Morphine action in grouped and isolated rats and mice

The present study determines the analgesic effects of morphine in grouped and isolated rats and mice. Isolated animals developed altered behavioral patterns, including mouse-killing in rats and mutual aggressiveness in mice. The analgesic effect of morphine was tested by tail compression in rats and by the hot plate for mice. Isolated rats developing mouse-killing behavior had a raised pain threshold, while indifferent animals (nonkillers) responded less to morphine. Isolated mice, particularly low aggressors, gave enhanced responses to morphine.     

Naltrexone-induced conditioned place aversion following a single dose of morphine in the rat

Limited preclinical research has been conducted investigating the motivational or "affective" properties of withdrawal from acute opioid dependence following a single morphine exposure. Therefore, the purpose of the present study was to pharmacologically characterize the motivational properties associated with naltrexone-precipitated withdrawal after a single injection of morphine using place conditioning. Conditioned place aversion was assessed using a biased two-compartment apparatus and procedure. Adult male Sprague-Dawley rats were given 15 min free access to the entire apparatus on day one to determine initial preferences. Beginning on the second day, combinations of either saline or morphine (1.0-10 mg/kg, s.c.) followed by naltrexone (0.003-3.0 mg/kg, s.c.) 3.75 h later were administered. Rats were then immediately confined to one compartment for 30 min. The next day, rats received the alternative treatment and were confined to the opposite compartment. Twenty-four hours later animals were tested again for 15 min while they had access to the entire apparatus. Morphine followed by naltrexone conditioned significant place aversion (CPA) with just one pairing. This effect was a function of the naltrexone and morphine doses. CPA was also dependent on morphine pretreatment time, with significant aversion only occurring 4 h after morphine pretreatment. Finally continuous morphine administration followed by a single injection of naltrexone resulted in CPA. These data extend the range of behavioral effects associated with antagonist-precipitated withdrawal from acutely administered morphine and suggest that place conditioning is an effective model in assessing motivational aspects of withdrawal from acute opioid dependence in rats.     

Ultra-low-dose naltrexone suppresses rewarding effects of opiates and aversive effects of opiate withdrawal in rats

Rationale Ultra-low-dose opioid antagonists enhance opiate analgesia and attenuate tolerance and withdrawal. Objectives To determine whether ultra-low-dose naltrexone (NTX) coadministration alters the rewarding effects of opiates or the aversive effects of opiate withdrawal. Methods We used the conditioned place preference (CPP) and conditioned place aversion (CPA) paradigms to assess whether ultra-low-dose NTX alters the acute rewarding effects of oxycodone or morphine, or the aversive aspect of withdrawal from either drug. To assess the dose response for ultra-low-dose NTX, a range of NTX doses (0.03–30 ng/kg) was tested in the oxycodone CPP experiment. In order to avoid tolerance or sensitization effects, we used single conditioning sessions and female rats, as females are more sensitive to the conditioning effects of these drugs. Results Ultra-low-dose NTX (5 ng/kg) blocked the CPP to morphine (5 mg/kg) and the CPA to withdrawal from chronic morphine (5 mg/kg, for 7 days). Coadministration of ultra-low-dose NTX (30 pg/kg) also blocked the CPA to withdrawal from chronic oxycodone administration (3 mg/kg, for 7 days). The effects of NTX on the CPP to oxycodone (3 mg/kg) revealed a biphasic dose response. The two lowest doses (0.03 and 0.3 ng/kg) blocked the CPP, the middle dose (3 ng/kg) was ineffective, and oxycodone combined with the highest dose (30 ng/kg) produced a trend toward a CPP. Conclusions Ultra-low-dose NTX coadministration blocks the acute rewarding effects of analgesic doses of oxycodone or morphine as well as the anhedonia of withdrawal from chronic administration.     

Conditioned flavor aversions for assessing precipitated morphine abstinence in rats.

Rats receiving twice-daily morphine injections acquired aversions to a saccharin solution which had been presented for 1 hr immediately prior to injections of naloxone. The degree of aversion was related to the maintenance dosage of morphine. Rats maintained on a regimen of daily saline injections did not show significant aversion to saccharin paired with naloxone, even at doses as high as 40 mg/kg. The sensitivity of the technique was such that significant aversions could be demonstrated in rats receiving doses of morphine as low as 1 mg/kg twice daily. It is suggested that conditioned flavor aversions provide a useful method for assessing the aversive quality of abstinence precipitated from low doses of morphine.     

