The role of lateral habenula NMDA receptors in tramadol-induced conditioning.

The role of the lateral habenula (LHb) as a hub for receiving and relaying signals from the limbic system to serotonergic, dopaminergic, and norepinephrinergic regions in the brainstem makes this area a critical region in the control of reward and addiction. Behavioral evidence reveals the vital role of the LHb in negative symptoms during withdrawal. In this investigation, we study the role of the LHb N-Methyl D-Aspartate receptor (NMDAR) in the modulation of tramadol reward. Male adult Wistar rats were used in this study. The effect of intra-LHb micro-injection of NMDAR agonist (NMDA, 0.1, 0.5, 2 µg/rat) and antagonist (D-AP5, 0.1, 0.5, 1 µg/rat) was evaluated in conditioned place preference (CPP) paradigm. The obtained results showed that intra-LHb administration of NMDA induced place aversion dose-dependently, while blockade of NMDAR in the LHb using D-AP5 micro-injection led to an increased preference score in the CPP task. Co-administration of NMDA (0.5 µg/rat) with tramadol (4 mg/kg) reduced preference score, while co-administration of D-AP5 (0.5 µg/rat) with a non-effective dose of tramadol (1 mg/kg) potentiate the rewarding effect of tramadol. LHb receives inputs from the limbic system and projects to the monoaminergic nuclei in the brainstem. It has been declared that NMDAR is expressed in LHb, and as obtained data revealed, these receptors could modulate the rewarding effect of tramadol. Therefore, NMDA receptors in the LHb might be a new target for modulating tramadol abuse.

Effect of tramadol on apoptosis and synaptogenesis in hippocampal neurons: The possible role of µ-opioid receptor.

Tramadol is a synthetic opioid with centrally acting analgesic activity that alleviates moderate to severe pain and treats withdrawal symptoms of the other opioids. Like other opioid drugs, tramadol abuse has adverse effects on central nervous system components. Chronic administration of tramadol induces maladaptive plasticity in brain structures responsible for cognitive function, such as the hippocampus. However, the mechanisms by which tramadol induces these alternations are not entirely understood. Here, we examine the effect of tramadol on apoptosis and synaptogenesis of hippocampal neuronal in vitro. First, the primary culture of hippocampal neurons from neonatal rats was established, and the purity of the neuronal cells was verified by immunofluorescent staining. To evaluate the effect of tramadol on neuronal cell viability MTT assay was carried out. The western blot analysis technique was performed for the assessment of apoptosis and synaptogenesis markers. Results show that chronic exposure to tramadol reduces cell viability of neuronal cells and naloxone reverses this effect. Also, the level of caspase-3 significantly increased in tramadol-exposed hippocampal neurons. Moreover, tramadol downregulates protein levels of synaptophysin and stathmin as synaptogenesis markers. Interestingly, the effects of tramadol were abrogated by naloxone treatment. These findings suggest that tramadol can induce neurotoxicity in hippocampal neuronal cells, and this effect was partly mediated through opioid receptors.

Dietary uptake of Salvia macilenta extract improves Nrf2 antioxidant signaling pathway and diminishes inflammation and apoptosis in amyloid beta-induced rats.

BACKGROUND: Studies showed the protective role of Salvia in traditional medicine against neurodegenerative diseases. Salvia macilenta is one of the potent antioxidant herbs among Salvia species against oxidative stress. In the current study, the effect of oral administration of S. macilenta in the antioxidant, anti-inflammatory activities of Aß-injected male albino Wistar rats was determined. METHODS: Rats were received S. macilenta (50 mg/kg/day) orally, for ten successive days and then some of them received Aß (10 ng/µl) in their hippocampus (CA1 region). Proteins involved in antioxidant defense system and inflammatory signaling pathways in the hippocampus and prefrontal cortex were evaluated using Western blotting technique. To study apoptosis, Western blotting technique and histological staining were used. Catalase activity, glutathione peroxidase (GSH) and nitric oxide levels were measured. RESULTS: Results demonstrated that S. macilenta increased Nrf2 protein level and decreased TNFα and IL-6 protein level in Aß-injected rats compared to the Aß-injected group in the hippocampus and prefrontal cortex. Histological analysis showed pretreatment with S. macilenta decreased apoptosis levels in the hippocampus and prefrontal cortex, about 41 and 42%, compared to Aß-injected rats, respectively. This study showed that catalase activity was changed in the S. macilenta + Aß group compared to the Aß-injected rats. Also, GSH level was increased in the S. macilenta + Aß group compared to the Aß-injected rat. CONCLUSION: Orally treatment of S. macilenta extract in Aß-injected rats could ameliorate protective pathways and, so, it can be one of the proposed dietary supplements for the prevention of Alzheimer's disease and dementia.

