N-acetylcysteine attenuates accumbal core neuronal activity in response to morphine in the reinstatement of morphine CPP in morphine extinguished rats.

Numerous studies have suggested that N-acetylcysteine (NAC), has the potential to suppress drug craving in people with substance use disorder and reduce drug-seeking behaviors in animals. The nucleus accumbens (NAc) plays a crucial role in the brain's reward system, with the nucleus accumbens core (NAcore) specifically implicated in compulsive drug seeking and relapse. In this study, we aimed to explore the impact of subchronic NAC administration during the extinction period and acute NAC administration on the electrical activity of NAcore neurons in response to a priming dose of morphine in rats subjected to extinction from morphine-induced place preference (CPP).We conducted single-unit recordings in anesthetized rats on the reinstatement day, following the establishment of morphine-induced conditioned place preference (7 mg/kg, s.c., 3 days), and subsequent drug-free extinction. In the subchronically NAC-treated groups, rats received daily injections of either NAC (50 mg/kg; i.p.) or saline during the extinction period. On the reinstatement day, we recorded the spontaneous activity of NAcore neurons for 15 min, administered a priming dose of morphine, and continued recording for an additional 45 min. While morphine excited most recorded neurons in saline-treated rats, it failed to alter firing rates in NAC-treated rats that had received NAC during the extinction period. For acutely NAC-treated animals, we recorded the baseline activity of NAcore neurons for 10 min before administering a single injection of either NAC (50 mg/kg; i.p.) or saline in rats with no treatment during the extinction. Following 30 min of recording and a priming dose of morphine (1 mg/kg, s.c.), the recording continued for an additional 30 min. The firing activity of NAcore neurons did not show significant changes after morphine or NAC injection. In conclusion, our findings emphasize that daily NAC administration during the extinction period significantly attenuates the morphine-induced increase in firing rates of NAcore neurons during the reinstatement of morphine CPP. However, acute NAC injection does not produce the same effect. These results suggest that modulating glutamate transmission through daily NAC during extinction may effectively inhibit the morphine place preference following the excitatory effects of morphine on NAcore neurons.

Systemic administration of N-acetylcysteine during the extinction period and on the reinstatement day decreased the maintenance of morphine rewarding properties in the rats.

Many animal studies and early clinical trials suggested that N-acetylcysteine (NAC) may benefit addiction treatment. The present study tried to evaluate whether chronic administration of systemic NAC during the extinction period and acute administration of systemic NAC on the reinstatement day could reduce the maintenance of the morphine rewarding properties in the conditioned place preference (CPP) paradigm in the rats. Ninety-six adult male Wistar rats (190-220 g) were examined with morphine (7 mg/kg; sc) and saline (1 mL/kg; sc) during the 3-day conditioning phase in the CPP paradigm. After the acquisition of morphine CPP, different doses of NAC were daily administered during the extinction period (5, 10, 25, and 50 mg/kg; ip), or 30 min before the CPP test on the reinstatement day (2, 5, 10, 25, and 50 mg/kg; ip). Conditioning score and locomotor activity were recorded by the video tracking system and Ethovision software after acquisition on the post-conditioning day, the extinction period, and reinstatement day. Daily NAC administration in high doses (25 and 50 mg/kg; ip) reduced extinction-responding compared with the vehicle-control group during the extinction period. Although a single injection of NAC in doses 10, 25, 50 mg/kg decreased the reinstatement of morphine-induced CPP, two lower doses (2 and 5 mg/kg) could not significantly reduce the CPP scores. These are the first data suggesting that NAC's application during the extinction period could attenuate the morphine reward-associated behaviors in the rats. Moreover, NAC could inhibit the reinstatement of morphine CPP, which adds to the growing appreciation that the NAC may have potential therapeutic use in combating morphine dependence. It can be consistent with the hypothesis of the involvement of the glutamatergic system in the pathophysiology of addiction.

Effect of histone acetylation on maintenance and reinstatement of morphine-induced conditioned place preference and ΔFosB expression in the nucleus accumbens and prefrontal cortex of male rats.