Levorphanol but not dextrorphan suppresses the foot-lifting response to an aversive thermal stimulus in the terrestrial snail, Cepaea nemoralis

The terrestrial snail, Cepaea nemoralis, when placed on a surface heated at 40 degrees C lifts the anterior portion of its foot from the source of heat. This stereotyped response, which suggests aversion, can be inhibited by injections of small doses of morphine and levorphanol, but is not affected by similar or larger doses of dextrorphan. It is suggested that the "analgesic" effect of the opiates in these animals involves interaction with stereochemically specific opiate receptors that may be fundamentally similar to those occurring in mammals.     

Cross-tolerance between morphine- and nicotine-induced conditioned place preference in mice

The acquisition of morphine and nicotine conditioned place preference (CPP) and cross-tolerance between the response of two drugs was studied in mice. A biased CPP paradigm was used to study the effect of the agents. Morphine (5 mg/kg) and nicotine (1 mg/kg) induced CPP. Naloxone (0.5, 1 and 2 mg/kg), but not mecamylamine (0.025, 0.05 and 0.1 mg/kg), induced conditioned place aversion (CPA). Both antagonists reversed CPP induced by morphine and nicotine. Administration of one daily dose of morphine (12.5, 25 or 50 mg/kg) for 3 days or nicotine (0.5, 1 or 2 mg/kg) three times a day for 12 days, in order to develop tolerance to the drugs, reduced the conditioning induced by morphine (5 mg/kg) or nicotine (1 mg/kg). CPA-induced by naloxone was reduced in animals, which were rendered tolerant to morphine (50 mg/kg) or nicotine (2 mg/kg). Mecamylamine, however, which did not induce any response in the nontolerant mice, elicited CPP in the tolerant animals. It is concluded that there may be a cross-tolerance between morphine- and nicotine-induced CPP.     

Modification of morphine-induced analgesia, tolerance and dependence by bromocriptine

The effect of two doses of bromocriptine, a dopamine agonist, on morphine-induced analgesia, tolerance and dependence was investigated in mice. Bromocriptine at doses of 0.04 and 0.08 mg/kg did not affect the baseline tail flick latency of mice but potentiated the morphine analgesia. Pretreatment of mice with 5 mg/kg of sulpiride, a D-2 antagonist, not only blocked the effect of 0.08 mg/kg of bromocriptine but also antagonized the morphine analgesia. Control animals given daily injections of 10 mg/kg of morphine rapidly developed tolerance to the analgesic effect. A combined treatment of bromocriptine with morphine given daily suppressed the development of tolerance to morphine analgesia. However, development of tolerance to morphine analgesia was not significantly modified in the animals treated daily with bromocriptine (0.08 mg/kg) plus sulpiride (5 mg/kg). Acute dependence was induced by the administration of 100 mg/kg of morphine. The administration of bromocriptine 30 min before naloxone significantly decreased the ED₅₀ value for naloxone for inducing jumping in mice. Coadministration of sulpiride and bromocriptine attenuated the ability of bromocriptine to potentiate the withdrawal syndrome of morphine dependence. The results indicate that bromocriptine potentiates morphine analgesia, suppresses the development of tolerance to morphine analgesia but exacerbates opiate withdrawal signs in morphine-dependent mice. These effects of bromocriptine appear to be mediated via D-2 receptors.     

Conditioned place aversion is a highly sensitive index of acute opioid dependence and withdrawal

Rationale Conditioned place aversion (CPA) is known to be a sensitive measure of the aversive motivational state produced by opioid withdrawal in rats made chronically dependent on opioids. Objective The purpose of the present study was to examine the sensitivity of the CPA model in detecting a possible aversive state associated with naloxone-precipitated withdrawal from acute treatment with morphine. Methods Doses of morphine and naloxone, as well as number of conditioning trials, were systematically varied to determine the minimum conditions that would result in a detectable CPA in male Wistar rats. Naloxone (0.003–16.7 mg/kg) was administered 4 h after an injection of vehicle or morphine (1.0, 3.3, or 5.6 mg/kg) and immediately prior to confinement to one compartment of the conditioning apparatus; rats received either one or two such naloxone-conditioning trials (separate by 48 h). Results Morphine (5.6 mg/kg) followed 4 h later by vehicle produced no significant preference or aversion. In morphine-naive rats, 10 mg/kg naloxone was required to produce a significant CPA with two cycles of conditioning. When increasing doses of morphine were administered (1.0, 3.3, 5.6 mg/kg), significant increases in naloxone potency to elicit a CPA were observed (16-, 211-, and 1018-fold potency shifts, respectively). Naloxone potency after two pretreatments with 5.6 mg/kg morphine was comparable to its potency to elicit a CPA after chronic exposure to morphine. Although naloxone was still effective in producing a CPA after a single conditioning cycle (and hence a single morphine exposure), its effects were dramatically reduced relative to those seen with two conditioning cycles. Conclusions CPA is a reliable and sensitive index of the aversive motivational state accompanying withdrawal from acute opioid dependence.     

