Behavioral Signs Of Morphine Withdrawal Research Articles

Dendrosomal Curcumin Nanoformulation Attenuates Naloxone Precipitated Morphine Withdrawal Signs in Rats

Curcuminis known to possess a wide range of therapeutic and pharmacological properties as anti-inflammatory, antitumor, antinociceptive and neuroprotective agents. The recent achievements of nanotechnology illustrate a novel approach in treatment of drug abuse and addiction. Curcumin as a new candidate can be exploited for drug abuse treatment, but its low bioavailability and water solubility is the most important obstacles and limits its application. Here, curcumin was efficiently encapsulated in a nontoxic nanocarrier, labeled dendrosome, to overcome these problems. The present study is designed to investigate the effect of intraperitoneal (i.p.) administration of Dendrosomal curcumin (DNC) on the behavioral signs of morphine withdrawal syndrome. Male Wistar rats weighing 250-300 g were rendered dependent on morphine by subcutaneous (s.c.) administration of morphine sulfate (6, 16, 26, 36, 46, 56 and 66 mg/kg, 2 ml/kg) at set intervals of 24 h for 7 days. The animals received i.p. administration of DNC(12.5 mg/kg) immediately after each morphine injection. On day 8, naloxone (2 mg/ kg, i.p.) was injected and behavioral signs of morphine withdrawal were assessed for 25 min. Our results show that i.p. injection of DNC significantly decreases naloxone precipitated morphine withdrawal signs. The present study indicates that intraperitoneal administration of Dendrosomal curcumin exerts a significant effect on morphine dependency in rats.

Orexin type 1 receptor antagonism in Lateral Paragigantocellularis nucleus attenuates naloxone precipitated morphine withdrawal symptoms in rats

Orexin neuropeptides have been reported to be involved in morphine induced physical dependence and withdrawal. The Lateral Paragigantocellularis (LPGi) is a key brain region implicated in the expression of somatic signs of morphine withdrawal syndrome. Orexin A and orexin type 1 receptor have been found in LPGi neurons but the effect of orexin on the expression of opiate dependence and withdrawal phenomena in this brain structure has not been studied yet. In this study, the effect of intra-LPGi administration of SB 334867 (selective orexin type 1 receptor antagonist) on the behavioral signs of morphine withdrawal syndrome was investigated. Male Wistar rats weighing 250–300g were rendered dependent by adding morphine sulfate (Temad, Tehran, Iran) to their drinking water in increasing concentrations of 0.1, 0.2, 0.3mg/ml for every 48h and 0.4mg/ml during the next 15 days. Behavioral signs of morphine withdrawal were assessed in a transparent cylindrical Plexiglas test chamber (30cm diameter, 50cm height) for 25min. One group of animals received intra-LPGi injection of SB 334867 (0.2μl, 100μM) immediately before naloxone. In the control group, SB-334867 vehicle (DMSO 1%, 0.2μl) was microinjected into LPGi. Our results indicate that intra-LPGi administration of SB 334867 significantly decreases naloxone precipitated morphine withdrawal signs. Thus, it seems that orexin might have a pivotal role in the expression of morphine withdrawal signs through affecting orexin type 1 receptor in LPGi nucleus.

Dual effect of cAMP agonist on ameliorative function of PKA inhibitor in morphine‐dependent mice

The present study shows interactive effects of bucladesine (db-cAMP) as a cyclic adenosine monophosphate (cAMP) agonist and H-89 as a protein kinase A (PKA) inhibitor on naloxone-induced withdrawal signs in morphine-dependent mice. Animals were treated subcutaneously with morphine thrice daily with doses progressively increased from 50 to 125mg/kg. A last dose of morphine (50mg/kg) was administered on the 4thday. Several withdrawal signs were precipitated by intraperitoneal (i.p.) administration of naloxone (5mg/kg). Different doses of bucladesine (50, 100, 200nm/mouse) and H-89 (0.05, 0.5, 1, 5mg/kg) were administered (i.p.) 60min before naloxone injection. In combination groups, bucladesine was injected 15min before H-89 injection. Single administration of H-89 (0.5, 1, 5mg/kg) and bucladesine (50, 100nm/mouse) significantly attenuated prominent behavioral signs of morphine withdrawal. Lower doses of bucladesine (50, 100nm/mouse) in combination with H-89 (0.05mg/kg) increased the inhibitory effects of H-89 on withdrawal signs while in high dose (200nm/mouse) decreased the ameliorative function of H-89 (0.05mg/kg) in morphine-dependent animals. It is concluded that H-89 and bucladesine could affect morphine withdrawal syndrome via possible interaction with cyclic nucleotide messengering systems, protein kinase A signaling pathways, and modified related neurotransmitters.