Recently, epigenetic mechanisms are considered as the new potential targets for addiction treatment. This research was designed to explore the effect of histone acetylation on ΔFosB gene expression in morphine-induced conditioned place preference (CPP) in male rats. CPP was induced via morphine injection (5 mg/kg) for three consecutive days. Animals received low-dose theophylline (LDT) or Suberoylanilide Hydroxamic acid (SAHA), as an histone deacetylase (HDAC) activator or inhibitor, respectively, and a combination of both in subsequent extinction days. Following extinction, a priming dose of morphine (1 mg/kg) was administered to induce reinstatement. H4 acetylation and ΔFosB expression in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were assessed on the last day of extinction and the following CPP reinstatement. Our results demonstrated that daily administration of SAHA (25 mg/kg; i.p.), facilitated morphine-extinction and decreased CPP score in reinstatement of place preference. Conversely, injections of LDT (20 mg/kg; i.p.) prolonged extinction in animals. Co-administration of LDT and SAHA on extinction days counterbalanced each other, such that maintenance and reinstatement were no different than the control group. The gene expression of ΔFosB was increased by SAHA in NAc and mPFC compared to the control group. Administration of SAHA during extinction days, also altered histone acetylation in the NAc and mPFC on the last day of extinction, but not on reinstatement day. Collectively, administration of SAHA facilitated extinction and reduced reinstatement of morphine-induced CPP in rats. This study confirms the essential role of epigenetic mechanisms, specifically histone acetylation, in regulating drug-induced plasticity and seeking behaviors.

Evaluation of smoking status: comparison of self-reports with exhaled carbon monoxide analysis in university students in the Islamic Republic of Iran.

BACKGROUND: Smoking is considered the leading risk factor for many chronic diseases and deaths worldwide. Thus, it is important to determine the number of smokers before implementing tobacco control initiatives. Due to stigma and deterrent measures, it is impossible to access smokers through a self-report questionnaire. AIMS: To compare exhaled carbon monoxide levels with self-reports among university students in the Islamic Republic of Iran. METHODS: This cross-sectional study included a convenience sample of 60 university students recruited in 2016 in Tehran. There were 30 women and 30 men with an average age of 23.1 (±15.6) years. They were interviewed using an adaptation of the International Union Against Tuberculosis and Lung Diseases questionnaire and further assessed by breath analysis. Smoking status was compared and then correlated with the resultant carbon monoxide levels at a cutoff of 6 ppm. RESULTS: Mean cigarette consumption was 4.7 (±1.8) each day and smoking status was reported as 19 (31.7%) current smokers and 41 (68.3%) nonsmokers of tobacco. Significant correlations were obtained between the exhaled carbon monoxide levels of the smoker and nonsmoker groups (P < 0.05). Irrespective of the measures of smoking status, the frequency of detecting smokers was comparable to that of detecting nonsmokers (P = 0.756). CONCLUSIONS: Similar to self-reports, the exhaled carbon monoxide measurement successfully distinguished smokers from nonsmokers. This allows healthcare providers and policy-makers to examine the effectiveness of tobacco cessation and prevention programmes.

New Biomarkers Based on Smoking-Related Phenotypes for Smoking Cessation Outcomes of Nicotine Replacement Therapy: A Prospective Study.

INTRODUCTION: Identifying a potent biomarker for smoking cessation can play a key role in predicting prognosis and improving treatment outcomes. This study aimed to evaluate the contribution of new biomarkers based on the levels of Cotinine (Cot) and carbon monoxide (CO) to the short- and long-term quit rates of nicotine replacement therapies (Nicotine Patch [NP] and Nicotine Lozenge [NL]). METHODS: In this prospective interventional study, 124 smokers under treatment with the 5A's method were selected from an outpatient smoking cessation center in district 18 of Tehran City, Iran. The study was conducted from April 2016 to December 2018. They were divided into NP (n=56) and NL (n=61) intervention groups. The levels of Cot and CO were measured using ELISA and breath analysis at the beginning of the study. Three markers were calculated: Cot/CO, Cot to cigarette per day ratio (Cot/CPD), and CO/CPD. Binary logistic regression models and generalized estimating equations models were analyzed by SPSS software, version 21 to determine the chances of quitting smoking. RESULTS: Of the NP participants, 30.4% and 19.6% were abstinent after 2 and 6 months, respectively, while NL was found less effective with 19.7% for 2-month follow-up and 13.1% for 6-month follow-up. The 6-month success of quitting attempts was significantly different for the NP participants at the second half of Cot/CO (P=0.029). Of the NL participants, CO/CPD would be a superior predictor for smoking cessation success (P>0.05). CONCLUSION: The findings of this study suggested two markers of Cot/CO and CO/CPD in this order for the optimum treatment outcomes of NP and NL.