The effects of lithium chloride and cathodal/anodal transcranial direct current stimulation on conditional fear memory changes and the level of p-mTOR/mTOR in PFC of male NMRI mice.

Lithium chloride clinically used to treat mental diseases but it has some side effects like cognitive impairment, memory deficit. Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that is able to change neural activity and gene transcription in the brain. The aim of the study is to provide a conceptual theoretical framework based on behavioral and molecular effects of tDCS on memory changes induced by lithium in male mice. we applied Anodal-tDCS and Cathodal-tDCS over the left PFC for 3 consecutive days tDCS for 20 min with 2 mA after injection of different doses of lithium/saline.Trained in fear condition and finally the day after that tested their memory persistency factors (freezing-latency) and other behavior such as grooming and rearing percentage time in the fear conditioning. P-mTOR/mTOR was analyzed using western blotting. The results obtained from the preliminary analysis of behavioral fear memory showed that lithium had destructive effect in higher doses and decreased freezing percentage time. However, both cathodal and anodal tDCS significantly improved memory and increased P-mTOR/mTOR level in the PFC. The results of this study indicate that cathodal and anodal tDCS upon the left prefrontal increased memory and reduced lithium side effects on memory consolidation and altered expression of plasticity-associated genes in the prefrontal cortex.

Altered D2 receptor and transcription factor EB expression in offspring of aggressive male rats, along with having depressive and anxiety-like behaviors.

MATERIALS AND METHODS: In this study we have evaluated the behavioral mood variations, and expression of DR-D2 and TFEB genes in the amygdala and PFC of aggressive male rats' offspring. RESULTS: Anxiety and depression-like behaviors were observed, but intra-ventricle injection of DR-D2 antagonist (Sulpiride) has shown to be efficient in reducing negative behavioral changes in offspring. Furthermore, DR-D2 gene expression was increased in the amygdala and PFC of aggressive male rats' offspring, which the injection of Sulpiride decreased it significantly. TFEB gene expression was also decreased in the amygdala and PFC of aggressive male rats' offspring, but the blockade of DR-D2 had no effect on it. CONCLUSIONS: The current data suggests the possible influence of dopaminergic receptors D2 and TFEB genes on the behavioral changes which is modified by having an aggressive father.

NMDA receptor subunits change in the prefrontal cortex of pure-opioid and multi-drug abusers: a post-mortem study.

Addiction is a chronic relapsing disorder and is one of the most important issues in the world. Changing the level of neurotransmitters and the activities of their receptors, play a major role in the pathophysiology of substance abuse disorders. It is well-established that N-methyl-D-aspartate receptors (NMDARs) play a significant role in the molecular basis of addiction. NMDAR has two obligatory GluN1 and two regionally localized GluN2 subunits. This study investigated changes in the protein level of GluN1, GluN2A, and GluN2B in the prefrontal cortex of drug abusers. The medial prefrontal cortex (mPFC), lateral prefrontal cortex (lPFC), and orbitofrontal cortex (OFC) were dissected from the brain of 101 drug addicts brains and were compared with the brains of non-addicts (N = 13). Western blotting technique was used to show the alteration in NMDAR subunits level. Data obtained using Western blotting technique showed a significant increase in the level of GluN1 and GluN2B, but not in GluN2A subunits in all the three regions (mPFC, lPFC, and OFC) of men whom suffered from addiction as compared to the appropriate controls. These findings showed a novel role for GluN1, GluN2B subunits, rather than the GluN2A subunit of NMDARs, in the pathophysiology of addiction and suggested their role in the drug-induced plasticity of NMDARs.

