|Year : 2015 | Volume
| Issue : 5 | Page : 194-200
Clinical manifestations of combined methamphetamine with morphine and their effects on brain dopamine and 5-hydroxytryptamine release in mice
Shing-Hwa Liu1, Shoei-Yn Lin-Shiau2, Alice Chien Chang3, Kuo-Cheng Lan4
1 Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
2 Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
3 Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan, ROC
4 Department of Emergency Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
|Date of Submission||21-Jun-2015|
|Date of Decision||03-Jul-2015|
|Date of Acceptance||13-Jul-2015|
|Date of Web Publication||22-Oct-2015|
No. 325, Section 2, Cheng-Kong Road, Taipei 114, Taiwan
Source of Support: None, Conflict of Interest: None
Background: Methamphetamine (MA) is often mixed with morphine by polydrug addicts, and polydrug abuse has become a serious health problem worldwide. The purpose of this study was to investigate the major signs and symptoms of combined MA and morphine abuse in the Emergency Department (ED). In addition, we used a mouse model to study their effects on the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the central nervous system. Materials and Methods: Seventy-two patients with combined MA and morphine abuse were collected during a 3-year period, and their medical records were reviewed. Mice were intraperitoneally administered MA (0.75 and 2.5 mg/kg/day) and morphine (5 mg/kg/day) either alone or in combination for 5 consecutive days. The mechanisms underlying the interaction between MA and morphine were explored by measuring the extracellular levels of DA and 5-HT in the shell of the nucleus accumbens using an in vivo microdialysis technique. Results: The most common manifestations of combined MA and morphine abuse included tachypnea, tachycardia, confusion, anxiety, delirium, insomnia, and diaphoresis in the ED. Of those, 25% of acute intoxication required hospitalization for intensive care. The group of mice treated with a combination of MA and morphine had higher concentrations of DA and 5-HT in the accumbens than with either drug alone. Conclusion: These findings suggest that MA pharmacologically interacts with morphine to induce characteristic signs and symptoms. Our preclinical results also implicate the involvement of increased DAergic and 5-HTergic neurotransmission among polydrug abusers with a combination of MA and morphine.
Keywords: Methamphetamine, morphine, combined abuse, dopamine, 5-hydroxytryptamine
|How to cite this article:|
Liu SH, Lin-Shiau SY, Chang AC, Lan KC. Clinical manifestations of combined methamphetamine with morphine and their effects on brain dopamine and 5-hydroxytryptamine release in mice. J Med Sci 2015;35:194-200
|How to cite this URL:|
Liu SH, Lin-Shiau SY, Chang AC, Lan KC. Clinical manifestations of combined methamphetamine with morphine and their effects on brain dopamine and 5-hydroxytryptamine release in mice. J Med Sci [serial online] 2015 [cited 2019 Oct 19];35:194-200. Available from: http://www.jmedscindmc.com/text.asp?2015/35/5/194/167740
| Introduction|| |
According to the epidemiologic survey of the National Bureau of Controlled Drugs in Taiwan from 2000 to 2003, amphetamines and opiates are frequently abused together by addicts [Table 1]. Moreover, patients with their combination abuse are more often than morphine abuse alone, and polydrug abuse has become a serious health problem in the Emergency Department (ED). Methamphetamine (MA) is a powerful psychostimulant that has more potent and more efficacious effects than amphetamine on the central nervous system (CNS). Repeated exposure to MA leads to drug dependence, which is characterized by drug-seeking behavior after withdrawal. Opiates such as morphine and heroin produce euphoric feelings and are highly addictive. Morphine (mu opioid receptors agonist) potentiates MA-induced behavioral responses, increases rewarding effects, and augments behavior sensitization in the mouse model.  Moreover, a behavioral study in humans showed that mixed regimens of d-amphetamine and morphine produced highly positive subjective responses in nondependent individuals. 