Carvacrol Protects Against 6-Hydroxydopamine-Induced Neurotoxicity in In Vivo and In Vitro Models of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons that project from the substantia nigra pars compacta to the striatum. Evidence from human and animal studies has suggested that oxidative damage critically contributes to neuronal loss in PD. Carvacrol (CAR), a monoterpenic phenol, is the main constituents in the essential oil of many aromatic plants and possesses some properties including anti-inflammatory and anti-oxidant effects. In this study, in vitro and in vivo experiments were performed with the CAR in order to investigate its potential neuroprotective effects in models of PD. Post-treatment with CAR in vitro was found to protect rat adrenal pheochromocytoma PC12 cells from toxicity induced by 6-hydroxydopamine (6-OHDA) administration in a dose-dependent manner by (1) increasing cell viability and (2) reduction in intracellular reactive oxygen species, intracellular lipid peroxidation, and annexin-positive cells. In vivo, post-treatment with CAR (15 and 20 mg/kg) was protective against neurodegenerative phenotypes associated with systemic administration of 6-OHDA. Results indicated that CAR improved the locomotor activity, catalepsy, akinesia, bradykinesia, and motor coordination and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Increased level of reduced glutathione content and a decreased level of MDA (malondialdehyde) were observed in the 6-OHDA rats post-treated with CAR. These findings suggest that CAR exerts protective effects, possibly related to an anti-oxidation mechanism, in these in vitro and in vivo models of Parkinson's disease.

The Effect of Pentoxifylline on Passive Avoidance Learning and Expression of Tumor Necrosis Factor-Alpha and Caspase-3 in the Rat Hippocampus Following Lipopolysaccharide-Induced Inflammation.

BACKGROUND: Peripheral inflammation is effective in the development of neurodegenerative diseases. Pentoxifylline (PTX) has an inhibitory effect on inflammatory cytokines; therefore, we aimed to evaluate the effect of PTX on passive avoidance learning and the expression of tumor necrosis factor-alpha (TNF-α) and caspase-3 in the rat hippocampus, following systemic lipopolysaccharide (LPS) injection. MATERIALS AND METHODS: Male Wistar rats were randomly divided into five groups: control, LPS, and LPS + PTX, receiving doses of 10, 25, and 50 mg/kg of PTX, respectively. The animals received daily injections of PTX (i.p.) 1 week before and 2 weeks after the LPS injection (5 mg/kg; i.p.). Learning and memory were evaluated by passive avoidance learning. Then, the expression of the associated genes was measured in the hippocampus. RESULTS: The results showed that the peripheral LPS injection had no significant effect on learning and memory. PTX only with a dose of 10 mg/kg shows an improvement (P < 0.05). Results from reverse transcription polymerase chain reaction showed that LPS had no significant effect on the expression of caspase-3 and TNF-α. PTX with a dose of 10 mg/kg decreased the caspase-3 expression in the LPS + PTX group (P < 0.001), but the expression of both genes increased, using other concentrations. CONCLUSIONS: Findings showed that systemic application of LPS after 2 weeks had no effect on learning and memory and the expression of inflammatory genes in the hippocampus, but PTX led to an increase in the expression of these genes, which could be due to its direct effects or possible exacerbation of LPS effects.