Tramadol induces changes in Δ-FosB, µ-opioid receptor, and p-CREB level in the nucleus accumbens and prefrontal cortex of male Wistar rat.

BACKGROUND: Besides the analgesic effect of tramadol, prolonged exposure to tramadol can induce adaptive changes thereby leading to dependence and tolerance. Tramadol induces its effect via µ-opioid receptor (MOR). However, tramadol has other targets such as serotonin and epinephrine transporters. OBJECTIVE: CREB and ΔFosB are transcriptional factors, which are involved in the behavioral abnormalities underlying drug abuse. In this study, the effects of acute and chronic tramadol treatments on MOR, ΔFosB, and CREB levels were studied. METHODS: For this purpose, 36 male Wistar rats were used. The animals were divided into two main groups. A total of 18 animals received tramadol (0, 5, and 10 mg/kg) acutely and 18 animals received the same doses for the following 14 days. One hour after the last injection, the NAC and PFC were dissected and kept at -80°C in liquid nitrogen. Using western blotting technique, the levels of MOR, ΔFosB, and p-CREB were evaluated. RESULTS: In the NAC, acute tramadol exposure increases the levels of MOR and p-CREB. Moreover, chronic tramadol administration in this region results in elevated levels of MOR, ΔFosB and p-CREB compared with saline-treated rats. The levels of MOR and p-CREB in the PFC increased in both acute and chronic tramadol exposure. Also, ΔFosB levels increased only following chronic tramadol administration. The results revealed that adaptive changes occurred during drug exposure. CONCLUSION: We concluded that both CREB and ΔFosB played a role in tramadol dependence. Additionally, increased MOR levels during tramadol treatments might be due to receptor desensitization.

Complexity of Compensatory Effects in Nrf1 Knockdown: Linking Undeveloped Anxiety-Like Behavior to Prevented Mitochondrial Dysfunction and Oxidative Stress.

Anxiety-related disorders are complex illnesses that underlying molecular mechanisms need to be understood. Mitochondria stand as an important link between energy metabolism, oxidative stress, and anxiety. The nuclear factor, erythroid-derived 2,-like 1(Nrf1) is a member of the cap "n" collar subfamily of basic region leucine zipper transcription factors and plays the major role in regulating the adaptive response to oxidants and electrophiles within the cell. Here, we injected small interfering RNA (siRNA) targeting Nrf1 in dorsal third ventricle of adult male albino Wistar rats and subsequently examined the effect of this silencing on anxiety-related behavior. We also evaluated apoptotic markers and mitochondrial biogenesis factors, along with electron transport chain activity in three brain regions: hippocampus, amygdala, and prefrontal cortex. Our data revealed that in the group that received Nrf1-siRNA, anxiety-related behavior did not show any significant changes compared to the control group. Caspase-3 did not increase in Nrf1-siRNA-injected rats even though Bax/Bcl2 ratio markedly elevated in Nrf1-knockdown rats in all three mentioned regions compared to control rats. Also, Nrf1 silencing of complex I and II-III did not alter, generally. In addition, Nrf1-knockdown affected mitochondrial biogenesis markers. The level of peroxisome proliferator-activated receptor gamma coactivator-1α and cytochrome-c increased, which indicates a possible role for mitochondrial biogenesis in anxiety.

Dose-dependent manner of luteolin in the modulation of spatial memory with respect to the hippocampal level of HSP70 and HSP90 in sleep-deprived rats.

