Cannabidiol and substance use disorder: Dream or reality.

BACKGROUND: Cannabidiol (CBD) is one of the major constituents of Cannabis sativa L. that lacks psychotomimetic and rewarding properties and inhibits the rewarding and reinforcing effects of addictive drugs such as cocaine, methamphetamine (METH), and morphine. Additionally, CBD's safety profile and therapeutic potential are currently evaluated in several medical conditions, including pain, depression, movement disorders, epilepsy, multiple sclerosis, Alzheimer's disease, ischemia, and substance use disorder. There is no effective treatment for substance use disorders such as addiction, and this review aims to describe preclinical and clinical investigations into the effects of CBD in various models of opioid, psychostimulant, cannabis, alcohol, and nicotine abuse. Furthermore, the possible mechanisms underlying the therapeutic potential of CBD on drug abuse disorders are reviewed. METHODS: The current review considers and summarizes the preclinical and clinical investigations into CBD's effects in various models of drug abuse include opioids, psychostimulants, cannabis, alcohol, and nicotine. RESULTS: Several preclinical and clinical studies have proposed that CBD may be a reliable agent to inhibit the reinforcing and rewarding impact of drugs. CONCLUSIONS: While the currently available evidence converges to suggest that CBD could effectively reduce the rewarding and reinforcing effects of addictive drugs, more preclinical and clinical studies are needed before CBD can be added to the therapeutic arsenal for treating addiction.

Cannabidiol Modulating the Expression of Neurotrophin Signaling Pathways in Chronic Exposure to Methamphetamine in Rats During Abstinence Period.

Introduction: Several neuropsychiatric disorders, such as addiction, have indicated variations in the levels of neurotrophic factors. As an extremely addictive stimulant, methamphetamine (METH) is associated with rising levels of abuse worldwide. We have recently demonstrated that repeated intracerebroventricular (ICV) of cannabidiol (CBD), the most important non-psychotomimetic compound, can lead to diminished impairing memory and hippocampal damage caused by chronic exposure to METH (CEM) in rats over the abstinence period. Furthermore, the results indicated a possible contribution of the neurotrophin signaling pathway (NSP) in regulating neurogenesis and survival. This study intends to evaluate whether these effects remained as measured in molecular pathways after the abstinence period. Methods: The animals were given 2mg/kg METH twice a day for 10 days. Then, we adopted real-time polymerase chain reaction (PCR) throughout the 10-day abstinence period to assess the CBD's effect (10 and 50µg/5µL) on the levels of the mRNA expression of the NSP. Results: The findings suggested that CEM, when compared to the control group in the hippocampus, downregulated mRNA expression of NSP. Moreover, a dosage of 50 µg/5µL CBD may possibly enhance the mRNA expression level of BDNF/TrkB and NGF/TrkA in the hippocampus. Besides, the expression of RAF-1 mRNA level could be reversed significantly by both doses of CBD. Conclusion: According to our results, CBD may partly bring about neuroprotective effects by modulating the NSP. These findings set forth solid evidence demonstrating that CBD is a protective factor attributed to neuropsychiatric disorders, such as METH addiction.

Effects of Morphine on Serum Reproductive Hormone Levels and the Expression of Genes Involved in Fertility-related Pathways in Male Rats.

The effects of morphine on serum reproductive hormone levels and markers involved in fertility-related pathways were evaluated. A total of 30 male Wistar rats were divided into three groups (n = 10) and intraperitoneally administered the following substances for 20 days: two single daily doses of morphine (10 mg/kg; morphine group), saline (healthy saline), and intact group. After confirming the morphine dependence of the experimental groups, all the animals were sacrificed and their total testis tissue was extracted and stored at -80 °C until use. Male reproductive parameters (blood serum of testosterone, luteinizing hormone, and follicle-stimulating hormone) and using Q-PCR and western blot, we evaluated mRNA and protein expression of CREM, TBP, CREB1, HDAC1, and FOS involved in fertility-related pathways were analyzed and compared in the testis samples. The luteinizing hormone and testosterone levels were significantly lower in the morphine-administered group than in the saline and intact groups (P < 0.05). Moreover, the expressions of all five target genes were downregulated in the morphine group (P < 0.05). The protein expression of all five target proteins was downregulated in the morphine group (P < 0.05). We concluded that morphine could decrease the reproductive parameters in male rats.