Spinal Matrix Metalloproteinase-9 Contributes to Physical Dependence on Morphine in Mice

Preventing and reversing opioid dependence continues to be a clinical challenge and underlying mechanisms of opioid actions remain elusive. We report that matrix metalloproteinase-9 (MMP-9) in the spinal cord contributes to development of physical dependence on morphine. Chronic morphine exposure and naloxone-precipitated withdrawal increase activity of spinal MMP-9. Spinal inhibition or targeted mutation of MMP-9 suppresses behavioral signs of morphine withdrawal and the associated neurochemical alterations. The increased MMP-9 activity is mainly distributed in the superficial dorsal horn and colocalized primarily with neurons and small numbers of astrocytes and microglia. Morphine exposure and withdrawal increase phosphorylation of NR1 and NR2B receptors, ERK1/2, calmodulin-dependent kinase II, and cAMP response element binding proteins; and such phosphorylation is suppressed by either spinal inhibition or targeted mutation of MMP-9. Further, spinal administration of exogenous MMP-9 induces morphine withdrawal-like behavioral signs and mechanical allodynia, activates NR1 and NR2 receptors, and downregulates integrin-beta1, while a function-neutralizing antibody against integrin-beta1 suppresses MMP-9-induced phosphorylation of NR1 and NR2B. Morphine withdrawal-induced MMP-9 activity is also reduced by an nNOS inhibitor. Thus, we hypothesize that spinal MMP-9 may contribute to the development of morphine dependence primarily through neuronal activation and interaction with NR1 and NR2B receptors via integrin-beta1 and NO pathways. The other gelatinase, MMP-2, is not involved in morphine dependence. Inhibiting spinal MMP-9 or MMP-2 reduces chronic and/or acute morphine tolerance. This study suggests a novel therapeutic approach for preventing, minimizing, or reversing opioid dependence and tolerance.

Effects of the cell type‐specific ablation of the cAMP‐responsive transcription factor in noradrenergic neurons on locus coeruleus firing and withdrawal behavior after chronic exposure to morphine

Repeated exposure to opiates leads to cellular and molecular changes and behavioral alterations reflecting a state of dependence. In noradrenergic neurons, cyclic AMP (cAMP)-dependent pathways are activated during opiate withdrawal, but their contribution to the activity of locus coeruleus noradrenergic neurons and behavioral manifestations remains controversial. Here, we test whether the cAMP-dependent transcription factors cAMP responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) in noradrenergic neurons control the cellular markers and the physical signs of morphine withdrawal in mice. Using the Cre/loxP system we ablated the Creb1 gene in noradrenergic neurons. To avoid adaptive effects because of compensatory up-regulation of CREM, we crossed the conditional Creb1 mutant mice with a Crem-/- line. We found that the enhanced expression of tyrosine hydroxylase normally observed during withdrawal was attenuated in CREB/CREM mutants. Moreover, the withdrawal-associated cellular hyperactivity and c-fos expression was blunted. In contrast, naloxone-precipitated withdrawal signs, such as jumping, paw tremor, tremor and mastication were preserved. We conclude by a specific genetic approach that the withdrawal-associated hyperexcitability of noradrenergic neurons depends on CREB/CREM activity in these neurons, but does not mediate several behavioral signs of morphine withdrawal.