Effect of Multiple Intraperitoneal Injections of Human Bone Marrow Mesenchymal Stem Cells on Cuprizone Model of Multiple Sclerosis

Background: Bone marrow mesenchymal stem cells (BM-MSCs) elicit neuroprotective effects, and their repair ability has been investigated in different experimental models. We aimed to investigate the effect of multiple i.p. BM-MSCs injections in the cuprizone model of multiple sclerosis in mice. Methods: Adult male C57BL/6 mice (n = 40) were fed a regular diet or a diet containing cuprizone (0.2% w/w) for 6 six weeks. Bone marrow samples were taken from patients with spinal cord injury. BM-MSCs (2 × 106 in 1 milliliter medium) were administered intraperitoneally for two consecutive weeks at the end of the forth weeks of cuprizone administration. Animals (n = 12) were perfused with 10% paraformaldehyde at the end of sixth week. The brains were sectioned coronally in 6-8-µm thickness (-2.3 to 1.8 mm from bregma). The sections were stained by luxol fast blue-cresyl violet, and images were captured via a microscope. Demyelination ratio was estimated in corpus callosum in a blind manner. A quantitative real-time PCR was used to measure the myelin basic protein gene expression at sixth week. Results: Histologically, cuprizone induced demyelination in the corpus callosum. Demyelinated area was diminished in the corpus callosum of cell-administered group. Cuprizone could decrease myelin-binding protein mRNAs expression in corpus callosum, which was significantly recovered after BM-MSCs injections. Conclusion: Our data indicated a remyelination potency of multiple i.p. BM-MSCs in the cuprizone model of multiple sclerosis in mice.

Protective effects of physical exercise on MDMA-induced cognitive and mitochondrial impairment.

Debate continues about the effect of 3, 4-methylenedioxymethamphetamine (MDMA) on cognitive and mitochondrial function through the CNS. It has been shown that physical exercise has an important protective effect on cellular damage and death. Therefore, we investigated the effect of physical exercise on MDMA-induced impairments of spatial learning and memory as well as MDMA effects on brain mitochondrial function in rats. Male wistar rats underwent short-term (2 weeks) or long-term (4 weeks) treadmill exercise. After completion of exercise duration, acquisition and retention of spatial memory were evaluated by Morris water maze (MWM) test. Rats were intraperitoneally (I.P) injected with MDMA (5, 10, and 15mg/kg) 30min before the first training trial in 4 training days of MWM. Different parameters of brain mitochondrial function were measured including the level of ROS production, mitochondrial membrane potential (MMP), mitochondrial swelling, mitochondrial outermembrane damage, the amount of cytochrome c release from the mitochondria, and ADP/ATP ratio. MDMA damaged the spatial learning and memory in a dose-dependent manner. Brain mitochondria isolated from the rats treated with MDMA showed significant increase in ROS formation, collapse of MMP, mitochondrial swelling, and outer membrane damage, cytochrome c release from the mitochondria, and finally increased ADP/ATP ratio. This study also found that physical exercise significantly decreased the MDMA-induced impairments of spatial learning and memory and also mitochondrial dysfunction. The results indicated that MDMA-induced neurotoxicity leads to brain mitochondrial dysfunction and subsequent oxidative stress is followed by cognitive impairments. However, physical exercise could reduce these deleterious effects of MDMA through protective effects on brain mitochondrial function.

Involvement of the dorsal hippocampal GABA-A receptors in histamine-induced facilitation of memory in the Morris water maze.

Several types of learning and memory processes are regulated by the hippocampus which is an important subcortical structure in the mammalians' brain. Previous investigations have shown that different receptor systems in the CA1 region of hippocampus are involved in learning and memory functions. Investigating the possible influence of dorsal hippocampal GABA-A receptors on histamine-induced spatial facilitation in adult male Wistar rats was the focus of the current study. Rats were bilaterally implanted with dorsal hippocampal (CA1) cannulae, recovered from surgery and then trained in Morris water maze (MWM) for 4 consecutive days. A block of four trials was given each day. All drugs were injected into CA1 regions, 5min before training. Pre-training intra-CA1 microinjection of muscimol, a GABA-A receptor agonist, at the dose of 0.01 or 0.02µg/rat, increased the traveled distance or the escape latency and traveled distance to the hidden platform, respectively, indicating a water maze spatial acquisition impairment. Intra-CA1 administration of bicuculline, a GABA-A receptor antagonist however, significantly decreased the escape latency and traveled distance to the hidden platform, suggesting a spatial learning facilitation. On the other hand, pre-training intra-CA1 microinjection of the subthreshold dose of muscimol plus different doses of histamine (0.025, 0.05 and 0.1µg/rat) did not alter the histamine response. Meanwhile, the co-administration of the ineffective dose of bicuculline together with histamine potentiated the spatial learning. Moreover, bilateral infusion of histamine (0.025, 0.05 and 0.1µg/rat) by itself, facilitated the spatial learning. Notably, the drug injections had no effect on swimming speed during the MWM training sessions. Our results suggest that the dorsal hippocampal (CA1) GABA-A mechanism(s) may influence the histamine-induced facilitation of spatial acquisition.