Cannabidiol microinjection into the nucleus accumbens attenuated nociceptive behaviors in an animal model of tonic pain.

Cannabidiol, the major non-psychoactive constituent of Cannabis, has attracted much attention as a therapeutic agent for intractable chronic pain in many conditions. Nucleus accumbens (NAc) as a major site of action of cannabinoids is one of the main mediators of several analgesic agents especially in the persistent pain condition. The present study aimed to investigate the effect of cannabidiol microinjection into the NAc on the modulation of nociception induced by formalin injection into the rat's paw. Adult male Wistar rats weighing 220-250 g were underwent stereotaxic surgery for unilateral (right or left side) cannula placement into the NAc. After one week recovery period, intra-NAc administration of the cannabidiol or its vehicle, DMSO was performed in a volume of 0.5 µl, five minutes before the formalin test. The formalin test was performed using 50 µl injection of formalin (2.5%) into the plantar surface of the rat's hind paw. Intra-accumbal administration of cannabidiol attenuated the nociceptive responses during the early and late phases of the formalin test in a dose-dependent manner. However, the antinociceptive effect of cannabidiol was significantly higher in the late phase of the formalin test than that in the early phase. Therefore, a non-psychoactive cannabinoid, cannabidiol may be developed as therapeutic agents in conditions, such as persistent inflammatory pain for which primary treatments are insufficient or not possible.

Cannabidiol promotes neurogenesis in the dentate gyrus during an abstinence period in rats following chronic exposure to methamphetamine.

Chronic methamphetamine (meth) abuse can lead to certain deficits in the hippocampal function by affecting the hippocampal neurogenesis and plasticity. To determine whether cannabidiol (CBD) can promote proliferation and maturation of neuronal progenitor cells, this study investigated the CBD effect on neurogenesis in the hippocampal dentate gyrus (DG) following chronic exposure to meth in rats. The rats received 2 mg/kg of meth twice a day for ten days. Next, immunofluorescence was performed to evaluate the effect of intracerebroventricular (ICV) administration of CBD (50 µg/5 µL) over an abstinence period (ten days) on the expression levels of neurogenesis markers, such as Ki67, NeuN, and doublecortin (DCX). Moreover, neuronal degeneration in the hippocampus was assessed using Nissl staining. According to our findings, repeated ICV administration of CBD improved cell proliferation and neurogenesis and increased the number of Ki-67 and DCX-positive cells in the abstinence period. Meanwhile, meth treatment subjects caused a significant decrease in the number of neurogenesis makers, as compared to the control group. The neurogenesis markers (Ki-67 and DCX) could be somewhat reversed, while NeuN did not show any significant increase in the CBD group. Our findings demonstrated that CBD can induce neuroprotective effects by modulating neurogenesis. Therefore, it can provide a promising therapeutic approach to improve cognitive performance following chronic exposure to psychostimulant drugs, including meth.

Therapeutic Effects of Cannabidiol on Methamphetamine Abuse: A Review of Preclinical Study.

As a strong and addictive psychostimulant, methamphetamine (METH) is often misused worldwide. Although relapse is the greatest challenge to the effective treatment of drug dependency, now, for METH addiction, there is not available accepted pharmacotherapy. To characterize a probable new target in this indication, a biological system comprised of endocannabinoids, known as the endocannabinoid system (ECS), has been advised. As a non-psychotomimetic Phytocannabinoid in Cannabis sativa, cannabidiol (CBD) has been used in preclinical and clinical studies for treating neuropsychiatric disorders. In this review article, we focus on the effects of CBD in the treatment of addiction in a preclinical investigation concerning the pharmaceutic effectiveness and the underlying mechanisms of action on drug abuse specially METH. Growing evidence shows that CBD is a potential therapeutic agent in reducing drug reward, as evaluated in conditioned place preference (CPP), brain-stimulation reward paradigms, and self- administration. Furthermore, CBD plays an effective role in decreasing relapse in animal research. Through multiple-mechanisms, there is a belief that CBD modulates brain dopamine responding to METH, resulting in a reduction of METH-seeking behaviors. As our studies indicate, CBD can decrease METH addiction-associated problems, for example, symptoms of withdrawal and craving. It is needed for conducting more preclinical investigations and upcoming clinical trials to entirely assess the CBD capability as interference for METH addiction.

