Chronic administration of nandrolone increases susceptibility to morphine dependence without correlation with LVV-hemorphin 7 in rats.

LVV-hemorphin 7 (LVVYPWTQRF; LVV-H7), an N-terminal fragment of the ß-chain of hemoglobin cleaved by cathepsin D/pepsin, is an atypical endogenous opioid peptide that is found in high concentration in blood. LVV-H7 acts as a µ-opioid agonist and an inhibitor of insulin-regulated aminopeptidase. Subchronic administration of anabolic androgenic steroids (AAS) has been clinically proven to induce the synthesis of erythrocytes and increase hemoglobin concentrations. Patients with a history of AAS abuse are more susceptible to opioid abuse. We hypothesized that this association could be at least partially attributed to the sensitization of the mesocorticolimbic dopaminergic pathway by LVV-H7. Using the conditioned place preference test and neurochemical analysis, we investigated the possible mechanism underlying the effect of chronic nandrolone administration on morphine-induced reward and its correlation with LVV-H7 in rats. Either LVV-H7 may not sensitize the rewarding neural circuits or its inhibition on locomotor activity could mask reward-related behaviors. Chronic nandrolone pretreatment indeed caused a significant reward by low dose morphine, which did not cause any reward in control rats. However, coadministration of anti-LVV-H7 antiserum with nandrolone did not block this effect. This may rule out the possibility of the involvement of LVV-H7 in the action of nandrolone to intensify morphine-induced reward. Moreover, the serum level of LVV-H7 was mildly increased in response to chronic nandrolone administration in our animal model. According to the current clinical observations, we may conclude that the chronic administration of nandrolone can increase susceptibility to morphine dependence, but that this effect is not related to elevated LVV-H7.

Morphine and nicotine addiction and withdrawal influence baroreflex sensitivity and blood pressure in two-kidney one clip hypertensive (2K1C) rats.

OBJECTIVE: This study aimed to investigate the effects of addiction to morphine and nicotine as well as their withdrawal on both baroreflex sensitivity and blood pressure in hypertensive rats. METHODS: In this experimental study 40 male rats were divided into two main groups as follows: in group I, hypertensive rats received saline for 8 weeks; in group II, hypertensive rats were treated with morphine and nicotine for 8 weeks. At the end of 8 weeks group II rats were divided into four sub-groups including, 3 sub-groups of those were put on drug withdrawal protocol. At the end of experiment, blood pressure, heart rate, plasma renin activity (PRA), serum NO concentration and baroreflex sensitivity (BRS) were measured. RESULTS: RESULTS demonstrated that BP and BRS were significantly lower in addicted to morphine and nicotine hypertensive rats compared to control (p < 0.05). Addiction withdrawal (in morphine and nicotine withdrawal rats) completely reversed BP and BRS to the pre-addiction levels (p < 0.05). Withdrawal in the only nicotine treated group lowered BP and BRS compared to group that had received morphine and nicotine together (p < 0.05). CONCLUSION: RESULTS of current study may propose simultaneous morphine and nicotine withdrawal can prevent cardiovascular complications raised due to withdrawal (Fig. 5, Ref. 58).

Codeine to morphine concentration ratios in samples from living subjects and autopsy cases after incubation.

The codeine to morphine concentration ratio is used in forensic toxicology to assess if codeine has been ingested alone or if morphine and/or heroin have been ingested in addition. In our experience, this interpretation is more difficult in autopsy cases compared with samples from living persons, since high morphine concentrations are observed in cases where only codeine is assumed to have been ingested. We have investigated if codeine and morphine glucuronides are subject to cleavage to the same extent in living and autopsy cases in vitro. We included whole blood samples from eight living subjects and nine forensic autopsy cases, where only codeine ingestion was suspected. All samples were incubated for 2 weeks at 37°C and analyzed for codeine and six codeine metabolites using liquid chromatography tandem mass spectrometry. A reduction in the codeine to morphine concentration ratio was found, both in samples from living subjects (mean 33%, range 22-50%) and autopsy cases (mean 37%, range 13-54%). The increase in the morphine concentrations was greater in the autopsy cases (mean 85%, max 200%) compared with that of the living cases (mean 51%, max 87%). No changes were seen for codeine or codeine-6-glucuronide concentrations. The altered ratios might mislead the forensic toxicologist to suspect morphine or heroin consumption in cases where only codeine has been ingested.