Sleep deprivation (SD) induces a variety of deleterious effects on different cognitive functions such as memory. Elevated neuroinflammation, oxidative stress, and apoptosis, and decreased synaptic plasticity and antioxidant capacity are involved in the deleterious effects of SD on memory. On the other hand, luteolin (a flavonoid compound) has antioxidant, neuroprotective, and anti-inflammatory properties. Also, Heat shock protein 70 (HSP70) and Heat shock protein 90 (HSP90) can be involved in modulating memory. In this study, we aimed to assess the effects of SD and luteolin on spatial learning and memory using Morris Water Maze apparatus in rats, with respect to the level of HSP70 and HSP90 in the hippocampus. Luteolin was injected intracerebroventricular (i.c.v.) at the doses of 0.5, 1, and 2 µg/rat. The results showed that SD impaired spatial memory, while luteolin dose-dependently restored SD-induced spatial memory impairment. SD increased the expression level of HSP90 in the hippocampus, whereas luteolin dose-dependently reversed the effect of SD. Furthermore, SD decreased the expression level of HSP70 protein in the hippocampus, while luteolin dose-dependently reversed the effect of SD. In conclusion, HSP70 and HSP90 may be involved in the deleterious effect of SD on memory, and in the improvement effect of luteolin on memory. This is a novel study reporting novel data and we suggest further detailed studies to better understand the interactions between SD, luteolin, and Heat shock proteins.

Does Morphine Exposure Before Gestation Change Anxiety-Like Behavior During Morphine Dependence in Male Wistar Rats?

Background: Anxiety is one of the comorbid disorders of opioid addiction, which leads to opioid abuse or persuades people to engage in opioid abuse. Evidence revealed that morphine exposure before conception changes the offspring's phenotype. The current study aimed to investigate the influence of morphine dependence and abstinence on anxiety-like behavior in morphine-exposed and drug-naïve offspring. Methods: Adult male and female rats were treated with morphine or vehicle for 21 days. Then, all rats were left without drug treatment for 10 days. A morphine-exposed female rat was mated with either a vehicle-exposed or morphine-abstinent male. According to parental morphine exposure, the offspring were categorized into four distinct groups: (1) control (both drug-naïve parents), (2) paternal morphine-exposed, (3) maternal morphine-exposed, and (4) biparental morphine-exposed. The anxiety-like behavior was measured in adult male offspring using open field and elevated plus-maze tests before morphine exposure (naïve), 21 days after morphine exposure (dependence), and ten days after the last morphine exposure (abstinence). Findings: The results indicated that anxiety-like behavior increased before morphine exposure in maternal and biparental morphine-exposed offspring (P<0.05). However, after morphine exposure, the anxiety level did not change among the groups. Ten days after the last morphine exposure, anxiety-like behavior increased only in biparental morphine-exposed offspring (P<0.05). Conclusion: The offspring of morphine-abstinent parents exhibited an anxious phenotype. Disruption of the HPA axis was seen in the progeny of maternal and biparental morphine-exposed rats. Indeed, morphine exposure for 21 days did not change anxiety-like behavior in these offspring which might be correlated to disruption of HPA axis in them.

Paternal Aggression in Early-life Impairs the Spatial Memory and Passive Avoidance Learning in Adulthood of Male Rats: The Possible Role of DRD2.

