scholarly journals Effects of Chlorogenic Acid on Epileptic Behavior and mRNA Expressions of Brain Derived Neurotrophic Factor in the Brain of Aged Rats

2021 ◽  
Author(s):  
Hossain Soleimani ◽  
Mohammad Ebrahim Rezvani ◽  
Zainab Hafizi-Barjin ◽  
Mansour Esmaeilidehaj ◽  
Fatemeh Zaremehrjerdi
Keyword(s):  
Chlorogenic Acid ◽  
Clonic Seizure ◽  
Tonic Clonic Seizure ◽  
Control Group ◽  
Mrna Levels ◽  
Aged Rats ◽  
Bdnf Gene ◽  
Histological Changes ◽  
Male Wistar Rats ◽  
The Brain

Introduction: The present study was conducted to evaluate the effect of chlorogenic acid (CA) and Diazepam (DZP) on epileptic complication that induced by repetitive intra-peritoneal injections of pentylenetetrazle (PTZ) in aged rats. Methods: Twenty-four month-old male Wistar rats (age > 12 months, 300-350 g) were divided in 4 experimental groups. Animal in control group (PTZ + Vehicle) received only PTZ. Animal in treated groups (PTZ + DZP, PTZ + CA10 and PTZ + 25) received diazepam 2 mg/kg, CA 10 mg/kg, or CA 25 mg/kg. All drugs injection were performed  30 min prior to each PTZ injection. Epilepsy was induced by injection of subconvulsive dose of PTZ every other day until the rats were completely kindled or epileptic. After each PTZ injection, animal was monitored for 40 min and epileptic behaviors were scored. At the end of the study, rats were sacrificed and the brains removed for evaluation of histological changes and Brain Derived Neurothrophic Factor (BDNF) gene expression. Results: CA at dose of 25 mg/kg reduced percent of generalized tonic-clonic seizure (GTCS) in aged rats (24%) in compared to control group (50%) (p < 0.05). The latencies to the start of GTCS were decreased in both dose of CA (p < 0.05). Also, the percent of survived neurons in rats treated with CA (154%) were significantly higher relative to that of control animals (100%) (p < 0.05). The mRNA levels of BDNF significantly increased in CA treated rats (p < 0.05). Conclusion: Hence, these findings revealed that CA have antiepileptic, neuroprotective and trophic effects in aged rats. CA can protect aged brain from deteriorative processes and save neurons during epilepsy in rats.

scholarly journals Sevoflurane and fentanyl exert protective effect on cognitive function in aged rats via regulation of inflammatory response in the brain

2021 ◽  
Vol 18 (5) ◽  
pp. 961-965
Author(s):  
Xin Zhao ◽  
Peixiang Li
Keyword(s):  
Cognitive Function ◽  
Inflammatory Response ◽  
Control Group ◽  
Vascular Endothelial ◽  
Aged Rats ◽  
Tnf Α ◽  
Male Wistar Rats ◽  
Factor Α ◽  
Endothelial Growth Factor ◽  
The Brain

Purpose: To investigate the effects of sevoflurane and fentanyl on cognitive function in aged rats, and to determine the mechanism of action. Methods: A total of 160 adult male Wistar rats were randomly assigned to four groups of 40 rats each. With the exception of control, the rats were surgically operated on. Sevoflurane group received sevoflurane (2 %) via inhalation for 2 h/day for 7 days, while the fentanyl group received fentanyl (50 µg/kg body weight) for 1 h via their tail veins for 7 days. The cognitive function of the rats was evaluated by shuttle box and Morris water maze (MWM) tests, while interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and tumor necrosis factor α (TNF-α) were evaluated using ELISA kits. Results: The learning and memory latencies of the rats were significantly prolonged in surgery, with prolongation greater in sevoflurane and fentanyl groups than in control group; however, the latencies were significantly shorter in sevoflurane and fentanyl groups than in surgery group (p < 0.05). The levels of VEGF, IL-6 and TNF-α were significantly higher in the surgery, sevoflurane and fentanyl groups than in control group (p < 0.05). Conclusion: Sevoflurane and fentanyl improve cognitive function in aged rats via a mechanism involving the regulation of inflammatory response in the brain.