A combined liquid three-phase micro-extraction and differential pulse voltammetric method for preconcentration and detection of ultra-trace amounts of buprenorphine using a modified pencil electrode.

A combination of polytetrafluorethylene membrane-based liquid three-phase micro-extraction and voltammetry was used for the micro-separation and determination of buprenorphine. Type of the organic solvent used, pH levels of the donor and acceptor phases, salt concentration, extraction time, stirring rate, and electrochemical parameters as the essential factors affecting the liquid three-phase micro-extraction of buprenorphine were investigated. Differential pulse voltammetry exhibited two linear dynamic ranges of 1.0-109.0 pmol L(-1) and 0.109 nmol L(-1)-0.11 µmol L(-1) of buprenorphine and the detection limit was found to be as low as 0.6 pmol L(-1) of buprenorphine. Also, the effects of a number of common substances potentially interfering with selectivity were studied. The results indicate that the proposed method is highly selective and sensitive for buprenorphine detection in real samples such as human urine and plasma of both drug-addict and non-addict human subjects.

Exposure to morphine affects the expression of endocannabinoid receptors and immune functions.

Compared with control rats, rats under morphine exposure exhibited cannabinoid receptor 2 (CB2-R) upregulation in the spleen and periphery blood mononuclear cells (PBMCs). IgG and IgM values in the plasma were also altered. In morphine abusers, cannabinoid receptors were upregulated in the PBMCs, and the expression of IL-4 mRNA in the PBMCs as well as the IgG and IgM values in the plasma were higher compared with those in healthy people. The expression of cannabinoid receptor 1 and CB2-R in culture cells was directly affected by morphine treatment. These findings indicate that the alteration in cannabinoid receptor expression could be disturbed by morphine exposure, and it may be involved in abnormal immune function.

Impaired emotional-like behavior and serotonergic function during protracted abstinence from chronic morphine.

BACKGROUND: Opiate abuse is a chronic relapsing disorder, and maintaining prolonged abstinence remains a major challenge. Protracted abstinence is characterized by lowered mood, and clinical studies show elevated comorbidity between addiction and depressive disorders. At present, their relationship remains unclear and has been little studied in animal models. Here we investigated emotional alterations during protracted abstinence, in mice with a history of chronic morphine exposure. METHODS: C57BL6J mice were exposed to a chronic intermittent escalating morphine regimen (20-100 mg/kg). Physical dependence (naloxone-precipitated withdrawal), despair-related behaviors (tail suspension test), and social behaviors were examined after 1 or 4 weeks of abstinence. Stress hormones and forebrain bioamine levels were analyzed at the end of morphine regimen and after 4 weeks of abstinence. Finally, we examined the effects of chronic fluoxetine during abstinence on morphine-induced behavioral deficits. RESULTS: Acute naloxone-induced withdrawal was clearly measurable after 1 week, and became undetectable after 4 weeks. In contrast, social and despair-related behaviors were unchanged after 1 week, but low sociability and despair-like behavior became significant after 4 weeks. Chronic morphine regimen increased both corticosterone levels and forebrain serotonin turnover, but only serotonergic activity in the dorsal raphe remained impaired after 4 weeks. Remarkably, chronic fluoxetine prevented depressive-like behavioral deficits in 4-week abstinent mice. CONCLUSIONS: During protracted abstinence, the immediate consequences of morphine exposure attenuate, whereas fluoxetine-sensitive emotional alterations strengthen with time. Our study establishes a direct link between morphine abstinence and depressive-like symptoms and strongly suggests that serotonin dysfunction represents a main mechanism contributing to mood disorders in opiate abstinence.

Social influences on plasma testosterone levels in morphine withdrawn adolescent mice and their drug-naïve cage-mates.