Introduction: Negative early-life experiences (e.g. having an aggressive father) can leave long-lastingimpacts on the behavior. However, it is not clear if they influence learning and memory. Methods: In this study, we investigated the influences that the presence of an aggressive father had on the level of passive avoidance learning and spatial memory. We also studied the changes in the dopamine receptor D2 (DRD2) and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) gene expression in the hippocampus. Then, we evaluated if a DRD2 antagonist (sulpiride, 0.125, 0.25, or 0.5 µg/rat) could modulate these changes. Results: We found that the subjects exposed to early-life stress made by aggressive fathers had impaired passive avoidance learning and spatial memory compared to subjects with normal fathers. Treatment with sulpiride improved passive avoidance learning and spatial memory in rats with aggressive fathers. The rats with aggressive fathers also had higher expression of the DRD2 gene in their hippocampus than those with normal fathers, while the PGC-1α gene expression was not different among groups. Treatment with sulpiride (0.125, 0.25, or 0.5 µg/rat) reduced the DRD2 gene expression in those with aggressive fathers to the normal level compared to those with normal fathers. Conclusion: These data suggest that having and living in a shared place with an aggressive father, even without any physical contact, can detrimentally affect passive avoidance learning and spatial memory which is accompanied by the increased expression of the DRD2 gene. Also, sulpiride as a dopaminergic antagonist could reverse this process. Highlights: Having and living with an aggressive father reduced learning and memory in offspring.Having and living with an aggressive father during early life increased DRD2 gene expression.Sulpiride improved learning and memory and also normalized DRD2 gene expression.A combination of genetic and environmental factors may modulate learning and memory. Plain Language Summary: In this study, we looked at how having an aggressive father, can affect behavior in the long term. We wanted to find out if this factor influences learning and memory. To do this, we investigated how the presence of an aggressive father affected passive avoidance learning and spatial memory in subjects. We also examined specific genes in the brain, called DRD2 and PGC-1α, which are known to be involved in learning and memory. Specifically, we wanted to see if the expression of these genes in the hippocampus (a region of the brain important for memory) was affected by having and presence of an aggressive father. To understand the role of the DRD2 gene further, we used a drug called sulpiride, which blocks the action of DRD2. We administered sulpiride to the subjects with aggressive fathers to see if it could reverse any negative effects on learning and memory. What we found was that subjects that had aggressive fathers had impaired passive avoidance learning and spatial memory compared to those with normal fathers. However, when we treated the subjects with sulpiride, their learning and memory improved. Additionally, we observed that rats with aggressive fathers had higher levels of the DRD2 gene in their hippocampus, while the PGC-1α gene expression was not different among the groups. The administration of sulpiride reduced the expression of the DRD2 gene in rats with aggressive fathers, bringing it back to normal levels similar to those with normal fathers. These findings suggest that having and living in the same environment as an aggressive father, even without direct physical contact, can negatively impact passive avoidance learning and spatial memory. This effect seems to be associated with increased expression of the DRD2 gene. However, using sulpiride as a dopaminergic antagonist can reverse this process and improve learning and memory in these subjects.

Investigating the effect of crocin on memory deficits induced by total sleep deprivation (TSD) with respect to the BDNF, TrkB and ERK levels in the hippocampus of male Wistar rats.

BACKGROUND: Sleep deprivation (SD) induces cognitive impairments such as memory deficit. Brain-derived neurotrophic factor (BDNF) is considered as the most critical neurotrophin in the central nervous system that is involved in sleep and memory. The main receptor of BDNF, tropomyosin receptor kinase B (TrkB), is dramatically expressed in the hippocampus. Also, extracellular signal-regulated kinase (ERK) has a significant role in memory function. Crocin is a carotenoid chemical compound and the active component of the flower Crocus sativus L. (saffron) that improves memory function and increases the level of BDNF, TrkB and ERK. AIMS: In this research, we aimed to investigate the effect of total SD (TSD, 24 h) and crocin on memory performance, and BDNF, TrkB and ERK hippocampal levels. METHODS: Passive avoidance memory was assessed using step-through, and working memory was measured using Y-maze tasks. The level of proteins in both hemispheres of the hippocampus was evaluated using Western blotting. Crocin was injected intraperitoneally at doses of 1, 5 and 15 mg/kg. RESULTS: Twenty-four-hour TSD impaired both types of memories and decreased the level of all proteins in both hemispheres of the hippocampus. Crocin at all doses restored TSD-induced memory deficits. Crocin (15 mg/kg) reversed the effect of TSD on levels of all proteins. CONCLUSIONS: The adverse effect of TSD on the level of proteins in the hippocampus may disrupt synaptic plasticity and transmission, which induces memory impairment. Additionally, the restoration effect of crocin on the decrease in protein levels may be involved in its improvement effect on memory performance.

Effects of Morphine and Maternal Care on Behaviors and Protein Expression of Male Offspring.