Chronic immobilization stress induces anxiety-related behaviors and affects brain essential minerals in male rats

2020 ◽  
pp. 1-8
Author(s):  
Zafer Sahin ◽  
Alpaslan Ozkurkculer ◽  
Omer Faruk Kalkan ◽  
Ahmet Ozkaya ◽  
Aynur Koc ◽  
...  
Keyword(s):  
Immobilization Stress ◽  
Central Area ◽  
Essential Elements ◽  
Male Rats ◽  
Control Group ◽  
Male Wistar Rats ◽  
Swimming Test ◽  
The Brain ◽  
Center Area ◽  
Chronic Immobilization Stress

Abstract. Alterations of essential elements in the brain are associated with the pathophysiology of many neuropsychiatric disorders. It is known that chronic/overwhelming stress may cause some anxiety and/or depression. We aimed to investigate the effects of two different chronic immobilization stress protocols on anxiety-related behaviors and brain minerals. Adult male Wistar rats were divided into 3 groups as follows ( n = 10/group): control, immobilization stress-1 (45 minutes daily for 7-day) and immobilization stress-2 (45 minutes twice a day for 7-day). Stress-related behaviors were evaluated by open field test and forced swimming test. In the immobilization stress-1 and immobilization stress-2 groups, percentage of time spent in the central area (6.38 ± 0.41% and 6.28 ± 1.03% respectively, p < 0.05) and rearing frequency (2.75 ± 0.41 and 3.85 ± 0.46, p < 0.01 and p < 0.05, respectively) were lower, latency to center area (49.11 ± 5.87 s and 44.92 ± 8.04 s, p < 0.01 and p < 0.01, respectively), were higher than the control group (8.65 ± 0.49%, 5.37 ± 0.44 and 15.3 ± 3.32 s, respectively). In the immobilization stress-1 group, zinc (12.65 ± 0.1 ppm, p < 0.001), magnesium (170.4 ± 1.7 ppm, p < 0.005) and phosphate (2.76 ± 0.1 ppm, p < 0.05) levels were lower than the control group (13.87 ± 0.16 ppm, 179.31 ± 1.87 ppm and 3.11 ± 0.06 ppm, respectively). In the immobilization stress-2 group, magnesium (171.56 ± 1.87 ppm, p < 0.05), phosphate (2.44 ± 0.07 ppm, p < 0.001) levels were lower, and manganese (373.68 ± 5.76 ppb, p < 0.001) and copper (2.79 ± 0.15 ppm, p < 0.05) levels were higher than the control group (179.31 ± 1.87 ppm, 3.11 ± 0.06 ppm, 327.25 ± 8.35 ppb and 2.45 ± 0.05 ppm, respectively). Our results indicated that 7-day chronic immobilization stress increased anxiety-related behaviors in both stress groups. Zinc, magnesium, phosphate, copper and manganese levels were affected in the brain.

Expression and Significance of Lipin1 and AMPKα in Hepatic Insulin Resistance in Diet-Induced Insulin Resistance Rats

2012 ◽  
Vol 120 (02) ◽  
pp. 84-88 ◽  
Author(s):  
S. Chen ◽  
X. Zhuang ◽  
Y. Liu ◽  
A. Sun ◽  
C. Chen
Keyword(s):  
Insulin Resistance ◽  
High Fat Diet ◽  
Diet Group ◽  
Hepatic Insulin Resistance ◽  
Control Group ◽  
Mrna Levels ◽  
Ampk Signaling ◽  
Protein Levels ◽  
Obese Rats ◽  
Male Wistar Rats

AbstractLipin1, a lately indentified adipokine, may link obesity with insulin resistance and diabetes. The present study aimed to investigate the changes and significance of lipin1 expression and lipin1-AMPK signaling in diet-induced hepatic insulin resistance.24 4-week-old Male Wistar rats were randomly divided into 2 groups: (1) control group (CO), (2) high-fat diet group (HF). Insulin sensitivity was evaluated by hyperinsulinemic-euglycemic clamp technique. The mRNA levels of α1 and α2 subunit of AMPKα as well as Lipin1 were measured using Real-time RT-PCR. The activities of AMPKα and Akt were evaluated by detection of p-AMPKα (Thr-172) and p-Akt (ser473) by Western blot.After treatment of 4 months, HF group showed significantly increased levels of body weight, fasting plasma glucose and insulin levels; Plasma and liver total cholesterol (TC), triglycerides (TG) levels were also markedly elevated; Lipin1 expression at both mRNA and protein levels were significantly deceased. Compared with CO group, the mRNA and protein levels of AMPKα1 and AMPKα2 were not changed, whereas the p-AMPK (Thr-172) and p-AKT (ser473) levels in liver were significantly decreased in HF group.These findings indicated that the decrease in lipin1 expression and AMPKα activation may contribute to hepatic insulin resistance in diet-induced obese rats.

