Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor,NG-nitro-L-arginine methyl ester

The hypothesis that an arginine-nitric oxide (NO) synthase-NO system mediates the morphine abstinence syndrome was tested in adult male rats implanted subcutaneosly for 3 days with one morphine (75 mg) pellet followed by naloxone-precipitated withdrawal (0.5 mg/kg). Injection with a NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (NAME, 100 mg/kg subcutaneous), shortly before naloxone-induced withdrawal significantly inhibited abstinence signs by 25–80%. Continuous infusion of NAME via subcutaneous osmotic pumps during the development of morphine physical dependence and during naloxone-precipitated withdrawal also inhibited morphine abstinence signs. In addition, treatment with isosorbide dinitrate, a NO donor, induced a quasi morphine-abstinence syndrome (QMAS) that was significantly suppressed by implantation of a morphine pellet 3 days before isosorbide dinitrate treatment. These results indicate that NO mediates part of the expression of the morphine abstinence syndrome.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Subcutaneous injection of an analog of neuropeptide FF precipitates morphine abstinence syndrome

Neuropeptide FF (NPFF) has been shown to exert various antiopiate actions, including precipitation of opiate abstinence syndrome by third ventricle injection in morphine dependent rats. In the present study, dansyl-Pro-Gln-Arg-Phe-amide, a lipophilic analog of NPFF, was injected into morphine dependent rats and appropriate sham controls at a dose of 9 mg/kg s.c. Comparison groups were injected with ethanol/water vehicle alone. The NPFF analog precipitated a vigorous opiate abstinence syndrome in morphine dependent rats, but not in sham controls.     

Methionine-enkephalin and beta-endorphin levels in spleen and thymus gland of morphine tolerant-dependent and abstinent rats

The effect of chronic administration of morphine and abrupt and naloxone-precipitated withdrawal on the levels of beta-endorphin and methionine-enkephalin in spleen, adrenals and thymus glands of Sprague-Dawley rats was determined. Rats were made tolerant to and dependent on morphine by subcutaneous implantation of 6 morphine pellets (75 mg morphine in each) during a 7-day period. The tolerant-dependent (with pellets intact) and abstinent (pellets removed 18 hours earlier) rats were sacrificed. In another group, rats with pellets intact were injected with naloxone and sacrificed 10 min later (precipitated abstinence). The weights of the tissues under any of the above treatments did not change nor did the levels of methionine-enkephalin and beta-endorphin in adrenals. The level of beta-endorphin was elevated in the spleen and thymus of morphine tolerant-dependent rats, while the levels of methionine-enkephalin in rats undergoing abrupt or naloxone-precipitated abstinence were significantly higher than in their respective placebo controls. The levels of methionine-enkephalin in the thymus gland of rats with placebo and morphine pellets left intact did not differ. It is concluded that in morphine tolerant-dependent rats the levels of beta-endorphin in spleen and thymus are elevated. During abrupt and naloxone-precipitated abstinence, the levels of methionine-enkephalin in the thymus gland are significantly elevated possibly due to an inhibition of their release. Since these opioid peptides have been implicated in immunomodulation, and alterations were seen in organs controlling immune function, the present results may be helpful in explaining altered immune function in morphine dependent and abstinent states.     

Changes in ribonuclease in the central nervous system of mice during morphine dependence development, withdrawal and naloxone administration

Ribonuclease has been studied in the whole brain homogenate of mice after a single dose of morphine (10 mg/kg), during the development of tolerance and dependence, during the course of withdrawal and naloxone administration. The enzyme increased dose dependently following the administration of morphine. Withdrawal caused a sudden fall in the enzyme with partial recovery by the 6th day of abstinence. Naloxone injections in normal, tolerant, dependent and deprived animals caused a reduction in the enzyme level. The morphine-induced increase in enzyme activity is suggested to be in direct correlation with a reduction in protein synthesis which can be ascribed to a disturbance of the translation processes.     

Suppression of GABA-induced abstinence behaviour in naive rats by morphine and bicuculline.