Changes in c-fos and p-CREB signaling following exposure to forced swim stress or exogenous corticosterone during morphine-induced place preference are dependent on glucocorticoid receptor in the basolateral amygdala.

Neural circuitry comprising the nucleus accumbens (NAc), prefrontal cortex (PFC), amygdala (AMY), and hippocampus (HIP) are the main components of the reward circuit. Our previous behavioral data showed that forced swim stress (FSS) and corticosterone administration could inhibit the acquisition of morphine-induced conditioned place preference (CPP), and this effect was blocked by intra-basolateral amygdala (BLA) administration of RU38486, glucocorticoid receptor (GR) antagonist. Therefore, we tried to evaluate the effect of intra-BLA administration of the GR antagonist during the conditioning phase on the c-fos and p-CREB/CREB ratio expression in the AMY, NAc, PFC, and HIP of rats that underwent FSS or received exogenous corticosterone (10 mg/kg; i.p.) before morphine injection (5 mg/kg; s.c.) during 3 conditioning days. Our results showed that morphine-induced CPP could increase c-fos level and p-CREB/CREB ratio in all regions (except in the HIP). In addition, c-fos expression was elevated by FSS in all regions and blockade of GR decreased this effect. In the PFC, in addition to FSS, corticosterone could raise c-fos expression, which was blocked by RU38486. In conclusion, it seems that the intra-BLA administration of RU38486 differently modulates the effect of morphine-induced CPP on the expression of c-fos and p-CREB/CREB ratio in animals that underwent FSS or corticosterone administration.

Neuroprotective effect of chronic administration of cannabidiol during the abstinence period on methamphetamine-induced impairment of recognition memory in the rats.

Neuropsychiatric disorders, such as addiction, are associated with cognitive impairment, including learning and memory deficits. Previous research has demonstrated that the chronic use of methamphetamine (METH) induces long-term cognitive impairment and cannabidiol (CBD), as a neuroprotectant, can reverse spatial memory deficits induced by drug abuse. The study aimed to evaluate the effect of CBD on METH-induced memory impairment in rats chronically exposed to METH (CEM). For the induction of CEM, animals received METH (2 mg/kg, twice/day) for 10 days. Thereafter, the effect of intracerebroventricular (ICV) administration of CBD (32 and 160 nmol) during the (10 days) abstinence period on spatial memory was evaluated using the Y-Maze test, while recognition memory was examined using the novel object recognition (NOR) test. The results revealed a significant increase in the motor activity of METH-treated animals compared with the control group and, after the 10-day abstinence period, motor activity returned to baseline. Notably, the chronic administration of METH had impairing effects on spontaneous alternation performance and recognition memory, which was clearly observed in the NOR test. Additionally, although the ICV administration of CBD (160 nmol) could reverse long-term memory, a lower dose (32 nmol) did not result in any significant increase in exploring the novel object during short-term memory testing. These novel findings suggest that the chronic administration of METH induces memory impairment and presents interesting implications for the potential use of CBD in treating impairment deficits after chronic exposure to psychostimulant drugs such as METH.

The synergistic effect of Paeonia spp and Glycyrrhiza glabra on polycystic ovary induced in mice.