scholarly journals Transcriptional Levels of Autophagy and UPR Markers in the Brain and Liver of Pregnant Rats

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Saeed Alizadeh ◽  
Ghasem Ghasempour ◽  
Elnaz Golestaneh ◽  
Yasaman Safian Isfahani ◽  
Arya Emami ◽  
...  
Keyword(s):  
Er Stress ◽  
Control Group ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Unfolded Protein ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Autophagy Pathway ◽  
Protein Response ◽  
The Brain

Background: Pregnancy is associated with oxidative stress that results in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Prolonged-unalleviated ER stress causes the activation of the autophagy pathway via UPR. Expression of genes encoding glucose-regulated protein 78 (GRP78) and BECLIN1 are induced in UPR and autophagy. Objectives: We studied the mRNA expression of the aforementioned genes in the liver and brain of Nulligravida versus saline and ethanol-treated pregnant rats. Methods: Control pregnant rats were orally treated with normal saline, and test animals received ethanol 250 mg/kg or resveratrol 120 mg/kg from day 1 to day 21 of gestation. Nulligravida rats treated by saline comprised the non-pregnant control group. On day 21, mRNAs encoding GRP78 and BECLIN1 were extracted from the liver and brain tissues and assessed using real-time PCR. Results: Our results showed that the level of transcripts encoding GRP78 and BECLIN1 was higher in the liver of pregnant rats compared to Nulligravida ones. Further, ethanol decreased the mRNA levels of GRP78 and BECLIN1 in the liver of pregnant rats, an effect that was reversed by resveratrol. Levels of GRP78 transcripts were decreased, and those of BECLIN1 remained unchanged in the brain of ethanol exposed pregnant rats. Conclusions: Levels of mRNAs for GRP78 and BECLIN1 are up-regulated during pregnancy. These levels are reduced in the liver of ethanol-treated rats, and resveratrol compensates these effects.

scholarly journals The Antioxidant, Anti-Inflammatory, Pathological, and Behavioural Effects of Medicago sativa L. (Alfalfa) Extract on Brain Injury Caused by Nicotine in Male Rats

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
M. Raeeszadeh ◽  
P. Mortazavi ◽  
R. Atashin-Sadafi
Keyword(s):  
Medicago Sativa ◽  
Inflammatory Responses ◽  
The Other ◽  
Male Rats ◽  
Control Group ◽  
Elevated Plus Maze Test ◽  
Male Wistar Rats ◽  
Medicago Sativa L ◽  
Histopathological Studies ◽  
The Brain

Nicotine is one of the most important compounds in cigarette which can cause changes in the concentration of neurotransmitters and damage to the nervous system. The aim of this study was to investigate the effect of the hydroalcoholic extract of Medicago Sativa L. (alfalfa) on controlling nicotine-induced brain damage and anxiety behaviour in rats. Forty-two male Wistar rats were randomly divided into six equal groups and treated daily as follows: a control group, T1 and T2 groups where animals were subcutaneously injected 250 and 500 mg/kg alfalfa extract, respectively, T3 and T4 groups where animals were injected subcutaneously 0.2 mg/kg nicotine and 250 and 500 mg/kg alfalfa extract, and T5 group in which only nicotine at the dose of 0.2 mg/kg was injected. At the end of the period after weighing, the elevated plus-maze test was taken from the animals. Serum assay was conducted to measure TCA, IL-1, and TNFα, and half of the brain tissue was used to measure oxidative stress parameters (GPx, SOD, TAC, and MDA) and the other parts were used for histopathological studies. Body weight in the T5 group was significantly different from that of the other groups. The time and number of open arms reduced in the T5 group. The duration and number of times in the open arm significantly decreased in the treated groups in a dose-depended manner. Malondialdehyde concentration was the highest in the nicotine group and the lowest in T2. The concentration of GPx and SOD was significantly increased in the presence of alfalfa extract in nicotine groups. TNFα and IL-1 in the T5 group showed a significant increase compared to the other groups. Moreover, the number of neurons and the level of necrotic neurons and gliosis significantly decreased and increased in the nicotine group, respectively, while these histopathological damages improved by treatment with alfalfa extract in T3 and T4 groups. Alfalfa extract can have a significant dose-dependent therapeutic effect on inducing oxidative damage and inflammatory responses of nicotine in the brain and reducing anxiety behaviours.