Opioid administration in males results in opioid-induced androgen deficiency which persists throughout the treatment. In adults, this quickly reverses once opioid administration is suspended. However, less is known about the duration of the effect following drug discontinuation in adolescents. Given the significant implications to sexual maturation in adolescent males, this study examined plasma testosterone levels in both morphine withdrawn mice and their drug-naïve (saline-injected) cage-mates as compared to drug-naïve mice housed physically and visually separate from the morphine-treated mice ('saline only'). Consistent with the literature, plasma testosterone levels in morphine withdrawn adults were reduced on withdrawal day 1 (WD1) and returned to baseline levels by WD9. No significant effects were observed in their saline cage-mates. In the adolescents, no significant differences were observed on WD1 between the morphine withdrawn mice, their saline cage-mates, and the saline only mice - all of which had significantly lower plasma testosterone levels than adults. By WD9, testosterone levels in the saline only adolescent mice had reached adult levels. Notably, plasma testosterone levels were reduced in both the morphine withdrawn adolescent mice and their saline cage-mates, as compared to saline only mice. The effect was not a drug effect per se, given that reduced plasma testosterone levels were not observed in individually housed morphine withdrawn mice. Moreover, our results also suggest that these social effects are not solely explained by stress. These results have numerous implications to the short term and long term health of both adolescents requiring pain management and of adolescent drug addicts.

Nitric oxide and renal protection in morphine-dependent rats.

Morphine treatment for 5 days protects heart against ischemia-reperfusion (IR) injury. This study evaluated the involvement of nitric oxide (NO) in morphine-induced renal protection. Three weeks after right nephrectomy, increasing doses of morphine were administered (20-30 mg kg(-1)day(-1), 5 days) to develop dependence in rats. The left kidney underwent 45-min ischemia and 24-h reperfusion. Some rats were pretreated with naloxone (5 mg kg(-1)) or L-NAME (20 mg kg(-1)). In one group, IR was induced 24h after the last dose of morphine during the withdrawal period. Plasma nitrite/nitrate levels and serum creatinine and BUN were measured. Creatinine clearance and fractional excretion of sodium (FE(Na)) were calculated. Myeloperoxidase (MPO) activity, malondialdehyde (MDA) level, and inducible NO synthase (iNOS) expression were determined and histopathology was studied in the left kidney. IR increased serum creatinine and BUN, plasma NO (p<0.01), FE(Na), iNOS expression (p<0.001), MPO activity, MDA level, and tissue damage and decreased creatinine clearance. Morphine decreased plasma NO (p<0.05 vs IR), serum creatinine and BUN (p<0.01), FE(Na), MPO activity, MDA level, iNOS expression, and tissue damage (p<0.05), but increased creatinine clearance (p<0.05). Pretreatment with naloxone significantly increased NO production and iNOS expression in morphine-treated rats after IR (p<0.01 vs morphine dependence+IR). Pretreatment with L-NAME in morphine-treated rats decreased NO production (10.7+/-1.9, p<0.01 vs morphine dependence+IR) but could not change iNOS expression after IR. Both naloxone and L-NAME significantly abolished the protective effects of morphine dependence on functional and histological factors. The protective effect of morphine dependence on serum creatinine, BUN, FE(Na), and creatinine clearance persisted during the withdrawal period, whereas iNOS expression decreased. NO production was not decreased during the withdrawal period (p>0.1 vs morphine dependence+IR group). Morphine dependence provided renal protection in the acute phase and during withdrawal. Excessive increase or decrease in NO production abolished the effects of morphine, which suggested a role for balanced NO production and iNOS expression.

Flexible dosing of tincture of opium in the management of opioid withdrawal: pharmacokinetics and pharmacodynamics.