The mGlu5 receptor antagonists MPEP and MTEP attenuate behavioral signs of morphine withdrawal and morphine-withdrawal-induced activation of locus coeruleus neurons in rats

N-Methyl- d-aspartate (NMDA) antagonists have been demonstrated to suppress the signs of opiate withdrawal; however, side effects limit their clinical use. Since the metabotropic glutamate (mGlu) 5 receptor has been shown to affect glutamate release and modulate NMDA receptor function, we examined the effects of two selective mGlu5 receptor antagonists, 2-methyl-6-(phenyl-ethynyl)-pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), on morphine withdrawal. Pretreatment with MPEP or MTEP (1, 3, and 10 mg/kg, i.p.) significantly attenuated behavioral signs of morphine withdrawal. Specifically, both MPEP and MTEP attenuated the occurrence/severity of chews, digging, salivation, and weight loss, and increased the occurrence of erections. Neither compound changed the occurrence of wet-dog shakes, ptosis, irritability, or lacrimation. Both MPEP and MTEP produced a modest, but significant, attenuation of morphine-withdrawal-induced activation of locus coeruleus neurons in anesthetized rats. These results indicate a role for mGlu5 receptors in morphine withdrawal and suggest the potential for mGlu5 antagonists in the treatment of withdrawal from opiates and other drugs of abuse.

The selective mGlu2/3 receptor antagonist LY341495 exacerbates behavioral signs of morphine withdrawal and morphine-withdrawal-induced activation of locus coeruleus neurons

Previous research has demonstrated that mGlu2/3 agonists can decrease many behavioral signs and the activation of locus coeruleus (LC) neurons observed during morphine withdrawal. However, it is not known if mGlu2/3 receptors are activated during morphine withdrawal by endogenous glutamate. Therefore, we investigated the effect of a novel metabotropic glutamate 2, 3 (mGlu2/3) receptor antagonist (LY341495) on naltrexone-precipitated behavioral signs of morphine withdrawal and withdrawal-induced activation of LC neurons. Three levels of severity of morphine withdrawal (mild, moderate, and strong) were operationally defined by varying the exposure to morphine. Pretreatment with LY341495 (1 mg/kg, sc) had no affect on behavioral signs at the mild level of withdrawal, but significantly increased behavioral signs at the moderate level of withdrawal. At the strong level of withdrawal, 3 and 10 mg/kg, but not 1 mg/kg, LY341495 significantly increased the behavioral signs of withdrawal. In in vivo recordings from anesthetized rats, pretreatment with 1 mg/kg LY341495 did not affect the morphine-withdrawal-induced activation of LC neurons at the mild level of withdrawal. At the moderate level of withdrawal, 1 and 10 mg/kg LY341495 did not affect morphine-withdrawal-induced activation of LC neurons. At the strong level of withdrawal, both 1 and 10 mg/kg LY341495 significantly increased morphine-withdrawal-induced activation of LC neurons. These results indicate that endogenous activation of mGlu2/3 receptors during morphine withdrawal acts to reduce the severity of morphine withdrawal and demonstrates that mGlu2/3 receptors are activated under a physiologically relevant, pathological condition.

The selective iGluR1-4 (AMPA) antagonist LY300168 attenuates morphine-withdrawal-induced activation of locus coeruleus neurons and behavioural signs of morphine withdrawal

Previously, we have shown that the AMPA (iGluR1-4) antagonist LY293558 attenuates the morphine-withdrawal-induced activation of locus coeruleus neurons and behavioral signs of morphine withdrawal. However, LY293558 has since been shown to also have affinity for one subtype of kainate receptor (iGluR5). In this study, we examined the effects of a selective antagonist of iGluR1-4 receptors, LY300168 (GYKI 53655), and a selective antagonist of iGluR5 receptors, LY382884, on the morphine-withdrawal-induced activation of locus coeruleus neurons and behavioral signs of morphine withdrawal. In in vivo recordings from anesthetized rats, pretreatment with LY300168 (0.3–3.0 mg/kg, s.c.), but not LY382884 (at a dose known to have central effects; 100 mg/kg, s.c.) attenuated the morphine-withdrawal-induced activation of LC neurons. In unanesthetized, morphine-dependent rats, pretreatment with LY300168 (0.3–3.0 mg/kg, s.c.), but not LY382884 (100 mg/kg, s.c.), suppressed the severity and occurrence of naltrexone-precipitated morphine-withdrawal signs. These results indicate iGluR1-4 (AMPA) receptors, but not iGluR5 receptors, play an important role the morphine-withdrawal-induced activation of LC neurons and a subset of behavioral signs of morphine withdrawal. In addition, selective AMPA antagonists may have therapeutic effects in man for the treatment of withdrawal from opiates and other drugs of abuse.