Paeonia spp and Glycyrrhiza glabra were traditionally used to treat female hormonal problems. This study was conducted to investigate the effect of root extract of these plants on ovarian follicles after induction of polycystic ovary syndrome (PCOS). 25-day-old NMRI mice were divided into 4 groups: control, dehydroepiandrosterone (DHEA), and DHEA plus 50mg/kg and 100mg/kg plant extract. Animals in the DHEA group received a daily dose of 6mg/100 g.b.w dissolved in 0.05ml sesame oil for 20 consecutive days through subcutaneous injection (0.2ml); the control group received sesame oil and saline only through i.p; and the other two groups received herbal extracts through i.p. At the end, blood samples were taken to examine hormonal changes and isolate ovarian tissue. There was a significant difference in the level of testosterone and fasting insulin between the polycystic group and treatment groups (50 and 100mg/kg doses). The results showed a significant difference between the control and DHEA treated groups in terms of the number of graph and primary follicles (P<0.05). The results of this study indicated that the mixture of paeonia spp and glycyrrhiza glabra extracts with the administered dosage had positive effects on the status of follicles in the ovaries in polycystic ovary syndrome.

The potential role of the orexin reward system in future treatments for opioid drug abuse.

Despite a history of more than a century of intense research in drug addiction, with currently available medication and behavioral therapy, the rate of relapse to drug use is 40-60 percent within a year after the cessation of treatment. The discovery of the neuropeptide orexin/hypocretin in 1998 and subsequent research during the past 20 years revealed an important role for the lateral hypothalamus (LH) in driving the reward pathway. The present review includes an overview of the orexinergic system and focuses on the role of LH orexin neurons targeting different components of the brain's reward pathway in addictive behaviors. Among major animal models of drug reinforcement and addictive behaviors, we narrowed our focus to include conditioned place preference (CPP) and self-administration methods. In this regard, studies on both orexin-1 receptors (OX1Rs) and orexin-2 receptors (OX2Rs) have shown some positive results, suggesting that single orexin receptor antagonists (SORAs) and dual orexin receptor antagonists (DORAs) may hold promising efficacy in the treatment of addiction compared to the currently used methods. We conclude that since current evidence is still preliminary, development of new SORA and DORA compounds and their evaluation in animal and clinical studies will guide us in our future efforts for developing effective medication.

Reproductive toxicity of manganese dioxide in forms of micro- and nanoparticles in male rats.

BACKGROUND: Manganese Dioxide (MnO 2 ) has long been used in industry, and its application has recently been increasing in the form of nanoparticle. Objective: The present study was an attempt to assess the effects of MnO 2 nanoparticles on spermatogenesis in male rats. MATERIALS AND METHODS: Micro- and nanoparticles of MnO 2 were injected (100 mg/kg) subcutaneously to male Wistar rats (150 ± 20 gr) once a week for a period of 4 weeks, and the vehicle group received only normal saline (each group included 8 rats). The effect of these particles on the bodyweight, number of sperms, spermatogonia, spermatocytes, diameter of seminiferous tubes, testosterone, estrogen, follicle stimulating factor, and the motility of sperms were evaluated and then compared among the control and vehicle groups as the criteria for spermatogenesis. RESULTS: The results showed that a chronic injection of MnO 2 nanoparticles caused a significant decrease in the number of sperms, spermatogonia, spermatocytes, diameter of seminiferous tubes (p < 0.001) and in the motility of sperms. However, no significant difference was observed in the weight of prostate, epididymis, left testicle, estradiol (p = 0.8) and testosterone hormone (p = 0.2). CONCLUSION: It seems that the high oxidative power of both particles was the main reason for the disturbances in the function of the testis. It is also concluded that these particles may have a potential reproductive toxicity in adult male rats. Further studies are thus needed to determine its mechanism of action upon spermatogenesis.

NMDA receptor dependent changes in c-fos and p-CREB signaling following extinction and reinstatement of morphine place preference.

Neural circuitry comprising the ventral tegmental area, nucleus accumbens (NAc), prefrontal cortex (PFC) and hippocampus (HIP) has a main role in reward phenomena. Previous behavioral studies indicated that intracerebroventricular administration of AP5 (NMDA glutamate receptor antagonist) and CNQX (AMPA/kainate glutamate receptor antagonist) during the extinction and before reinstatement of morphine-induced conditioned place preference (CPP) reduced the extinction period and reinstatement of morphine-CPP. Therefore, in the present study, we tried to evaluate the effect of antagonism of NMDA glutamate receptors on the p-CREB/CREB ratio and c-fos expression in the NAc, PFC and HIP during these two phases of morphine-CPP in male adult albino Wistar rats. The p-CREB/CREB ratio and c-fos levels were estimated by Western blot analysis. The results revealed that these two factors decreased by antagonism of NMDA glutamate receptors (different doses of AP5) compared to saline-control group in aforementioned regions. The reduction of molecular markers, especially the p-CREB/CREB ratio, after AP5 administration was more during the extinction period. Therefore, it can be assumed that consolidation and reconsolidation of morphine memory via intra-PFC, -NAc and -HIP NMDA glutamate receptors are in accordance with changes in p-CREB/CREB ratio and c-fos levels.