Genotype and allele frequencies of N-acetyltransferase 2 and glutathione S-transferase in the Iranian population.

1. Xenobiotic-metabolizing enzymes constitute an important line of defence against a variety of carcinogens. Many are polymorphic, constituting the basis for the wide interindividual variation in metabolic capacity and possibly a source of variation in the susceptibility to chemical-induced carcinogenesis. The aim of the present study was to determine the frequencies of important allelic variants in the N-acetyltransferase 2 (NAT2) and glutathione S-transferase (GST) genes in the Iranian population and compare them with frequencies in other ethnic populations. 2. Genotyping was performed in a total of 229 unrelated healthy subjects (119 men, 110 women) for NAT2 and 170 unrelated healthy subjects (89 men, 81 women) for GST from the general Tehran population. A combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was applied for typing of NAT2 polymorphisms. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. 3. The frequencies of specific NAT2 alleles were 0.299, 0.314, 0.380, 0.007 and 0.000 for 4 (wild-type), 5 (C481T, M1), 6 (G590A, M2), 7 (G857A, M3) and 14 (G191A, M4), respectively. The most prevalent genotypes were NAT2 5/6 (29.70%) and 4/6 (21.40%). The GSTM1- and GSTT1-null alleles were detected in 44.7 and 21.2% of subjects, respectively. 4. We found that Iranians resemble Indians with regard to allelic frequencies of the tested variants of NAT2. The predominance of slow (49.36%) and intermediate (41.47%) acetylation status compared with wild-type rapid acetylation status (9.17%) in the study group suggests the significant prevalence of the slow acetylator (SA) phenotypes in the Iranian population. Our data confirmed that Iranians are similar to other Caucasian populations in the frequency of both GSTM1- and GSTT1-null alleles.

The effects of adenosine receptor agonists and antagonists on morphine state-dependent memory of passive avoidance.

Pretraining administration of morphine (5 mg/kg, intraperitonically) in a step-down passive avoidance task led to state-dependent learning with impaired retrieval on the test day that was dose-dependently restored by pretest administration of morphine (0.5, 1, 3, and 5 mg/kg). This restoration was reversible by pretest naloxone administration. Pretest administration of adenosine receptor antagonists theophylline or 8-phenyltheophylline (8-PT) did not alter morphine-induced amnesia. However, both the antagonists inhibited the restoration of memory by pretest morphine (5 mg/kg). Adenosine A(1) receptor agonists N(6)-cyclohexyladenosine (CHA) or N(6)-phenylisopropyladenosine (R-PIA) only at the higher doses used, and adenosine A(2) receptor agonist 5'-N-ethylcarboxaminoadenosine (NECA), at all doses used, decreased morphine-induced amnesia in a dose-dependent manner. Pretest administration of low doses of CHA, R-PIA, or NECA significantly showed additive effects with low dose pretest morphine (1 mg/kg) in restoring memory. The promnestic effects of high-dose CHA and R-PIA were inhibited by theophylline or 8-PT but not by naloxone. The additive effects of low-dose CHA or R-PIA and morphine were inhibited by theophylline, 8-PT, or a higher dose of naloxone. The promnestic effect of NECA and its additive effect with low-dose morphine were both inhibited by theophylline and naloxone but not by 8-PT. It is concluded that activation of the adenosinergic system, through both A(1) and A(2) receptors, can reverse morphine-induced amnesia and is involved in morphine state of memory.