Intraperitoneal administration of n-dipropylacetate (DPA) to naive rats produced abstinence behaviour including shaking, digging, hunchback posture, piloerection and ptosis during 15 min and increased motor activity considerably. Treatment with a subconvulsive dose of the GABA antagonist bicuculline suppressed this DPA-induced abstinence behaviour, indicating that GABA was increased at receptor sites. Also morphine in a low dose of 1 mg/kg suppressed this behaviour, while administration of naloxone after morphine treatment could release the abstinence behaviour. Simultaneous treatment with morphine and naloxone or naloxone alone were without effect. The administration to DPA treated rats of doses higher than 1 mg/kg morphine resulted in a severe depression of motor activity. It is concluded that an increased availability of GABA at its receptor sites plays an important role in the behaviour observed after DPA administration. The experiments with morphine and naloxone suggest that morphine receptors are involved in DPA-induced abstinence behaviour.     

Proteomic Data in Morphine Addiction Versus Real Protein Activity: Metabolic Enzymes

Drug dependence is an escalating problem worldwide and many efforts are being made to understand the molecular basis of addiction. The morphine model is widely used in these investigations. To date, at least 29 studies exploring the influence of morphine on mammals’ proteomes have been published. Among various proteins indicated as up‐ or down‐regulated, the expression changes of enzymes engaged in energy metabolism pathways have often been confirmed. To verify whether proteomics‐indicated alterations in enzyme levels reflect changes in their activity, four enzymes: PK, MDH, Complex I, and Complex V were investigated in morphine addiction and abstinence models. After analyses of the rat brain mitochondria fraction in the model of morphine dependence, we found that one of the investigated enzymes (pyruvate kinase) showed statistically significant differences observed between morphine, control, and abstinence groups. J. Cell. Biochem. 118: 4323–4330, 2017. © 2017 Wiley Periodicals, Inc.     

Morphine dependence is associated with changes in neuropeptide S receptor expression and function in rat brain

Neuropeptide S (NPS) is a newly identified ligand for the previously discovered G-protein coupled receptor 154 now named NPSR. Recently, it has been found that NPSR gene expression is altered during ethanol withdrawal. In this study we tried to elucidate if NPSR gene expression is modified in response to morphine withdrawal and its protracted abstinence. To induce opioid dependence Wistar rats were treated for 7 days with morphine. Twelve hours and 7 days after the last morphine administration brains were removed and the expression of NPSR mRNA was analyzed by in situ hybridization (ISH). Successful induction of opioid dependence was confirmed by the naloxone-precipitated withdrawal test 2 h after the last morphine administration. Moreover, 7 days after the last morphine dose animals were checked for signs of anxiety and for intracerebroventricular (ICV) NPS (0.3 and 1.0 nmol) induced anxiolytic effects by elevated plus maze (EPM). Results showed that in morphine treated rats strong somatic signs of naloxone-precipitated withdrawal occurred. ISH data revealed changes in NPSR gene expression in the ventral tegmental area as well as in the basolateral amygdaloid and bed nucleus of stria terminalis at 12 h and 7 days into abstinence, respectively. At 7 days into abstinence post dependent animals showed higher levels of anxiety than controls which were significantly attenuated by NPS. These results demonstrated that morphine dependence induction led to (i) changes in NPSR mRNA expression; (ii) increased anxiety; and (iii) more potent anxiolytic-like effect of NPS.     

Spinal cord thyrotropin releasing hormone receptors of morphine tolerant-dependent and abstinent rats

The effect of chronic administration of morphine and its withdrawal on the binding of 3H-[3-MeHis2]thyrotropin releasing hormone (3H-MeTRH) to membranes of the spinal cord of the rat was determined. Male Sprague-Dawley rats were implanted with either 6 placebo or 6 morphine pellets (each containing 75-mg morphine base) during a 7-day period. Two sets of animals were used. In one, the pellets were left intact at the time of sacrificing (tolerant-dependent) and in the other, the pellets were removed 16 hours prior to sacrificing (abstinent rats). In placebo-pellet-implanted rats, 3H-MeTRH bound to the spinal cord membranes at a single high affinity binding site with a Bmax of 21.3 +/- 1.6 fmol/mg protein, and an apparent dissociation constant Kd of 4.7 +/- 0.8 nM. In morphine tolerant-dependent or abstinent rats, the binding constants of 3H-MeTRH to spinal cord membranes were unaffected. Previous studies from this laboratory indicate that TRH can inhibit morphine tolerance-dependence and abstinence processes without modifying brain TRH receptors. Together with the present results, it appears that the inhibitory effect of TRH on morphine tolerance-dependence and abstinence is probably not mediated via central TRH receptors but may be due to its interaction with other neurotransmitter systems.     