Orexin A induced antinociception in the ventral tegmental area involves D1 and D2 receptors in the nucleus accumbens.

Previous studies have shown that there are functional interactions among the lateral hypothalamus (LH), ventral tegmental area (VTA) and the nucleus accumbens (NAc), implicating pain modulation in the central nervous system. It has been shown that the LH-VTA orexinergic projecting neurons play an important role in mediating the suppression of nociception in animal models. However, little is known about the function of intra-VTA orexin receptors and involvement of D1/D2 receptors within the NAc in this antinociception. In the present study, we investigated the effect of direct administration of orexin A into the VTA, and examined the role of intra-accumbal dopamine receptors in tail-flick test as a model of acute nociceptive responses in rats. Adult male Wistar rats were unilaterally implanted by two separate cannulae into the VTA and NAc. The results showed that intra-VTA orexin A (0.055, 0.55, 5.5 and 55ng/0.3µl saline) could induce antinociception in a dose-dependent manner. In two separated supergroups, rats received intra-accumbal infusions of D1 and D2 receptor antagonists, SCH-23390 and sulpiride (0.125, 0.25, 1 and 4µg/rat) prior to intra-VTA orexin A (5.5ng/rat) administration respectively. Antinociceptive responses of drugs are represented as maximal possible effect in 5, 15, 30, 45 and 60min after their administrations. Our findings showed that intra-accumbal SCH-23390 and sulpiride dose-dependently prevented intra-VTA orexin-induced antinociception. Nevertheless, this effect is more potent in animals that received D2 receptor antagonist. It is supposed that orexin A can induce the antinociception through activation of orexinergic receptors which activate the dopaminergic inputs to the NAc in rats.

Effect of physical stress on the alteration of mesolimbic system apoptotic factors in conditioned place preference paradigm.

Mesocorticolimbic (MCL) dopaminergic system has an essential role in the rewarding action of opiates and is proved to be influenced by stress. Additionally, both morphine and stress can induce apoptosis. In this study, we investigated the effects of morphine-induced conditioned place preference (CPP) in the presence and absence of stress on the changes of apoptotic factors (Bax/Bcl-2 ratio, caspase-3 activation and PARP degradation) in the MCL system. Male Wistar rats were divided into two saline- and morphine-treated supergroups, each of which consisted of control, acute stress (AS) and subchronic stress (SS) subgroups. In all groups, the CPP paradigm was performed; thereinafter alternations of apoptotic factors in the nucleus accumbens, amygdala, striatum and prefrontal cortex were measured. The results indicated that in the saline-treated animals, AS and SS increased apoptotic factors significantly in the mentioned areas (except for the Bax/Bcl-2 ratio after AS in the Str). In addition, in these animals, conditioning scores decreased after SS but not after AS. In the morphine-treated animals, AS and SS increased apoptotic factors remarkably (except for the Bax/Bcl-2 ratio after AS and SS in the Str and caspase-3 activation after AS in the NAc) and also decreased conditioning scores. Our findings suggest that in the saline- or morphine-treated animals, AS and SS can increase the apoptotic factors in the MCL system and it is more prominent in the morphine-treated animals.

Cannabinoid receptors in the basolateral amygdala are involved in the potentiation of morphine rewarding properties in the acquisition, but not expression of conditioned place preference in rats.