AIMS: The aim was to evaluate the clinical effectiveness, pharmacodynamics and pharmacokinetics of a range of Tincture of Opium (TOP) doses in the management of opioid withdrawal. METHODS: Forty-five opium-dependent Thai subjects were allocated to three dosing groups (6.66, 13.3 and 20 mg morphine equivalents, twice daily) depending on their self-reported prior opium use. On day 5 of dosing subjects underwent an interdosing interval study where blood, withdrawal scores, heart rate and blood pressure (BP) were collected at 0, 1, 3 and 8 h. Plasma morphine concentrations were quantified by high-performance liquid chromatography, and plasma morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) concentrations by LCMS. RESULTS: Thirty-two subjects completed the study. Withdrawal scores were low for all subjects (range 9-23% of maximum response). There were dose-dependent changes in both systolic and diastolic BP (P = 0.021 and P = 0.01, respectively), but these were not considered clinically significant. There were no effects of dose on respiratory rate. Plasma morphine concentrations changed significantly across the interdosing interval (P = 0.0001), rising to a maximum at 1 h after dosing. Plasma morphine concentrations also differed according to dose (P < 0.05). The mean ratios of the morphine glucuronides were found to be: M3G/M6G = 7.7, M3G/morphine = 35.6 and M6G/morphine = 4.9, values comparable to those previously reported. CONCLUSION: The management of opioid withdrawal can be achieved, with minimal adverse effects, by using flexible dosing of TOP.

Morphine blood levels, dependence, and regulation of hippocampal subgranular zone proliferation rely on administration paradigm.

Chronic morphine, administered via s.c. pellet, decreases the number of proliferating cells in the dentate gyrus subgranular zone (SGZ) in both rats and mice. This robust morphine-induced decrease could be used to better understand mechanisms regulating adult hippocampal neurogenesis, as well as to explore the relationship between neurogenesis and drug dependence, withdrawal, and relapse behaviors. Such research would benefit enormously from identifying a route of morphine administration that produces addiction-relevant blood levels of morphine, results in a high degree of dependence, translates to both rat and mouse, and is free of the behavioral confounds of s.c. pellets. Therefore, we examined a classic chronic morphine pellet paradigm (two s.c. pellets over 5 days) versus three chronic morphine injection paradigms (escalating dose i.p. injections over 2, 5, or 10 days) for their effect in adult male C57BL/6J mice. We assessed blood morphine levels, SGZ proliferation, and drug dependence as assessed by tolerance to locomotion sensitization and naloxone-precipitated withdrawal. The pellet paradigm produced high and relatively stable blood levels of morphine, a high degree of dependence, and a significant decrease in SGZ proliferation. In contrast, the three injection paradigms produced transient spikes in morphine blood levels, significantly less dependence than the pellet paradigm, and no significant decrease in SGZ proliferation. These data show that regulation of mouse SGZ proliferation requires high and relatively stable blood levels of morphine, and provide critical knowledge for the design of future studies to probe the relationship between addiction and neurogenesis.

Pharmacokinetic aspects of naloxone-precipitated morphine withdrawal in male and female prepubertal mice.

It has been shown that the expression of the morphine (MOR) withdrawal syndrome precipitated by naloxone (NAL) is more intense in male mice than in females, but the reasons for this phenomenon remain uncertain. The purpose of the present study was to evaluate whether this sexual dimorphism might be due to differences in MOR and/or NAL plasma levels after a chronic treatment with MOR. Prepubertal Swiss male and female mice were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg), twice daily for 9 days. On day 10 dependent mice received NAL (6 mg/kg, i.p.) 60 min after MOR injection. Blood samples were taken at different times in order to determine MOR and NAL plasma levels by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC), respectively. Pharmacokinetic analysis showed no differences between male and female mice either for MOR or for NAL. In conclusion, although males and females respond differentially to NAL-precipitated withdrawal, this dimorphic behavior would not be influenced by a pharmacokinetic factor.

Area-specific increased density of mu-opioid receptor immunoreactive neurons in the cerebral cortex of drug-related fatalities.