Genes and environment interact during development to alter gene expression and behavior. Parental morphine exposure before conception has devastating effects on the offspring. In the present study, we evaluated the role of maternal care in the intergenerational effect of maternal morphine exposure. Female rats received morphine or saline for ten days and were drugfree for another ten days. Thereafter, they were allowed to mate with drug-naïve male rats. When pups were born, they were cross-fostered to assess the contribution of maternal care versus morphine effects on the offspring. Adult male offspring were examined for anxiety-like behavior, spatial memory, and obsessive-compulsive-like behavior. To determine the mechanisms underlying the observed behavioral changes, protein levels of acetylated histone H3, BDNF, Trk-B, NMDA subunits, p-CREB, and 5-HT3R were measured in the brain. Our results indicate that maternal caregiving is impaired in morphine-abstinent mothers. Interestingly, maternal care behaviors were also affected in drug-naïve mothers that raised offspring of morphine-exposed mothers. In addition, the offspring of morphine abstinent and non-drug dependent mothers, when raised by morphine abstinent mothers, exhibited more anxiety, obsessive-compulsive behaviors and impaired spatial memory. These altered behaviors were associated with alterations in the levels of the above-mentioned proteins. These data illustrate the intergenerational effects of maternal morphine exposure on offspring behaviors. Moreover, exposure to morphine before gestation not only affects maternal care and offspring behavior, but also has negative consequences on behaviors and protein expression in adoptive mothers of affected offspring.

Tramadol Treatment Induces Change in Phospho-Cyclic Adenosine Monophosphate Response Element-Binding Protein and Delta and Mu Opioid Receptors within Hippocampus and Amygdala Areas of Rat Brain.

BACKGROUND: Tramadol induces its unique effects through opioid pathways, but the exact mechanism is not known. The study aims to evaluate changes in the level of mu-opioid receptor (µOR), delta-opioid receptor (δOR), and phosphorylated cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB) in the hippocampus (HPC) and amygdala (AL) areas of tramadol-treated rats. METHODS: For this purpose, a total of 36 male rats were divided into two main groups for chronic or acute tramadol exposure. The animals were then exposed to 5 mg.kg-1 of tramadol, 10 mg.kg-1 of tramadol, and normal saline. The HPC and AL areas of the animals were dissected upon completion of the period. The levels of p-CREB and µOR were quantified using the western blotting technique. The data were subjected to analysis of variance (ANOVA) followed by Tukey's post-hoc analysis. The differences with the P-value lower than 0.05 were considered as significant. FINDINGS: In the HPC and AL areas of the brain, the level of µOR was decreased by acute tramadol exposure, while no significant difference was observed by chronic tramadol exposure. Moreover, results showed that the level of p-CREB dose-dependently increased by acute and chronic tramadol exposure. CONCLUSION: HPC and AL are essential in the control of tramadol abuse. Tramadol abuse affects gene expression and transcription factors such as CREB. With acute drug tramadol treatments, the level of cAMP response element-binding protein (CREB) rapidly increases, while by chronic tramadol treatment, "peak and trough pattern is observing". The activation of the rewarding mechanism is a precise instance of addictive behavior in tramadol-treated individuals.

The effect of fish oil on social interaction memory in total sleep-deprived rats with respect to the hippocampal level of stathmin, TFEB, synaptophysin and LAMP-1 proteins.

Fish oil (FO) is one of the richest natural sources of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). DHA is essential for brain functions and EPA has been approved for brain health. On the other hand, stathmin, TFEB, synaptophysin and LAMP-1 proteins are involved in synaptic plasticity, lysosome biogenesis and synaptic vesicles biogenesis. In this study, we aimed to investigate the effect of FO on social interaction memory in sleep-deprived rats with respect to level of stathmin, TFEB, synaptophysin and LAMP-1 in the hippocampus of rats. All rats received FO through oral gavage at the doses of 0.5, 0.75 and 1 mg/kg. The water box was used to induce total sleep deprivation (TSD) and the three-chamber paradigm test was used to assess social behavior. Hippocampal level of proteins was assessed using Western blot. The results showed, FO impaired social memory at the dose of 1 mg/kg in normal and sham groups. SD impaired social memory and FO did not restore this effect. Furthermore, FO at the dose of 0.75 mg/kg decreased social affiliation and social memory in all groups of normal rats, compared with related saline groups, and at the dose of 1 mg/kg impaired social memory for stranger 2 compared with saline group. In sham groups, FO at the dose of 1 mg/kg impaired social memory for stranger 2 compared with saline group. SD decreased hippocampal level of all proteins (except stathmin), and FO (1 mg/kg) restored these effects. In conclusion, FO negatively affects social interaction memory in rats.