Several studies show the role of the basolateral amygdala (BLA) in drug-seeking, relapse and the brain׳s emotional systems. Several lines of evidence indicate a functional interaction between opioid and endogenous cannabinoid systems. In the present study, we investigated the role of intra-BLA cannabinoid CB1 receptors in the potentiation, acquisition and expression of morphine-induced conditioned place preference (CPP). One-hundred and forty-two adult male Wistar rats weighing 230-280g were bilaterally implanted by two separate cannulae into the BLA. The CPP paradigm was done, and conditioning score and locomotor activity were recorded by Ethovision software. Results showed that intra-BLA administration of different doses of WIN55,212-2 (1, 2 and 4mmol/0.3µl DMSO) as a cannabinoid receptor agonist during the conditioning phase induced place preference in animals that received the ineffective (2mg/kg) dose of morphine compared to respective control group in saline-treated animals. On the other hand, intra-BLA injection of the cannabinoid CB1 receptor antagonist AM251 (45 and 90µmol/0.3µl DMSO) during the 3-day conditioning phase reduced morphine-induced CPP. Furthermore, microinjection of both AM251 (15, 45 and 90µmol) and WIN55,212-2 (1-4mmol), into the BLA had no effect on the expression of morphine (5mg/kg)-induced CPP. Our findings suggest that cannabinoid CB1 receptors in the BLA are involved in the development of reward-related behaviors and they can potentiate the rewarding effects of morphine. It seems that the glutamatergic projection from the BLA to the nucleus accumbens and reward-related learning in the hippocampus may be involved in the acquisition and expression of opioid reward-related behaviors in rats.

Forced swim stress but not exogenous corticosterone could induce the reinstatement of extinguished morphine conditioned place preference in rats: involvement of glucocorticoid receptors in the basolateral amygdala.

Addiction is a common psychiatric disease and stress has an important role in the drug seeking and relapse behaviors. The involvement of basolateral amygdala (BLA) in the effects of stress on reward pathway is discussed in several studies. In this study, we tried to find out the involvement of glucocorticoid receptors (GRs) in the BLA in stress-induced reinstatement of extinguished morphine-induced conditioned place preference (CPP) in rats. The CPP paradigm was done in adult male Wistar rats weighing 220-320 g, and conditioning score and locomotor activity were recorded by Ethovision software. Animals received effective dose of morphine (5mg/kg) daily, during the 3-day conditioning phase. In extinction phase, rats were put in the CPP box for 30 min a day for 8 days. After extinction, animals were injected by corticosterone (10 m/kg) or exposed to forced swim stress (FSS) 10 min before subcutaneous administration of ineffective dose of morphine (0.5mg/kg) in order to reinstate the extinguished morphine-CPP. To block the glucocorticoid receptors in the BLA, after stereotaxic surgery and placing two cannulae in this area bilaterally, animals received GR antagonist mifepristone (RU38486; 0.3, 3 and 30 ng/0.3 µl DMSO per side) prior to exposure to FSS then each animal received ineffective dose of morphine (0.5mg/kg) as drug-induced reinstatement. The results revealed that physical stress (FSS) but not exogenous corticosterone can significantly induce reinstatement of extinguished morphine-CPP, and intra-BLA mifepristone prevents the stress-induced reinstatement. It can be proposed that stress partially exerts its effect on the reward pathway via glucocorticoid receptors in the BLA.

Morphine-induced apoptosis in the ventral tegmental area and hippocampus after the development but not extinction of reward-related behaviors in rats.

