance; there was a non-dose-related hyperthermia (1.5 hr post injection) to which rapid sensitization occurred. All changes reached asymptote and the rate and extent of change varied with the test. Plots of log response vs test day for tailflick and swimming indicated an early steep component and a later less steep component of decline. Subsequent testing indicated that the log-dose/response (LDR) curves for tailflick latency and time to maximum hyperthermia shifted to the right by an amount dependent on the treatment dose; there was no change in the curve for hyperthermia duration. In high dose groups no further shift occurred, but the tailflick LDR curves became flattened. The tailflick LDR curve changes were replicated in rats treated for 24 days with 0, 8, 24, 48, 96, or 240 mg/kg/day. Subsequently, a constellation of withdrawal signs precipitated by naloxone HCl (1 mg/kg, IP) was measured. On the basis of the relation between treatment dose and the magnitude of the various measures, there was a parallel between analgesia tolerance and some, but not all, signs of physical dependence.     

The influence of learning on morphine analgesia and tolerance development in rats tested on the hot plate

The influence of learning on the development of tolerance to the analgesic effect of morphine in rats was examined employing the hot plate procedure. A tested-reinforced (Tr) group and its yoked-control, a tested-non-reinforced (Tnr) group, received identical exposure to the testing procedure; the Tr group was reinforced daily for its behavior on the heated plate whereas the Tnr group was reinforced only on the last day of the experiment. Paired statistical comparisons between these two groups on the last day of the experiment revealed that: 1. premorphine control reaction times on the heated plate were significantly lower in Tr than in Tnr animals; and 2. post-morphine increases in reaction time did not differ between Tr and Tnr animals. It was concluded that whereas some learning does occur in this testing procedure, learning does not influence the behavioral tolerance to morphine which develops in this analgesiometric method. An hypothesis which accommodates this behavioral tolerance and a mechanistic scheme is offered.     

Indomethacin facilitates acute tolerance to and dependence upon morphine as measured by changes in fixed-ratio behavior and rectal temperature in rats

The effects of indomethacin, a prostaglandin (PG) synthetase inhibitor, on acute tolerance to and dependence on morphine were investigated. Twelve mature, male Long-Evans rats were trained to lever press for food reinforcement on a fixed-ratio 30 schedule (FR 30 behavior) and have their rectal temperature taken. The experimental protocol began with taking the rat's temperature followed by a 30 minute behavioral session. Immediately after this session the animal was injected with indomethacin or its vehicle. Two-and-a-half hours later this procedure was repeated, except that morphine or saline was administered. After an additional 2.5 hours had elapsed, a 60 minute behavioral session occurred. Half-way through the session the rat was injected with morphine (tolerance), naloxone (dependence), or saline. Immediately after the session the rat's temperature was recorded. Indomethacin potentiated the acute tolerance to the behavioral suppressant and hyperthermic effects of morphine. Indomethacin pretreatment also greatly enhanced the capacity of naloxone to decrease temperature and suppress FR 30 behavior in morphine-treated rats. These effects were not due to indomethacin altering the acute effects of morphine or the amount of morphine in the brain. These data suggest that indomethacin is inhibiting synthesis of PGs which are important in morphine tolerance and dependence.     

The development of tolerance to morphine in the rat

Tolerance to various effects of morphine in the rat can be quantified by means of a shift of semilogarithmic dose-response curves. Tolerance to analgesia (hot plate, acetic acid writhing), catalepsy, and the tilted plane develops in a closely similar manner. Also, the stimulating effects of about 1 mg/kg morphine-HCl tested in an open-field procedure are somewhat less pronounced in chronically treated rats than in naive ones. There is no correlation between tolerance development and the acute ED₅₀ of different tests.