Multiple brain regions and neurotransmitter systems are associated with drug addiction to MA and morphine. The reinforcing effects of MA on behavior are mainly mediated through dopamine (DA) and 5-hydroxytryptamine (5-HT) neurotransmission in the brain and primarily depend on its ability to increase DA and 5-HT release in the terminal regions of the mesoaccumbens and neostriatum. , Pharmacological evidence also indicates that systemic administration of morphine increases the extracellular levels of DA and 5-HT in the nucleus accumbens (NAc) and striatum of mice.  Furthermore, a series of studies to explore neural correlates of rewarding effects and drug-seeking behavior induced by MA and morphine focused on the interaction between the dopaminergic and opioidergic systems. , For example, systemic administration of MA enhances endorphin neurotransmission and expression of opioid peptide mRNA in the NAc.  In addition to DA pathways, a number of investigators provided significant evidence for the interaction between the central 5-HTergic and opioidergic systems. , Indeed, 5-HT release in the NAc was modified when the NAc was receiving projections from the dorsal raphe nucleus after systemic morphine administration in rats. 
In the present study, we investigated and characterized the signs and symptoms of combined MA and morphine abuse in the ED. Decoding the interactions between MA and morphine relating to brain neurotransmission may unveil the mechanisms leading to the high prevalence of polydrug abuse among humans. We hypothesize that the mesolimbic pathway plays a critical role in the reinforcement of drug abuse, and the NAc is a crucial brain region associated with drug reward. Therefore, we applied brain microdialysis to investigate alterations of individual and combined administration of MA and morphine in mice. Furthermore, we characterized their combined effects on extracellular levels of DA and 5-HT in the mouse NAc shell.
| Materials and Methods|| |
Selection of patients
The Committee on Human Research for Tri-Service General Hospital approved this observational study (TSGHIRB: 1-103-05-103). We retrospectively reviewed the medical records of patients from January 2011 to December 2013 who were consecutively registered to Military Poison Control Center and treated for combined MA and morphine abuse in the ED of a university-affiliated teaching hospital.
The diagnosis of combined MA and morphine abuse was based on patients' significant history, clinical features, and toxicological confirmation of the drugs in their urine. Urine samples were collected on ED arrival and then submitted for preliminary drug screening using an enzyme-multiplied immunoassay technique. The same samples with positive findings of amphetamines and opiates were further confirmed with gas chromatography/mass spectrometry (HP 5890, 5971A MSD, Hewlett-Packard Co., Palo Alto, CA, USA). Data were collected, including gender, age, route of exposure, signs and symptoms, physical findings, laboratory abnormalities, and associated complications.
Animals and drug treatments
All procedures for animal care were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee of National Taiwan University College of Medicine. The obtained male Institute of Cancer Research mice (an albino mouse strain stock originally established in the ICR), weighing 30-35 g from in the Animal Center of National Taiwan University. Mice were maintained in an animal room with a 12-h light/dark cycle and at a constant temperature (22 ± 2°C) with food and water available ad libitum.
MA hydrochloride and morphine hydrochloride were purchased from the National Bureau of Controlled Drugs in Taipei, Taiwan, ROC. In this study, dosing of 0.75 mg/kg and 2.5 mg/kg of MA (MA0.75 and MA2.5, respectively) and the dose of 5 mg/kg of morphine (M5) were chosen on the basis of our behavioral experiments (appendices [Additional file 1]) in which their combination proved to induce conditioned place preference [Supplementary Figure S1]. Mice (n = 6 for each group) were given MA or morphine alone or in combination once a day for 5 consecutive days. MA and morphine were dissolved in physiological-saline (0.9% NaCl) and administered intraperitoneally (i.p.) to mice with separate syringes at the same time on test days. Control mice received 10 ml/kg i.p. injections of physiological saline.