In animal experiments and in cell culture, chronic morphine treatment has been followed by "up-regulation" as well as "down-regulation" of the mu-opioid receptor (OR) number. The present postmortem morphometric study of morphine-related fatalities of drug-addicts (n=13, 20-35 years old, with blood unconjugated morphine levels from 27.1 ng/ml to 458 ng/ml, m.v. 198.5 ng/ml) versus a non-addicted control group (n=13, 10-44 years old) was intended to examine, whether chronic opiate exposure affects the numerical density of mu-OR expressing neurons in the human neocortex (areas 11, 24 and 25 according to Brodmann). For the immunohistochemical procedure, vibratome sections (100 microm) were incubated with a monoclonal antibody against the mu-OR, diluted 1:100, and immunolabelled sites were visualized using an immunoperoxidase protocol. The numerical densities of OR immunoreactive neuronal profiles and Nissl-stained central profiles were assessed morphometrically (camera lucida-drawings). In both groups, the anti-mu-OR-immunoreactivity was mainly localized in pyramidal neurons of layers (L) II/III and V and in multiform neurons of L VI. In the areas 24 and 25, the density of the immunoreactive neuronal profiles did not display a significant difference between the two examined groups. In the area 11, however, the number of immunolabelled neuronal profiles amounted to 2777+/-206 mm(3) in the drug-related fatalities and to 2320+/-124 mm(3) in the control group and thus was significantly increased.

[Inhibitory effect of melatonin on morphine withdrawal syndromes and the content of NO in plasma and brain tissue in morphine dependent mice].

AIM: To observe the effect of melatonin (MT) on morphine withdrawal syndromes and determine the content of NO in plasma and brain tissue in morphine dependent mice. METHODS: A physical dependent model in mice was established by subcutaneous injection of morphine. MT (15 mg.kg-1, qd x 3) was given by intragastric infusion (ig) for three days. Withdrawal syndromes were induced by intraperitoneal injection of naloxon (5 mg.kg-1). The intensity of withdrawal syndromes was evaluated according to the jumping latency, the jumping times and the body weight loss. The content of NO was detected with Griess method. RESULTS: The jumping latency of morphine withdrawal reaction was prolonged and the jumping times were reduced obviously by ig MT. The increased NO content in plasma and brain tissue in morphine dependent mice was reduced by ig MT. CONCLUSION: The physical withdrawal syndromes and the content of NO in plasma and brain tissue in morphine dependent mice are inhibited by MT.

Effects of morphine on T-cell recirculation in rhesus monkeys.

A 2-yr study on effects of morphine on lymphocyte circulation in rhesus monkeys (Macaca mulatta) showed that, over time, a well-maintained morphine-dependency caused biphasic depressive effects on circulating lymphocyte levels. Depression of T cell circulation by opiates actually was a relative effect. Morphine exposure basically stabilized T cell circulation in the context of concurrent increases in controls. Biphasic effects of morphine were attributable to distinctions in circulation kinetics of CD4+/CD62L (+ & -) T cells. That is, levels of CD4+/CD62L+ T cells were selectively depressed by opiates through the first 32wk after initiation of drug, and levels of CD4+/CD62L- T cells were selectively depressed thereafter. Regression analyses also showed that morphine stabilized lymphocyte recirculation. Circulating levels of resting and activated-memory types of T cells were positively correlated in opiate-exposed monkeys during the first 32wk after opiate exposure--an effect not seen with control monkeys. Considerations of changes in the types of experimental stressors extant during the study suggested that temporally differential effects of opiates on T cell recirculation were connected with changes in the stress environment and the ability of morphine to modulate these changes. Thus, morphine, and by inference the endogenous opioid system, are involved in homeostasis of lymphocyte recirculation, probably through effects on central mediation of the stress axis.

Numerical density of mu opioid receptor expressing neurons in the frontal cortex of drug related fatalities.