Some data suggest that morphine induces apoptosis in neurons, while other evidences show that morphine could have protective effects against cell death. In this study, we suggested that there is a parallel role of morphine in reward circuitry and apoptosis processing. Therefore, we investigated the effect of morphine on modifications of apoptotic factors in the ventral tegmental area (VTA) and hippocampus (HPC) which are involved in the reward circuitry after the acquisition and extinction periods of conditioned place preference (CPP). In behavioral experiments, different doses of morphine (0.5, 5, and 10 mg/kg) and saline were examined in the CPP paradigm. Conditioning score and locomotor activity were recorded by Ethovision software after acquisition on the post-conditioning day, and days 4 and 8 of extinction periods. In order to investigate the molecular mechanisms in each group, we then dissected the brains and measured the expression of apoptotic factors in the VTA and HPC by western blotting analysis. All of the morphine-treated groups showed an increase of apoptotic factors in these regions during acquisition but not in extinction period. In the HPC, morphine significantly increased the ratio of Bax/Bcl-2, caspases-3, and PARP by the lowest dose (0.5 mg/kg), but, in the VTA, a considerable increase was seen in the dose of 5 mg/kg; promotion of apoptotic factors in the HPC and VTA insinuates that morphine can affect the molecular mechanisms that interfere with apoptosis through different receptors. Our findings suggest that a specific opioid receptor involves in modification of apoptotic factors expression in these areas. It seems that the reduction of cell death in response to high dose of morphine in the VTA and HPC may be due to activation of low affinity opioid receptors which are involved in neuroprotective features of morphine.

Chemical stimulation of the lateral hypothalamus potentiated the sensitization to morphine in rats: involvement of orexin-1 receptor in the ventral tegmental area.

Orexin plays a crucial role in drug-seeking behavior. The lateral hypothalamus (LH) is a central region that produces orexin, and its projections to the ventral tegmental area (VTA) play an important role in reward and addiction-related behaviors. In this study, we investigated the role of LH stimulation and the involvement of the orexin-1 receptor (Ox1r) in the VTA in relation to morphine sensitization. In all animals, cannulae were implanted unilaterally into the LH and VTA to inject different doses of carbachol (62.5, 125 and 250 nmol/0.5 µl saline) as a cholinergic agonist and SB334867 (1, 10 and 20 nmol/0.3 µl DMSO) as a selective Ox1r antagonist for three consecutive days (sensitization period) respectively. These drugs were injected five minutes before administration of an ineffective dose of morphine (0.5 mg/kg; sc) during the sensitization period. In all groups, the sensitization period occurred in a separate room from which the conditioning occurred. After this period, all groups exceeded five days under the conditioned place preference (CPP) paradigm without any treatment. For evaluation of morphine sensitization, place preference was induced by ineffective dose of morphine (0.5 mg/kg) and the CPP score was represented by the difference in time spent in drug- and saline-paired compartments. The results revealed that concurrent intra-LH administration of carbachol (125 nmol/0.5 µl saline) and an ineffective dose of morphine (0.5 mg/kg) significantly induce CPP. Additionally, the blockade of Ox1r in the VTA by SB334867 can attenuate the conditioning score induced by concurrent administration of carbachol and an ineffective dose of morphine. Our findings suggest that LH stimulation potentiates the effect of an ineffective dose of morphine, and induces morphine sensitization. It seems that the chemical stimulation of LH potentiates sensitization to morphine through the orexinergic system in the VTA in rats.

Morphine could increase apoptotic factors in the nucleus accumbens and prefrontal cortex of rat brain's reward circuitry.

The nucleus accumbens (NAc) and prefrontal cortex (PFC) are two parts of neuronal reward circuit involved in motivated and goal-directed behaviors. Some data suggest that morphine is toxic to neurons and induces apoptosis, while other evidence shows that morphine could have beneficial effects against cell death. This study was designed to evaluate the effect of morphine on apoptosis by measuring the expression of apoptotic proteins in two important regions, the NAc and PFC, in the rat brain's reward circuitry. Morphine subchronic administration in different doses (0.5, 5 and 10mg/kg) in conditioned place preference (CPP) paradigm (3 times in 3 days, for each dose in each group of rats) was used to induce its rewarding effect. Then, the expression of four apoptotic factors; Bax, Bcl2, caspase3 and PARP, in the NAc and PFC were assessed using the Western blot technique. All of morphine-treated groups showed increase of apoptotic factors in these regions. In the NAc, morphine significantly increased the Bax/Bcl-2 ratio, caspase3 and PARP in the lowest dose (0.5mg/kg) but in the PFC considerable increase was seen in dose of 5mg/kg. Elevation of apoptotic factors in the NAc and PFC implies that morphine can affect the molecular mechanisms which interfere with apoptosis through different receptors. Our findings suggest that the NAc and PFC may have a different distribution of receptors which become active in different doses of morphine.