Inhibitory effect of neuropeptide Y on morphine withdrawal is accompanied by reduced c-fos expression in specific brain regions.

Neuropeptide Y (NPY) was previously shown in our laboratory to attenuate behavioral signs of morphine withdrawal. To further characterize the anti-withdrawal effect of NPY, the present study attempted to identify specific brain regions where NPY inhibits neuronal activity during withdrawal. Morphine dependence was induced in male Wistar rats by two daily subcutaneous injections of morphine at increasing doses, and the withdrawal syndrome was precipitated acutely by intraperitoneal administration of naloxone. Rats were pre-treated with an intracerebroventricular (icv) injection of NPY (12 nmol) or vehicle 30 min before the naloxone challenge. Withdrawal behavior was quantified using a point scoring technique based on motor- and non-motor-related signs. Brain areas involved in the attenuation of morphine withdrawal were delineated by radioactive in situ hybridization for the immediate early gene c-fos, which is a marker for neuronal activity. The present study confirmed the inhibitory effect of NPY on withdrawal behavior. Inhibition of behavioral signs of naloxone-precipitated morphine withdrawal was accompanied by significantly reduced c-fos expression in the locus coeruleus, lateral septal nucleus, ventral part of the periaqueductal grey, cingulate and frontal cortices, and septohippocampal nucleus. Our data suggest that neo- and allo-cortical areas as well as specific brainstem nuclei are involved in the anti-withdrawal effects of NPY.

Sensitivity to naloxone of the behavioral signs of morphine withdrawal and c-Fos expression in the rat CNS: a quantitative dose-response analysis.

Several studies have used c-Fos expression to delineate the neural substrate underlying naloxone-precipitated morphine withdrawal (MW). However, because behavioral manifestations of MW depend on both the degree of dependence and the doses of naloxone (NAL), a comprehensive study would require examining c-Fos expression in relation with the degree of MW. Here, changes in behavior and in c-Fos-like immunoreactivity (FLI) were studied in the same rats after injection of three doses of NAL to precipitate various degrees of MW. Fifteen established signs of MW were examined for 1 hour after NAL injection, and FLI was quantified in 52 regions of the brain and in the lumbosacral spinal cord. Linear regression analyses were used to examine changes in numbers of signs and FLI neurons with the doses of NAL, and data were considered dose-related for a statistical level of significance of P < 0.05. In summary, autonomic signs of MW increased in a dose-related manner, whereas somatomotor signs did not. After MW, 33 central nervous system regions exhibited significant increases in FLI and were, thus, considered as important neural correlates of MW. Twenty of them displayed dose-related increases in c-Fos expression and correspond to regions related to autonomic functions. Low c-Fos expression was detected in some regions involved in motor control or in reward, suggesting either their minor role in MW or a limitation of the technique. This dose-response analysis suggests that the increase in the severity of autonomic manifestations of MW is associated with a gradual activation of major structures of the autonomic nervous system.