Morphine dependent rats: Blockade of precipitated abstinence by calcium

In rats made dependent on morphine, brain levels of calcium were significantly lowered. Naloxone administration to such rats produced characteristic abstinence signs. Prior treatment with calcium markedly reduced naloxone-induced abstinence signs and prevented morphine-induced lowering of brain calcium. It is suggested that the antagonistic actions of calcium and morphine may be related to their effects on morphine-sensitive adenylate cyclase activity.     

Anxiety level during morphine abstinence correlates with the status of nitrergic system in the rat hippocampus

Opiate addiction is accompanied by long-term structural and functional changes in brain regions persisting during abstinence, this status being an experimental model of the aberrant neuroplasticity. Nitric oxide is known to be involved in mechanisms of psychopathological events during opiate abstinence. In this study, indices of a nitregic system (nitric synthase activity--NOS, nitrites and nitrates concentration--NOx-) were measured in the rat brain region during morphine abstinence. Prior to this, the rats were tested for anxiety in an elevated plus maze. NOS activity increased in hippocampus 3 days after morphine withdrawal, while NOx--6 days after withdrawal. No changes of the nitrergic system could be revealed in other brain regions under study. Six days (but not 3 days) after morphine withdrawal, rats visited the open arms of the plus maze more frequently and spent more time in these arms as compared with respective controls. The data suggest that nitrergic system changes in the hippocampus may be involved in molecular mechanisms of behavioural alteration during morphine abstinence in rats.     

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response.     

Effects of hypophysectomy and ACTH on opiate tolerance and physical dependence.

In opiate-naive hypophysectomized rats, the enhanced potency of parenterally injected morphine was almost completely normalized by chronic ACTH in doses that maintained adrenal weight. Following morphine pellet implantation, the amount of morphine required for antinociception, catalepsy, and colonic temperature effects in tolerant rats was identical among hypophysectomized and sham-operated rats whether or not chronic ACTH was administered. However, brain levels of morphine were greater in tolerant, hypophysectomized rats, an effect blocked by chronic ACTH. Hypophysectomy thus appears to enhance the magnitude of tolerance development by a mechanism that is reversed by doses of ACTH that maintain adrenal weight. On the other hand, in sham-control rats this dose and schedule of ACTH were without effect on morphine potency in opiate-naive or tolerant rats or on brain levels of morphine. In general, hypophysectomy and/or ACTH did not modify the intensity of abstinence signs following naloxone-precipitated withdrawal. Therefore, little evidence was obtained to suggest a direct, commanding role of the pituitary in chronic opiate effects. Instead, a secondary adrenal involvement may be important.     

Attenuation of morphine tolerance and dependence by alpha-melanocyte stimulating hormone(alpha-MSH).

Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.     

Changes in succinic dehydrogenase activity in the central nervous system of mice during morphine dependence development, withdrawal and naloxone treatment

The possible role of succinic dehydrogenase (SD) in producing physical dependence to morphine by affecting tissue respiration was investigated in Swiss albino mice during the development of morphine tolerance through a period of addiction and naloxone withdrawal therapy. Tolerance and physical dependence were induced by injecting the mice with morphine sulfate subcutaneously at 8-hour intervals, increasing the dose from 10 mg/kg BW every 24 h for 15 days. The animals were considered to be addicted when they were able to tolerate an otherwise lethal dose of 150 mg/kg 3 times a day. Results indicated that succinic dehydrogenase was inhibited throughout the 15-day period of morphine administration and that this effect was greatest in tolerant animals. Increasing the dose and duration of treatment did not cause further decreases in enzyme activity; instead, after 15 days levels of enzyme activity increased in addicted animals compared with tolerant mice. Furthermore, morphine abstinence for 2 days, markedly increased the levels of SD activity, while 6 days of abstinence had little effect. Naloxone withdrawal at each stage was associated with increased SD activity, but the increase was significant only in tolerant mice.     

Interactions of thyrotropin releasing hormone and morphine sulfate in rodents.

Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ⁹-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$, and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats.     

The effect of an endogenous compound 1-methyl-1,2,3,4,-tetrahydroisoquinoline on morphine-induced analgesia, dependence and neurochemical changes in dopamine metabolism in rat brain structures.