Surgical procedures and microdialysis study
A guide cannula was implanted into each test animal's left NAc shell 5 days prior to drug administration [Figure 1]. The coordinates for the implantation relative to the bregma were as follows: Anteriorly 1.4 mm, and laterally 0.7 mm and 3.6 mm below the dorsal skull surface [Supplementary Figure S2].  After repeated administration of drugs or saline, microdialysis procedures, and high-performance liquid chromatography analyses were conducted according to the method reported by our laboratory previously.  A freshly calibrated microdialysis probe (CMA/7, membrane length = 1 mm) was placed into the implanted guide cannula. The collection of consecutive 20 min dialysate samples for the determination of basal values of DA and 5-HT began after equilibration for a period of 2-3 h. Following a collection of three basal dialysate samples (with no more than 10% inter-sample concentration variation), a single i.p. injection of drugs or saline was administered to the mice, and consecutive 20 min dialysate samples were again collected for the next 120 min.
|Figure 1: Time schedule for surgery, drug administration and high-performance liquid chromatography analyses. Mice were injected with methamphetamine at either 0.75 or 2.5 mg/kg and morphine at 5 mg/kg alone or in combination once a day for 5 consecutive days. Surgical implantation of the guide cannula was performed fi ve days prior to drug administration; surgery (♦), dosage (↑) and high-performance liquid chromatography analysis (•)|
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All values were expressed as means ± standard error of means and statistical analyses were performed using SPSS for Windows (Version 20, SPSS Inc., Chicago, IL, USA). The changes in dialysate concentrations of NAc DA and 5-HT subsequent to the administration of MA and morphine alone or together were expressed as a proportion (percentage) of the corresponding basal value for each mouse. We examined the differences in the microdialysate levels of DA and 5-HT in the NAc shell for six treatment groups during a 120 min perfusion period using repeated measures analysis of variance (ANOVA). These statistical analyses were followed by post-hoc multiple comparisons using Dunnett's test. A value of P < 0.05 was considered to indicate a statistically significant difference between data sets.
| Results|| |
Patients data analysis
Of 2580 total urine drug screens in the ED, 387 (15%) were positive for MA and of those, 89 (23%) were positive for morphine. ED physicians treated 88 patients for acute intoxication of combined MA and morphine. We excluded 16 patients because alcohol was also detected in their blood. Subsequently, 72 typical patients with combined MA and morphine abuse were collected during a 3-year period. Forty-four patients (61%) had a cigarette smoking habit. Several routes of administration of MA and morphine were reported by the patient, including oral ingestion (51/72, 71%), nasal snorting (11/72, 15%), intravenous injection (6/72, 8%), or a combination of these (4/72, 6%). There were 46 men and 26 women, with a mean age of 26.8 years (range: 20-52 years).
The patients most often manifested neuropsychiatric signs and symptoms due to drug abuse with a combination of MA and morphine. These signs and symptoms included confusion (66/72, 92%), anxiety (66/72, 92%), delirium (65/72, 90%), and insomnia (62/72, 86%), which were observed in over 80% of the patients [Table 2]. In addition, the majority of the patients (43/72, 60%) with insomnia persisted to abuse with a combination of MA and morphine more than 3 months. These patients experienced tachypnea (49/72, 68%), tachycardia (48/72, 67%), and diaphoresis (47/72, 65%), which occurred in over 60% of them in our study. Eighteen of 72 patients (25%) with acute intoxication had serious complications related to combined MA and morphine abuse such as acute pulmonary edema, myocardial ischemia, seizure, coma, rhabdomyolysis, and acute renal failure; these patients were admitted to Intensive Care Unit of the hospital. The laboratory findings of these critically ill patients always showed leukocytosis (white blood cell count > 11000/μl), high serum concentrations of aspartate transaminase, creatine kinase, and troponin-I, and low values of arterial pH and bicarbonate. Five patients (7%) died of respiratory compromise and circulatory instability, and the predominant cause of death was multiple organs failure.