Prenatal cocaine exposure alters spontaneous and cocaine-induced motor and social behaviors

The abuse of cocaine in pregnant women could affect emotional behaviors in their descendents. The aim of this work was to evaluate the effects of prenatal cocaine exposure on spontaneous and cocaine-induced motor and social behaviors in mice. Three kinds of prenatal treatment were used: non-treated animals; mice treated daily with physiological saline during the last week of pregnancy; and finally, those treated with cocaine (25 mg/kg) during the same period. Behavioral studies took place on adult males, which were housed in two different conditions: grouped (non-aggressive), or isolated (aggressive). Cocaine-pretreated animals exhibited slight differences in spontaneous motor activity, but alterations in their social relationship with conspecifics were presented, with decreases in isolated but increases in grouped mice. The cocaine challenge increased aggression specifically in grouped prenatally cocaine-treated mice, but increases in motor activity or avoidance and flee behavior were presented in those animals pretreated with either saline or cocaine. Isolated saline-or cocaine-treated animals exhibited greater concentrations of DA and DOPAC than those grouped. A decrease in 5-HIAA concentrations was presented in pretreated animals, irrespective of their housing conditions. In conclusion, cocaine administration during pregnancy induces long lasting effects on the offspring, for both behavioral abnormalities and cocaine response, which last to adult life.     

GHB ameliorates naloxone-induced conditioned place aversion and physical aspects of morphine withdrawal in mice

Rationale Gamma-hydroxybutyric acid (GHB) is a naturally occurring substance in the brain, the administration of which has proved useful in the treatment of the opiate withdrawal symptoms in humans.Objectives The aim of the present work was to validate this beneficial effect on the physical and motivational aspects of morphine withdrawal in mice.Methods In a first experiment, animals rendered morphine-dependent were conditioned to develop a place aversion (CPA) to the compartment paired with naloxone administration in a two-chamber apparatus. The conditioning phase consisted of three pairings of either naloxone (0.250 mg/kg) or vehicle in one compartment, both with similar time allotments during the preconditioning test. During the testing phase, mice were again allowed to explore the entire apparatus. GHB (6, 12.5, 25, and 50 mg/kg) was administered during either the acquisition or expression phase of this conditioning. In a second experiment, the capacity of GHB to ameliorate the intensity of physical signs of morphine withdrawal was evaluated.Results GHB blocked CPA in both phases: administered during acquisition (from 12.5 mg/kg and higher) as well as in the expression phase (from 6 mg/kg, except for 25 mg/kg). It also decreased the intensity of physical signs of morphine withdrawal to near control levels measured by the modified Gellert–Holtzman scale (25 mg/kg and higher). Decreases in jumping, body shakes, and paw tremor were also observed.Conclusions Our results support the idea that GHB ameliorates both aspects of morphine withdrawal, physical as well as motivational signs.     

Inhibition of the cyclooxygenase pathway attenuates morphine-induced conditioned place preference in mice

Prostanoids are shown to be important lipid mediators, not only in periphery but also in the brain, where they appear to modulate synaptic transmission. Recent studies have demonstrated that cyclooxygenase (COX) pathway might modulate the neurotransmission of gamma-aminobutyric acid and dopamine in the central nervous system. In this study, we have evaluated the effects of indomethacin (a non-selective COX inhibitor) and celecoxib (a selective COX-2 inhibitor) on the acquisition of morphine-induced conditioned place preference (CPP) in male Swiss mice. Our data shows that morphine (2.5-7.5 mg/kg) induces place preference conditioning in a dose-dependent manner. Celecoxib (0.01-5 mg/kg) and indomethacin (1 mg/kg) fail to produce a significant CPP or conditioned place aversion (CPA); however, higher doses of celecoxib (10 mg/kg) and indomethacin (5 mg/kg) induce CPA. Co-administration of celecoxib (0.5-5 mg/kg) or indomethacin (1-5 mg/kg) with morphine during the conditioning phase, blocked the acquisition of morphine CPP. These results indicate that the reward properties of morphine can be modulated by inhibiting COX activity in mice.     