In animal and cell culture experiments, chronic morphine treatment has been followed by 'up'- as well as 'down-regulation' of the mu opioid receptor (mu OR) number. The present postmortem morphometric study of morphine-related fatalities of drug addicts (n=12, and 22-35 years old, with blood unconjugated morphine levels from 27.1 to 458 ng/ml, m.v. 198.5 ng/ml) versus a non-addicted control group (n=13 and 10-44 years old) was intended to examine whether chronic opiate exposure affects the numerical density of mu OR expressing neurons in the human neocortex (area 10 according to Brodmann). For the immunohistochemical procedure, thick (100 microm) vibratome sections were incubated with a monoclonal antibody against the mu OR [Arvidsson et al., J. Neurosci. 15 (1995) 3328] and immunoreactive sites were visualized using an immunoperoxidase protocol. The numerical densities of mu OR-expressing and Nissl-stained neurons were assessed morphometrically (camera lucida-drawings). In both collectives, the anti-mu OR immunoreactivity was mainly found in pyramidal neurons of layers (L) II/III and V and in multiform neurons of L VI. In the drug-related fatalities and the control group, the density of neurons expressing mu OR protein was similar, amounting for 2698 +/- 153 and 2688 +/- 172/mm(3), respectively. These findings extend the binding studies of opioid ligands in postmortem brains of heroin addicts [Gabilondo et al., Psychopharmacology 115 (1994) 135] revealing similar receptor densities and affinities by showing no difference in the density of mu OR-positive neurons.

Numerical density of delta-opioid receptor expressing neurons in the frontal cortex of drug-related fatalities.

In animal experiment and in cell culture, chronic morphine treatment has been followed by a reduction as well as an increase of the delta-opioid receptor (OR) number. The present postmortem morphometric study of morphine-related fatalities of drug addicts (n=12, 22-35 years old, with blood unconjugated morphine levels from 27.1 to 407 ng/ml, m.v. 176.9 ng/ml) versus a non-addicted control group (n=13, 10-44 years old) is intended to examine whether chronic opiate exposure also affects the numerical density of deltaOR expressing neurons in the human neocortex (area 10 according to Brodmann (Vergleichende Lokalisationslehre der Grosshirnrinde (1909) Johann Ambrosius Barth, Leipzig)). For the immunohistochemical procedure, vibratome sections (100 microm) were incubated with a monoclonal antibody against the deltaOR diluted 1:100, and immunoreactive sites were visualized using an immunoperoxidase protocol. The numerical densities of OR expressing and Nissl-stained neurons were assessed morphometrically (camera lucida drawings). In both collectives, the anti deltaOR immunoreactivity was predominantly localized in pyramidal neurons of layers (L) II/III and V as well as in round and ovoid neurons of L VI. In the drug-related fatalities, the density of neurons expressing deltaOR protein amounted for 2515+/-240/mm(3), in the control group for 2616+/-204/mm(3), thus displaying no statistically significant difference. These findings go along with the binding behavior of opioid ligands in postmortem brains of heroin addicts revealing similar receptor densities and affinities in the control subjects and addicts.

[The inhibitory effect of melatonin on morphine withdrawal syndromes and serum monoamines in morphine dependent mice].

OBJECTIVE: To observe the inhibitory effect of melatonin on morphine withdrawal syndromes and serum monoamines in morphine dependent mice. METHODS: A physical dependent model in mice was established by subcutaneous injection of morphine. The intensity of withdrawal syndromes was evaluated according to the jumping latency and jumping times. The concentration of serum monoamines was detected with HPLC-ECD. RESULTS: The physical withdrawal syndromes in morphine dependent mice were inhibited partly by four different doses (25, 50, 100, 200 mg/kg) of melatonin and showed a significant dose-dependent manner. The increased concentration of serum norepinephrine and dopamine in morphine-dependent mice could be reduced by large dose (100 mg/kg) of melatonin. CONCLUSION: The jumping withdrawal syndromes and serum monoamiues in morphine-dependent mice could be inhibited partly by melatonin.

Possible involvement of the total amount of morphine infused in the development of acute morphine dependence in rats.