Alteration of orexin-A and PKCα in the postmortem brain of pure-opioid and multi-drug abusers.

Finding changes induced by the drug of abuse is one of the most important approaches to design new drugs for the treatment of substance use disorders (SUD). Postmortem study is the most reliable method for detecting alteration in the brain of SUD patients. Recently, the role of orexinergic system in SUD is in consideration. In the current study, we evaluated the level of orexin-A in the CSF and protein kinase Cα (PKCα) in the brain of pure-opioid (POA) and multi-drug abusers (MDA). A total of 56 POA, 45 MDA, and 13 matched control brains were collected from the legal medicine center, Tehran, Iran. The CSF was gathered from the third ventricle immediately after opening the skull and kept at -80 °C. The medial prefrontal cortex (mPFC), lateral prefrontal cortex (lPFC), orbitofrontal cortex (OFC), nucleus accumbens (NAc), and amygdala were dissected from fresh brain, frozen with liquid nitrogen and kept at -80 °C. The level of orexin-A evaluated in the CSF. Using western blotting, the level of PKCα assessed in the brain. Obtained data revealed that the level of orexin-A increased in POA and MDA compared with the control group (p < 0.05). In addition, the level of PKCα increased in the prefrontal cortex and amygdala of the abusers compared with the control group, although we did not detect changes in the level of PKCα in the NAc. Along with animal studies, the current results showed that the level of orexin increased in the CSF of drug abusers, which might be related to increases in the activation of lateral hypothalamic orexinergic neurons faced with the drug of abuse. Enhancement in the level of PKCα in the drug reward circuits might be adaptational changes induced by orexin and drugs of abuse.

The role of cannabinoid 1 receptor in the nucleus accumbens on tramadol induced conditioning and reinstatement.

AIMS: Previous investigations demonstrated that tramadol, as a painkiller, similar to morphine induces tolerance and dependence. Furthermore, the cannabinoid receptor 1 (CB1R) located in the nucleus accumbens (NAc) plays a critical role in morphine-induced conditioning. Therefore, the main objective of this study was to evaluate the role of NAc CB1R in tramadol induced conditioning and reinstatement. MAIN METHODS: In the present experiment, the effect of NAc CB1 receptors on tramadol induced conditioning was tested by microinjecting of arachidonylcyclopropylamide (ACPA, CB1R agonist) and AM 251 (CB1R inverse agonist) in the NAc during tramadol-induced conditioning in the adult male Wistar rats. In addition, the role of NAc CB1R in the reinstatement was also evaluated by injecting ACPA and AM 251 after a 10-days extinction period. KEY FINDINGS: The obtained data revealed that the administration of tramadol (1,2, and 4 mg/kg, ip) dose-dependently produced conditioned place preference (CPP). Moreover, intra-NAc administration of ACPA (0.25, 0.5, and 1 µg/rat) dose-dependently induced conditioning, while the administration of AM-251 (30, 60, and 120 ng/rat) induced a significant aversion. In addition, the administration of a non-effective dose of AM251 during tramadol conditioning inhibited conditioning induced by tramadol. On the other hand, the administration of ACPA after extinction induced a significant reinstatement. Notably, the locomotor activity did not change among groups. SIGNIFICANCE: Previous studies have shown that tramadol-induced CPP occurs through µ-opioid receptors. The data obtained in the current study indicated that CB1R located in the NAc is involved in mediating conditioning induced by tramadol. Besides, CB1R also plays a vital role in the reinstatement of tramadol-conditioned animals. It might be due to the effect of opioids on enhancing the level of CB1R.

Effect of morphine exposure on novel object memory of the offspring: The role of histone H3 and ΔFosB.