SEX DIFFERENCES AND THE EFFECT OF GONADECTOMY ON MORPHINE-INDUCED ANTINOCICEPTION AND DEPENDENCE IN RATS AND MICE

1. Differences between sexes and the effect of bilateral surgical gonadectomy on the response to morphine analgesia and dependence were examined in rats and mice. 2. The analgesic response to morphine (5 mg/kg) was assessed by the hot plate and the abdominal constriction tests. Dependence was induced by injecting morphine at increasing doses (5–160 mg/kg) for 6 consecutive days and withdrawal signs elicited by injecting naloxone (2.5 mg/kg). 3. The base line reaction times in the intact control, shamoperated and gonadectomized animals of either sex were not significantly different from each other. 4. After treatment with morphine, the percentage increase in the reaction time in gonadectomized male and female rats and in gonadectomized male mice was significantly higher than in their respective controls. 5. The increase in the reaction time, after morphine treatment, was significantly higher in gonadectomized female rats than in gonadectomized male rats. 6. Naloxone-induced withdrawal signs in morphine-dependent gonadectomized rats and mice were not significantly different from those in sham-operated controls. However, female rats in both groups exhibited a significantly higher number of escape jumping responses than males.     

Constitutively active micro opioid receptors mediate the enhanced conditioned aversive effect of naloxone in morphine-dependent mice.

Naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at mu opioid receptors. This aversive response is potentiated by prior exposure to morphine. In vitro studies indicate that morphine treatment may promote constitutive activity of mu opioid receptors. We hypothesized that such enhanced constitutive activity in vivo may underlie the increased aversive property of naloxone by uncovering the inverse agonist property of this drug. The CPA produced by naloxone was compared with that produced by the neutral antagonists 6-alpha- and 6-beta-naloxol in mice with and without prior morphine exposure. While all three drugs produced CPA, only naloxone CPA was enhanced by morphine given 20 h prior to each naloxone injection. Furthermore, only naloxone produced withdrawal jumping when given 20 h after morphine, even though 6-alpha-naloxol was able to produce jumping when given 4 h after morphine. These data suggest that morphine may enhance naloxone CPA by increasing levels of constitutively active mu receptors and further support the role of such constitutive activity in mediating naloxone-precipitated physical withdrawal. Such long-term changes in constitutive activity of the mu receptor induced by exogenous opiate exposure may thus be an important factor in hedonic homeostatic dysregulation proposed to underlie the addictive process.     

Involvement of the amygdala on place aversion induced by naloxone in single-dose morphine-treated rats

Signs characteristic of opiate withdrawal symptoms can be precipitated by an opiate antagonist after short-term infusion or even a single dose of an opiate both in humans and in animals. This phenomenon has been referred to as acute dependence. In contrast to extensive studies on chronic dependence, less is known about the neural mechanisms mediating acute dependence. It will benefit the development of appropriate therapies to facilitate opiate abstinence and reduced craving to better understand the mechanisms underlying acute opiate dependence and to determine whether there are dissociation and similarity between the early and fully developed stages of dependence. In the present study, we examined the influence of c-Fos expression in the amygdala in acquisition of conditioned place aversion (CPA) induced by naloxone-precipitated withdrawal from a single morphine exposure 24 h earlier. The effect of microinjection into the central amygdaloid nucleus (CeA) of various kinds of glutamatergic neurotransmission inhibitors was also investigated. Findings showed that CeA displayed significant increase in c-Fos expression in the acquisition of CPA. Furthermore, CPA was attenuated significantly and dose-dependently by microinjection into CeA of all glutamatergic neurotransmission inhibitors (NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine maleate (MK-801), AMPA receptor antagonist 1-(4-aminophenyl)4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI52466), metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), and glutamate release inhibitor riluzole). These findings suggest that CeA involves the acquisition of CPA induced by naloxone-precipitated withdrawal from a single morphine exposure, and the function of the glutamatergic system projected from the amygdala to nucleus accumbens plays a facilitative role in formation of morphine dependence.     