The severity of naloxone-precipitated withdrawal in rats infused intravenously with morphine at the rates of 2.5, 5 and 10 mg/kg/hr over various time periods was investigated. Plasma morphine concentration reached a constant and rate-dependent level at 1 hr after the start of morphine infusion, and this level was maintained until the termination of infusion. Naloxone (2.0 mg/kg, s.c.) was challenged 18 hr after infusion was stopped, and the withdrawal was evaluated by plasma corticosterone (PCS) increase, diarrhea and body weight loss. The incidence of naloxone-precipitated withdrawal signs was related to both the infusion rate and duration of morphine infusion. The duration of morphine infusion (ET50) needed to elicit naloxone-precipitated PCS increase and diarrhea in 50% of the rats was inversely related to the morphine infusion rates, but the total amount of infused morphine (EA50) that elicited naloxone-precipitated withdrawals in 50% of rats was the same at all infusion rates. These results suggest that the total amount of morphine infused may play an important role in the development of acute physical dependence on morphine rendered by continuous intravenous morphine infusion for 1-8 hr.

Pharmacological analysis of acute morphine dependence in infant rats: close molecular relationship of head-shaking precipitated by opiate antagonists and cholinergic neurotransmission

The influence of drugs affecting different neurotransmitter systems on an acute abstinence heanshaking (AHS) model induced by nalorphine or naloxone was studied in 9-day-old rat pups pretreated (3 h before) with morphine (10mg/kg, i.p.). One hour after the injection of nalorphine (10 mg/kg, i.p.) AHS was stopped by a second dose of morphine (10 mg/kg, i.p.) and reinitiated 1 h later by a higher dose of nalorphine (20 mg/kg, i.p.). In other groups AHS was blocked by spiroperidol (0.1 mg/kg, i.p.), clonidine (0.01 mg/kg, i.p.) or scopolamine (50 mg/kg, i.p.). In these groups a second injection of nalorphine did not reinitiate AHS. In dose-effect curve experiments the AHS induced by naloxone or nalorphine was significantly reduced by previous injections of scopolamine, spiroperidol, metergoline or phentolamine in the corresponding groups. Scopolamine was the only antagonist which displaced the AHS dose-effect curves to the right without affecting the maximal response. Since no common receptors exist for a direct competitive interaction between opiate antagonists and scopolamine, these experiments suggest that a direct molecular relationship exists between the tissue concentration of nalorphine (or naloxone) and the endogenous ACh release during abstinence. Thus, the AHS model in 9-day-old rats clearly differentiates specific from non-specific blockade of the abstinence syndrome, and confirms a distinct or primary role of cholinergic neurotransmission in morphine abstinence

Acute physical dependence: time course and relation to human plasma morphine concentrations.

OBJECTIVES: To characterize the postmorphine time course of precipitated withdrawal responses in comparison with the time course of opioid agonist effects and of plasma morphine concentrations. BACKGROUND: The study provides a more detailed and comprehensive assessment of the postagonist time course of acute dependence effects in humans than previously available. DESIGN: Opioid agonist effects, morphine plasma levels, and withdrawal effects precipitated by naloxone (10 mg/70 kg, administered intramuscularly) were examined at 1, 3, 6, 12, 18, 24, 30, 36, and 42 hours after a single dose of morphine (18 mg/70 kg, administered intramuscularly) in 10 nondependent opioid-experienced subjects. RESULTS: The intensity of subjectively reported precipitated withdrawal effects was greatest when testing was conducted at 6 hours after morphine administration, whereas peak intensity of agonist effects (pupil constriction and subjective ratings) and highest plasma morphine concentrations (57.3 ng/ml) were observed at the shortest test interval (1 hour) after morphine. Offset time course of naloxone-precipitated effects differed across specific measures, with hot and cold feelings elevated for the longest time after morphine (36 hrs), but significant effects were generally apparent for up to 24 hours after morphine pretreatment. Agonist effects lasted through only 12 hours; trace amounts of morphine were detected in plasma for up to 30 hours after administration. CONCLUSIONS: Results show that acute physical dependence engendered by a single dose of morphine peaks later and persists over a longer duration after morphine administration than do other agonist effects. This suggests that neuronal adaptations underlying physical dependence develop and decay gradually over time during a single episode of receptor occupancy. The presence of detectable morphine in plasma is consistent with a competitive displacement mechanism of precipitated effects, although noncompetitive actions of morphine or its metabolites are not ruled out.