Effects of methamphetamine and morphine on the dialysate levels of dopamine in the nucleus accumbens of mice
There were no significant differences among the experimental groups of mice in the basal concentrations (mean of three individual samples) of DA in the NAc prior to the final injection of drugs or saline [Table 3]. The repeated measures ANOVA model revealed a main effect of treatment on DA levels (treatment: F (5,30) = 170.48, P < 0.001) and an interaction between treatment and every 20 min period (treatment × time: F (25,150) = 34.22, P < 0.001). A post-hoc analysis revealed that repeated administration of combined MA and morphine significantly increased DA levels in the NAc compared with repeated administration of MA or morphine alone [Figure 2]. Treatment with MA produced a dose and time-dependent increase in DA levels compared with saline controls. The time points of maximal impact of treatment with MA in combination with M5 on DA levels were quite similar to MA alone during the 40 min and 60 min perfusion periods. Treatment with M5 did not produce significant differences in DA levels compared to the saline controls.
|Figure 2: Time course of changes in the release of dopamine in the dialysate of the nucleus accumbens after repeated administration of methamphetamine, morphine or their combination in mice. The regimens of pretreatment with methamphetamine (0.75 mg/kg/day, MA0.75 and 2.5 mg/kg/day, MA2.5), morphine (5 mg/kg/day, M5) or their combination (MA0.75 + M5 and MA2.5 + M5) are described in the methods. The drugs were administered on day 5 as indicated by the arrow. Dialysate levels of dopamine in the nucleus accumbens were measured once every 20 min prior to and after treatment with drugs or saline. The combination of methamphetamine and morphine signifi cantly increased dopamine levels compared with methamphetamine or morphine alone. Each experimental group included six mice. The data are expressed as a percentage (mean ± standard error of mean) of the respective basal values. Repeated measures analysis of variance followed by Dunnett's test was used to analyze the signifi cance of differences between treatments during the 120 min perfusion period in levels of dopamine (MA0.75 + M5 vs. MA0.75, *P < 0.001; MA2.5 + M5 vs. MA2.5, #P < 0.001)|
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|Table 3: Basal values (pg/20 ìL) of the concentrations of DA and 5-HT prior to the administration of MA, M, and combination of MA and M measured in the nucleus accumbens of mice|
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Effects of methamphetamine and morphine on the dialysate levels of 5-hydroxytryptamine in the nucleus accumbens of mice
The basal concentrations of 5-HT in the NAc did not significantly differ among the experimental groups [Table 3]. The repeated measures ANOVA model revealed a main effect of treatment on 5-HT levels (treatment: F (5,30) = 54.64, P < 0.001) and an interaction between treatment and every 20 min period (treatment × time: F (25,150) = 37.78, P < 0.001). A post-hoc analysis revealed that repeated administration of combined MA and morphine significantly increased 5-HT levels in the NAc compared with repeated administration of MA or morphine alone [Figure 3]. Treatment with MA produced a dose and time-dependent increase in 5-HT levels compared with saline controls. The time points of maximal impact of treatment with MA in combination with M5 on 5-HT levels were quite similar to MA alone during the 40 min and 60 min perfusion periods. Treatment with M5 did not produce significant differences in 5-HT levels compared with saline controls.