The effect of morphine pretreatment on the sensitivity of mouse and guinea-pig ileum to acetylcholine and to morphine

Guinea-pigs and mice were subjected to morphine pretreatment for at least 14 days culminating in subcutaneous injections of 1 g/kg daily in 2 divided doses. In mice, tolerance to the effects of morphine on gastrointestinal propulsion had developed by the end of the pretreatment course. The sensitivity to acetylcholine of the ilia from pretreated animals and the effects of morphine on transmural stimulation were compared with the ilia from normal animals. Ilia from pretreated guinea-pigs were marginally more sensitive to frequency of stimulation but significantly less sensitive to the depressant effect of morphine than ilia from normal animals. There was no significant difference in the sensitivity to acetylcholine. There was a highly significant decrease in sensitivity to morphine of ilia from pretreated mice, but this also was not associated with any alteration in sensitivity to acetylcholine. It is concluded that on the isolated ileum preparation the development of tolerance to morphine is not associated with an increased sensitivity to the transmitter whose release morphine inhibits.     

Isolation housing decreases the effectiveness of morphine in the conditioned taste aversion paradigm

Male Long Evans rats were obtained at 21 days of age and were housed in either an aggregated (four per double cage) or isolated (one per single cage) condition for 6 weeks. They were then placed on a fluid deprivation schedule that allowed them access to fluids for 20 min daily. This schedule was maintained for the remainder of the experiment. Following habituation, sensitivity to morphine-induced conditioned taste aversion (CTA) was compared in the differentially housed rats. On the 1st day and every 5 days thereafter the rats were presented with a 0.1% solution of sodium saccharin for the 20-min drinking period, followed immediately by an injection of morphine (0, 2.5, 5.0, 10.0, or 20.0 mg/kg). On intervening days they received water as the fluid. No drugs were given on these days. There was no difference in baseline saccharin consumption as a function of housing condition. In comparison with the isolated rats, the grouped animals were more sensitive to the CTA-inducing properties of low doses of morphine. These data strengthen the already existing evidence for the influence of the early housing environment on drug sensitivity and provide additional support for the conclusion that variability in response to a number of drugs of abuse can be reduced by environmental means. Possible mechanisms for the differences between isolation and aggregation housed rats are discussed.     

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.     

Influence of isolation and of fighting on adrenal tyrosine hydroxylase and phenylethanolamine-N-methyltransferase activities in three strains of mice

Mice (BALB/C or NIH, C57 BR/cdJ and A/HeJ) were isolated for 2 weeks then exposed to another mouse for 10 min daily for 1 week. Isolated, unexposed and grouped controls were also studied. Aggression, defined as stereotyped attack behavior, was produced only in the NIH mice which were subsequently used as aggressors. When attacked, the victims, C57 BR/cdJ mice defended themselves whereas A/HeJ mice remained submissive.Isolation alone did not alter adrenal medullary levels of tyrosine hydroxylase (TOH), phenylethanolamine-N-methyltransferase (PNMT) or catecholamines. NIH mice showed an increase in TOH but not in PNMT when they fought with NIH or C57 BR/cdJ mice, whereas both victims showed increases in TOH and PNMT. It was concluded therefore that fright or active fighting induces increases in adrenal medullary enzymes.Drugs were injected into C57 BR/cdJ mice at doses which did not impair their defense behavior, using untreated NIH mice as aggressors. Phenobarbital fully inhibited both TOH and PNMT rise while chlorpromazine only partly suppressed PNMT increase. Methamphetamine was less effective than phenobarbital. Tranylcypromine increased both enzymes in control animals and partly suppressed TOH rise induced by defense.Thus, a reliable model of mild stress was produced which appears suitable for screening psychoactive drugs.     

Differential sexual activity of isolated and group-housed male mice: Influence of acute d-Amphetamine sulfate administration

It is established that group housing can impair sexual activity of male mice, and that central catecholamines are involved in male sexual response, but it is not known whether catecholamine mechanisms are involved in sexual impairment in grouped males. Injections of 0, 0.22, 0.67, 2.0, or 6.0 mg/kg of d-amphetamine sulfate were administered 1 h before testing to individually and group-housed male C57BL/6J mice. Isolated mice showed more mounts, intromissions, and ejaculations and shorter response latencies than did group-housed mice. The latencies to first mount, intromission, and ejaculation were nonmonotonically related to dosage, being shortest at the lowest dosage in isolated mice, but significantly elevated by the higher dosages in both isolated and grouped males. The number of ejaculations was significantly elevated by moderate dosages in both isolated and grouped mice, peaking at the 2.0 mg/kg dosage in isolated mice and at 0.67 mg/kg in grouped mice. Nevertheless, amphetamine treatment generally failed to eliminate differences between males from isolated and grouped backgrounds.