The selective mGlu2/3 receptor agonist LY354740 attenuates morphine-withdrawal-induced activation of locus coeruleus neurons and behavioral signs of morphine withdrawal

Naltrexone-precipitated morphine withdrawal induces hyperactivity of locus coeruleus (LC) neurons, as well as a plethora of behavioral withdrawal signs. Previous research has demonstrated that an increased release of glutamate and activation of AMPA receptors, particularly in the LC, play an important role in opiate withdrawal. LY354740 is a novel Group II metabotropic glutamate mGlu2/3 receptor agonist that decreases the release of glutamate. Therefore, we investigated the effect of LY354740 on naltrexone-precipitated morphine-withdrawal-induced activation of LC neurons and behavioral signs of morphine withdrawal. In in vivo recordings from anesthetized rats, pretreatment with LY354740 (3–30 mg/kg, s.c.) dose-dependently attenuated the morphine-withdrawal-induced activation of LC neurons. In unanesthetized, morphine-dependent animals, pretreatment with LY354740 (3–30 mg/kg, s.c.) dose-dependently suppressed the severity and occurrence of many naltrexone-precipitated morphine-withdrawal signs. These results indicate mGlu2/3 receptor agonists: (1) can attenuate the morphine-withdrawal-induced activation of LC neurons and many behavioral signs of morphine withdrawal; and (2) may have therapeutic effects in man for the treatment of opiate withdrawal.

Opposite Modulation of Opiate Withdrawal Behaviors on Microinfusion of a Protein Kinase A Inhibitor Versus Activator into the Locus Coeruleus or Periaqueductal Gray

Chronic opiate administration upregulates the cAMP pathway in the locus coeruleus (LC). This adaptation is thought to increase the electrical excitability of LC neurons and contribute to the dramatic increase in LC firing induced by opioid receptor antagonists in opiate-dependent animals. The goal of the present study was to evaluate directly a role of the cAMP pathway in opiate withdrawal behaviors by studying, in vivo, whether withdrawal is influenced by intra-LC infusion of compounds known to activate or inhibit protein kinase A (PKA). Infusions into amygdala or periaqueductal gray (PAG) were studied for comparison. In one series of experiments the effect of intra-LC, intra-amygdala, or intra-PAG infusions of the PKA inhibitor Rp-cAMPS on naloxone-precipitated withdrawal from morphine was examined. Intra-LC infusions of Rp-cAMPS significantly attenuated several prominent behavioral signs of morphine withdrawal. Intra-PAG infusions of Rp-cAMPS also significantly attenuated opiate withdrawal behaviors, although different behaviors were affected. In contrast, intra-amygdala infusions of Rp-cAMPS were without significant effect. In a second series of experiments the effect of intra-LC or intra-PAG infusions of the PKA activator Sp-cAMPS on behavior in nondependent drug-naive animals was determined. Sp-cAMPS infusions into either brain region induced a quasi-withdrawal syndrome, but the observed behaviors differed between the two groups. Analysis of the phosphorylation state of tyrosine hydroxylase, a well characterized substrate for PKA, confirmed the ability of Rp-cAMPS and Sp-cAMPS to inhibit and activate, respectively, PKA activity in vivo. Together, these data provide direct evidence for involvement of the cAMP-PKA system in the LC, as well as in the PAG, in opiate withdrawal and withdrawal-related behaviors.

A selective AMPA antagonist, LY293558, suppresses morphine withdrawal-induced activation of locus coeruleus neurons and behavioral signs of morphine withdrawal

The glutamate receptor subtype that mediates the morphine withdrawal-induced activation of locus coeruleus (LC) neurons was examined in this study using in vitro and in vivo single-unit electrophysiologic recordings. For LC neurons recorded in vitro in rat brain slices, the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) antagonist, LY293558, showed a greater than 10-fold selectivity for inhibiting the excitatory effects of AMPA vs kainate, and a greater than 30-fold selectivity for inhibiting the excitatory effects of AMPA vs NMDA. LY293558 also greatly reduced the response of LC neurons to glutamate in a concentration-dependent manner. In in vivo recordings in anesthetized rats, pretreatment with LY293558 (0.1 to 10 mg/kg, i.p.) dose dependently suppressed the morphine withdrawal-induced activation of LC neurons. In unanesthetized, morphine-dependent animals, pretreatment with LY293558 (1 to 30 mg/kg, i.p.) dose dependently suppressed naltrexone-precipitated morphine withdrawal signs. These results indicate: (1) AMPA receptors mediate a large component of the excitatory effects of glutamate on LC neurons; (2) activation of AMPA receptors plays an important role in the morphine withdrawal-induced activation of LC neurons; (3) AMPA antagonists can suppress many signs of morphine withdrawal in awake animals; and (4) AMPA antagonists may have therapeutic effects in humans for the treatment of opiate withdrawal.