|Figure 3: Time course of changes in the release of 5-hydroxytryptamine in the nucleus accumbens dialysate after repeated administration of methamphetamine, morphine or their combination in mice. The regimens of pretreatment with methamphetamine (0.75 mg/kg/day, MA0.75 and 2.5 mg/kg/day, MA2.5), morphine (5 mg/kg/day, M5) or their combination (MA0.75 + M5 and MA2.5 + M5) are as described in Figure 1. The administration of the respective drugs was given on day 5 as indicated by an arrow. Dialysate levels of 5-hydroxytryptamine in the nucleus accumbens were measured once every 20 min prior to and after treatment with drugs or saline. The combination of Methamphetamine and morphine signifi cantly increased 5-hydroxytryptamine levels compared with methamphetamine or morphine alone. Each experimental group included six mice. Data are expressed as a percentage (mean ± standard error of mean) of the respective basal values. Repeated measures analysis of variance followed by Dunnett's test was used to analyze the signifi cance of differences between treatments during the 120 min perfusion period in levels of 5-hydroxytryptamine (MA0.75 + M5 vs. MA0.75, *P < 0.01; MA2.5 + M5 vs. MA2.5, #P < 0.001)|
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| Discussion|| |
The clinical presentation of MA and morphine is reported in several phenomenological surveys of abused drugs, but it is a serious problem to recognize the characteristic signs and symptoms of co-administration of MA with morphine. The drug effects of MA in combination with morphine apparently differ from those of either drug alone. For example, most patients with a combination of MA and morphine had normal pupil size and did not exhibit mydriasis typical of MA abuse or pinpoint pupils typical of morphine abuse. In addition, the physical findings did not show psychomotor hyperactivity as with MA abuse or analgesic sedation as with morphine abuse in this study. Instead, our patients with combination of MA and morphine manifested with increased neuropsychiatric toxicity in the CNS and catecholamine hyperstimulation in the respiratory, cardiovascular, and peripheral nervous systems including seizure, tachypnea, tachycardia, hypertension, and diaphoresis. In the ED, observations of clinical features are more relevant to the severity of drug intoxication than estimates of abused doses because repeated administration of the drugs results in tachyphylaxis. 
Approximately, 25% of the study patients experienced serious complications related to acute intoxication with a combination of MA and morphine including acute pulmonary edema, myocardial ischemia, coma, rhabdomyolysis, and acute renal failure. In fact, these complications were also observed in ED patients with high doses of acute MA intoxication resulting in fatal outcomes.  According to our preliminary study in ICR mice, systemic co-administration of MA and morphine (10 mg/kg) was more toxic than administration of MA alone (LD 50 : 22.2 mg/kg vs. 49.2 mg/kg). This observation is consistent with previous a report showing that systemic administration of MA produced greater toxicity for morphine-dependent mice than for saline-treated mice. 
In the mouse model, the combined effects of MA and morphine are mediated by potentiating the release of DA and 5-HT in the mesolimbic system of the brain, especially as it relates to polydrug abuse. It is very interesting to research why patients favor to abuse combination of MA and morphine than either drug alone. Of particular importance is the high prevalence of drug abuse of combined MA and morphine among polydrug addicts; whether DAergic and 5-HTergic neurotransmission in the NAc play critical roles in addiction to co-administered MA with morphine still awaits elucidation. A mixture of equal amounts of amphetamine and morphine with a dose of 12.5 or 20 mg in 70 kg male adults enhanced the abuse liability, which was greater than that of either drug alone.  Similarly, several behavioral studies in mice have demonstrated that the effects of combined MA and morphine on conditioned rewarding effects and locomotor activity exhibited a synergistic interaction and are seen at low-dose combinations. , The neurochemical effect of combined MA and morphine leading to increased levels of DA and 5-HT in the NAc was expected by either additive or synergistic interactions. The mesolimbic DA system in the CNS is thought to play a prominent role in the development of drug reward and is strongly modulated by other neurotransmitter systems, including the opioidergic, glutamatergic, and γ-aminobutyric acid (GABA-ergic) systems. The most possible neural mechanism by which morphine enhances MA-induced DA release in the NAc is by dis-inhibition of DA neurons in the ventral tegmental area. That is, μ-OR stimulation inhibited local GABA-ergic interneurons,  resulting in subsequent increase in DA outflow in the NAc. There are many factors that affect the extracellular concentrations of DA and 5-HT induced by morphine including genetic factors, doses of drugs, interval between injections, and the duration of drug administration. , Although behavioral interaction between MA and morphine produced the greatest effect at 5 mg/kg of morphine in combination with MA,  morphine itself elicited not apparently increased dialysate concentrations of accumbal DA and 5-HT in this study. The drug effects of a broad dosage range on the interaction between MA and morphine remains to be evaluated.
| Conclusion|| |
The present study provides an important basis for understanding the major toxidromes of combined MA and morphine abuse in the ED. Our results demonstrated that the co-administration of MA with morphine not only resulted in significantly increased levels of DA in the NAc, but also produced profoundly increased 5-HT release in the NAc compared to the corresponding doses of MA in a dose-dependent manner. Our findings support the hypothesis that opioidergic systems participate in the potentiation of the effects of MA on brain DA and 5-HT release.
| References|| |
Lan KC, Chang AC, Liu SH, Ho IK, Lin-Shiau SY. Enhancing effects of morphine on methamphetamine-induced reinforcing behavior and its association with dopamine release and metabolism in mice. J Neurochem 2009;109:382-92.