It has been demonstrated that alteration in histone acetylation in the regions of the brain involved in the reward which may have an important role in morphine addiction. It is well established that epigenetic changes prior to birth influence the function and development of the brain. The current study was designed to evaluate changes in novel object memory, histone acetylation and ΔFosB in the brain of the offspring of morphine-withdrawn parents. Male and female Wistar rats received morphine orally for 21 following days. After ten days of abstinent, they were prepared for mating. The male offspring of the first parturition were euthanized on postnatal days 5, 21, 30 and 60. The novel object recognition (NOR) test was performed on adult male offspring. The amount of acetylated histone H3 and ΔFosB were evaluated in the prefrontal cortex (PFC) and hippocampus using western blotting. Obtained results indicated that the discrimination index in the NOR test was decreased in the offspring of morphine-withdrawn parents as compared with morphine-naïve offspring. In addition, the level of acetylated histone H3 was decreased in the PFC and hippocampus in the offspring of morphine-withdrawn parents during lifetime (postnatal days 5, 21, 30 and 60). In the case of ΔFosB, it also decreased in these regions in the morphine-withdrawn offspring. These results demonstrated that parental morphine exposure affects NOR memory, and decreased the level of histone H3 acetylation and ΔFosB in the PFC and hippocampus. Taken together, the effect of morphine might be transmitted to the next generation even after stop consuming morphine.

Effects of Maternal Deprivation on Anxiety, Depression, and Empathy in Male and Female Offspring of Wistar Rats in the Face of Novel Objects.

BACKGROUND: Early life stress (ELS) models such as maternal deprivation (MD) are used to investigate behavioral changes in rodents under stressful situations. MD is a situation in which rat pups are separated from the dam; MD has different paradigms. The purpose of this research is to evaluate the effects of maternal deprivation on anxiety, depression, and empathy in adult Wistar rats. MATERIALS AND METHODS: MD was applied to pups as per specifically designed protocol to compare rats of the control group with maternal deprivation rats and also the group, which faced novel objects. Each group consisted of eight rats. In this study, separation started from postnatal day (PND) 14 for various periods up to PND 60. EPM test was undertaken to measure anxiety; moreover, FST was used to indicate levels of depression. Also, changes in the empathy ratio were also demonstrated. One-way analysis of variance (ANOVA), Tukey's post hoc analysis, and t-test were applied to analyze the results. RESULTS: MD-treated rats showed a significant decrease in anxiety and empathy indexes compared with those in the control group (P<0.05). However, MD significantly increased depression in both male and female rats (P<0.05). Finally, exposure to novel objects decreased depression but did not have any effect on anxiety and empathy levels in MD rats (P<0.05). CONCLUSION: ELS may lead to various states of mood and behavior in adulthood. According to the findings of this study, depression increases due to MD, though both anxiety and empathy decrease in both male and female Wistar rats. Moreover, exposure to novel objects decreases depression, while anxiety and empathy do not change significantly with exposure to novel objects.

The role of calcium-calmodulin-dependent protein kinase II in modulation of spatial memory in morphine sensitized rats.

It has been shown that drug addiction and memory system are related but the signaling cascades underlying this interaction is not completely revealed yet. It has been demonstrated that binding of Calcium-calmodulin-dependent protein kinase II (CaMKII) to NMDA receptor is important in the memory process. The main objective of the study was to evaluate the role of CaMKII on the spatial memory of rats which previously were sensitized by morphine. The effect of CaMKII inhibitor (KN-93) on memory changes was investigated by hippocampal microinjection of KN-93 on the morphine-sensitized rats. Also, the role of the NMDA receptor in memory retention by KN-93 on the morphine sensitized rat was investigated with NMDA agonist and antagonist. Sensitization was induced by morphine injection (once daily for 3 days) followed by 5 days free of the drug before the trial phase. For the evaluation of spatial memory, the Morris Water Maze test (MWM) was used. Results showed that pre-trial administration of morphine, induced amnesia in MWM (p < 0.05). Also, three days pretreatment with morphine (20 mg/kg) followed by five days washout period, caused to enhance memory retrieval in confront with a pre-trial challenging dose of morphine (5 mg/kg). In addition, KN-93 administration during induction phase in morphine sensitization phenomena facilitated morphine-induced memory retention. In addition, inhibition of the NMDA receptor and KN-93 during the induction phase did not improve memory. However; intra-CA1 co-administration of KN-93 and NMDA during the induction phase of morphine sensitization resulted in improving spatial memory. It can be concluded that the effect of CaMKII on memory retention in morphine-sensitized rats depends on NMDA receptor.