NMDA receptor antagonists suppress behaviors but not norepinephrine turnover or locus coeruleus unit activity induced by opiate withdrawal

Pretreatment with the non-competitive NMDA (N-methyl-D-aspartate) antagonist MK801 (0.5, 1.0 mg/kg, s.c.) suppressed the behavioral signs of withdrawal in morphine-dependent rats. However, the same doses of MK801 that suppressed morphine withdrawal also simultaneously produced phencyclidine (PCP)-like behaviors. Pretreatment with the competitive NMDA antagonist LY274614 (25, 50, 100 mg/kg i.p.) also suppressed the behavioral signs of withdrawal in morphine-dependent rats but did not produce PCP-like behavioral effects. Single unit recordings were made from noradrenergic neurons in the locus coeruleus (LC) and, at doses that suppressed morphine withdrawal behaviors, neither MK801 nor LY274614 blocked the withdrawal-induced activation of LC neurons. Biochemical analysis indicated that, at the same behaviorally relevant doses, neither MK801 nor LY274614 blocked the withdrawal-induced increase in norepinephrine turnover in the hippocampus, cerebral cortex, or hypothalamus. These results indicate that NMDA antagonists attenuate the behavioral signs of morphine withdrawal without blocking the withdrawal-induced increase in norepinephrine turnover or the withdrawal-induced increase in LC unit activity. In addition, non-competitive NMDA antagonists, like MK801, may not be useful to alleviate opiate withdrawal symptoms in man because of their PCP-like side effects. However, competitive NMDA antagonists, like LY274614, could be of great benefit for alleviating opiate withdrawal symptoms in man.

Dose effects of naloxone on fixed morphine dependence: simultaneous behavioral and 2-deoxyglucose study in the rat

We have simultaneously studied regional cerebral glucose utilization (RCGU) and behavior during naloxone precipitated morphine withdrawal. For RCGU studies, 25 brain regions were analyzed that previously had been shown to participate in morphine withdrawal. Four established behavioral signs of morphine withdrawal were recorded: wet shakes, jumping, weight loss, and autonomic signs. Using a 10 4 range of naloxone dose (0.0005–5.0 mg/kg), dose dependent effects were found for 3 behaviors: jumping, weight loss and autonomic signs. The incidence of wet shakes did not correlate with naloxone dose. Increases in RCGU in several specific brain sites were also naloxone dose dependent. Naloxone dose dependent increases in RCGU during precipitated morphine withdrawal may be divided into 3 classes of responses: Class I structures (paraventricular, ventromedial, and lateral hypothalamus) exhibited their largest RCGU increases between 0.005 and 0.05 mg/kg of naloxone; Class II structures (preoptic areas, basal ganglia, anterior and intralaminar thalamic nuclei, mammillary nuclei, and certain midbrain regions) showed gradual RCGU increases across the 10 4 range of naloxone dose; and, Class III structures (diagonal band, medial and lateral septum, and the central amygdaloid nucleus) displayed large RCGU increases across 0.5–5.0 mg/kg of naloxone. Regression analysis of RCGU vs behavior showed correlations between Class I responses and autonomic signs ( p<0.010); weight loss was correlated with all 3 classes of naloxone dose dependent RCGU responses during withdrawal ( p<0.05). The strong positive correlation among these RCGU increases and certain morphine withdrawal behaviors supported the use of RCGU measurements in specific brain sites as a sensitive and objective biochemical indicator of the presence and severity of morphine dependence. In addition, this study demonstrates the changes in RCGU in different brain regions are heterogeneous with respect to naloxone dose and appear reproducibly along a continuum from mild to severe withdrawal.