Jasinski DR, Preston KL. Evaluation of mixtures of morphine and d-amphetamine for subjective and physiological effects. Drug Alcohol Depend 1986;17:1-13.
Seiden LS, Sabol KE, Ricaurte GA. Amphetamine: Effects on catecholamine systems and behavior. Annu Rev Pharmacol Toxicol 1993;33:639-77.
Kuczenski R, Segal DS, Cho AK, Melega W. Hippocampus norepinephrine, caudate dopamine and serotonin, and behavioral responses to the stereoisomers of amphetamine and methamphetamine. J Neurosci 1995;15:1308-17.
Fadda P, Scherma M, Fresu A, Collu M, Fratta W. Dopamine and serotonin release in dorsal striatum and nucleus accumbens is differentially modulated by morphine in DBA/2J and C57BL/6J mice. Synapse 2005;56:29-38.
Hnasko TS, Sotak BN, Palmiter RD. Morphine reward in dopamine-deficient mice. Nature 2005;438:854-7.
Häggkvist J, Lindholm S, Franck J. The opioid receptor antagonist naltrexone attenuates reinstatement of amphetamine drug-seeking in the rat. Behav Brain Res 2009;197:219-24.
Tien LT, Ho IK, Loh HH, Ma T. Role of mu-opioid receptor in modulation of preproenkephalin mRNA expression and opioid and dopamine receptor binding in methamphetamine-sensitized mice. J Neurosci Res 2007;85:673-80.
Marek GJ. Behavioral evidence for mu-opioid and 5-HT2A receptor interactions. Eur J Pharmacol 2003;474:77-83.
Nemmani KV, Mogil JS. Serotonin-GABA interactions in the modulation of mu- and kappa-opioid analgesia. Neuropharmacology 2003;44:304-10.
Tao R, Auerbach SB. Involvement of the dorsal raphe but not median raphe nucleus in morphine-induced increases in serotonin release in the rat forebrain. Neuroscience 1995;68:553-61.
Paxinos G, Franklin KB. The Mouse Brain in Stereotaxic Coordinates. San Diego, CA: Academic Press; 2001.
McGuigan MA. Toxicology of drug abuse. Emerg Med Clin North Am 1984;2:87-101.
Lan KC, Lin YF, Yu FC, Lin CS, Chu P. Clinical manifestations and prognostic features of acute methamphetamine intoxication. J Formos Med Assoc 1998;97:528-33.
Ginawi OT, al-Shabanah OA, Bakheet SA. Increased toxicity of methamphetamine in morphine-dependent mice. Gen Pharmacol 1997;28:727-31.
Mori T, Ito S, Narita M, Suzuki T, Sawaguchi T. Combined effects of psychostimulants and morphine on locomotor activity in mice. J Pharmacol Sci 2004;96:450-8.
Johnson SW, North RA. Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 1992;12:483-8.
Saito H. Inhibitory and stimulatory effects of morphine on locomotor activity in mice: Biochemical and behavioral studies. Pharmacol Biochem Behav 1990;35:231-5.
Maisonneuve IM, Warner LM, Glick SD. Biphasic dose-related effects of morphine on dopamine release. Drug Alcohol Depend 2001;65:55-63.
Trujillo KA, Smith ML, Guaderrama MM. Powerful behavioral interactions between methamphetamine and morphine. Pharmacol Biochem Behav 2011;99:451-8.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]