1,2,3,4-Tetrahydroisoquinolines, among them the most interesting neuroprotective substance, an inhibitor of MAO, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), are endogenous compounds present in the central nervous system of mammals and humans. In this study, we investigated the effect of 1MeTIQ on morphine-induced analgesia, tolerance and abstinence syndrome as well as its effect on morphine-induced changes in dopamine metabolism in rat brain structures (nucleus accumbens, striatum, substantia nigra) using HPLC methodology. The experiments were carried out on male Wistar rats. Morphine analgesia was measured in the "hot-plate" test. To induce tolerance, morphine was given chronically (20 mg/kg i.p.) alone or following 1MeTIQ (50 mg/kg i.p.) injection. The development of dependence was assessed in the naloxone (2 mg/kg i.p.) precipitation test, after 10 days of morphine administration. The behavioral studies have shown that an endogenous compound, 1MeTIQ produced strong potentiation of morphine analgesia, prevented the development of morphine tolerance and inhibited expression of morphine abstinence syndrome in morphine-dependent rats. In neurochemical studies, we have demonstrated that 1MeTIQ antagonized morphine-induced changes in dopamine metabolism observed in rat brain structures. The main finding of this study was demonstration for the first time of an anti-abuse effect of an endogenous compound, 1MeTIQ, and its efficiency in counteracting morphine-induced addiction in the way useful from clinical point of view. The obtained results suggested a possibility of clinical application of 1MeTIQ in morphine addiction.     

Chronic morphine exposure and its abstinence alters dendritic spine morphology and upregulates Shank1

Exposure to chronic drugs of abuse has been reported to produce significant changes in postsynaptic protein profile, dendritic spine morphology and synaptic transmission. In the present study we demonstrate alterations in dendritic spine morphology in the frontal cortex and nucleus accumbens of mice following chronic morphine treatment as well as during abstinence for two months. Such alterations were accompanied with significant upregulation of the postsynaptic protein Shank1 in synaptosomal enriched fractions. mRNA levels of Shank1 was also markedly increased during morphine treatment and during withdrawal. Studies of the different postsynaptic proteins at the protein and mRNA levels showed significant alterations in the morphine treated groups compared to that of saline treated controls. Taken together, these observations suggest that Shank1 may have an important role in the regulation of spine morphology induced by chronic morphine leading to addiction.     

Morphine challenges in levo-alpha-acetylmethadol (LAAM) post-addict rats

LAAM dependence was established in female Sprague-Dawley rats over a two week period by a series of twice daily oral intubations. LAAM was then withdrawn. These LAAM post-addict rats along with controls were challenged with 10 mg/kg of i.p. morphine at 15 days, one month, three months, and six months post-withdrawal. In control rats morphine challenges produced, as expected, a biphasic response consisting of 60–90 minutes of EEG slow-wave bursts and behavioral depression followed by 60–90 minutes of EEG and behavioral arousal. In LAAM post-addict rats on day 15 of abstinence morphine challenges produced primarily EEG and behavioral arousal for two to three hours with little or no depression. At one and three months of LAAM abstinence morphine challenges still produced more immediate and sustained EEG and behavioral arousal during the initial two hours than in control rats. At six months in LAAM post-addict rats responses to morphine challenges were biphasic and were similar to those seen in control rats. These results are indicative of protracted abstinence in LAAM post-addict rats, and these results are analogous to those previously reported from our laboratory for morphine post-addict rats.     

Morphine withdrawal modifies antinociceptive effects of acute morphine in rats

Repeated opioid use is known to cause tolerance of antinociceptive effects. Whether opioid abstinence modifies antinociceptive effects is unknown. Here we reported that morphine withdrawal for 18 h and 4 days after repeated morphine treatment largely reduced tail-flick latencies compared with control, while the rats showed severe withdrawal syndromes. However, the latencies and withdrawal syndromes were restored to control level at 20 days withdrawal. Similarly, antinociceptive effects of acute morphine were decreased at 18 h and further decreased at 4 days but restored to control level at 20 days withdrawal. Behavioral stress that was given to the rats at 18 h withdrawal further reduced tail-flick latencies and antinociceptive effects. Conversely, the glucocorticoid receptor antagonist RU38486 increased tail-flick latencies and antinociceptive effects at 4 days withdrawal. These results suggest that morphine withdrawal could evoke behavioral stress to modify antinociceptive effects, implicating a significant influence of opioid abstinence on chronic pain treatment.