scholarly journals D-limonene in diabetic rats

2021 ◽  
Vol 10 (3) ◽  
pp. e24-e24
Author(s):  
Shahrokh Bagheri ◽  
Mostafa Moradi Sarabi ◽  
Mohammadreza Gholami ◽  
Vahideh Assadollahi ◽  
Reza Mohammadrezaei Khorramabadi ◽  
...  
Keyword(s):  
Real Time ◽  
Kidney Tissue ◽  
Serum Levels ◽  
Diabetic Control ◽  
Serum Glucose ◽  
Diabetic Rats ◽  
Tissue Homogenate ◽  
Control Group ◽  
Mrna Levels ◽  
Male Wistar Rats

Introduction: Diabetes mellitus (DM) is a multi-factorial condition associated with oxidative stress. Limonene, as a plant-derived antioxidant, can be used for treating DM. Objectives: An investigation on antioxidant effects in diabetic rats exposed to D-limonene. Materials and Methods: Sixty male Wistar rats were categorized into six groups as follows: control (healthy rats), diabetic control (untreated diabetic rats), sham glibenclamide, diabetic glibenclamide, sham limonene, and finally diabetic limonene. Alloxan (100 mg/dL) was infused intraperitoneally to induce type 1 diabetes in rats. Rats in certain groups were given limonene (100 mg/dL) and glibenclamide (10 mg/dL) orally for 8 weeks. Subsequently, animals were killed, and their kidneys were removed. Serum levels of biochemical factors (including serum creatinine, urea, and glucose) were determined, and factors such as nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), and myeloperoxidase (MPO) were measured in kidney tissue homogenate. The gene expression and enzymatic activity of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) in the kidney were measured by real-time polymerase chain reaction (real-time PCR) and spectrophotometry, respectively. Results: Limonene treatment significantly decreased serum glucose, creatinine, and urea. Additionally, MDA, MPO, and NO significantly decreased while GSH increased after treatment with limonene. Real-time RT-PCR showed significant elevation (P<0.05) in mRNA levels of GPx, CAT, and SOD in the limonene-treated compared with the diabetic control group. Conclusion: Our results demonstrated that limonene as an herbal antioxidant had better effects on antioxidant markers compared to glibenclamide in rat models of diabetes.

Evaluation of Prophylactic and Therapeutic Roles of NAcetylcysteine on Biochemical and Oxidative Changes Induced by Acute Poisoning of Diazinon in Various Rat Tissues

2020 ◽  
Vol 14 (2) ◽  
pp. 100-116
Author(s):  
Kavoos Tahmasebi ◽  
Mahvash Jafari ◽  
Farideh Izadi ◽  
Alireza Asgari ◽  
Hoosein Bahadoran ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Biochemical Parameters ◽  
Corn Oil ◽  
Acetylcholinesterase Activity ◽  
Acute Poisoning ◽  
Control Group ◽  
Rat Tissues ◽  
Oxidative Stress Biomarkers ◽  
Male Wistar Rats ◽  
The Brain

Background: Exposure to diazinon (DZN) as an organophosphorus insecticide is associated with reducing the antioxidant capacity of cells. N-acetyl cysteine (NAC) is widely used in clinics to treat several diseases related to oxidative stress. Objective: The current study was aimed to evaluate the prophylactic and therapeutic roles of NAC on biochemical and oxidative changes induced by acute poisoning of DZN in various tissues of male Wistar rats. Methods: Thirty rats were divided into five groups: control group received corn oil as DZN solvent; DZN group received 100 mg/kg of DZN; NAC group received 160 mg/kg of NAC; NAC-DZN and DZN-NAC groups received 160 mg/kg of NAC before and after 100 mg/kg of DZN injection, respectively. Plasma and various tissues were prepared and evaluated for the measurement of the biochemical parameters and oxidative stress biomarkers. Results: Both prophylactic and therapeutic treatments by NAC ameliorated the increased lipid peroxidation and decreased glutathione level and superoxide dismutase, catalase and glutathione S-transferase activities in tissues (P<0.05). Moreover, treatment with the NAC caused a significant reduction in DZN-induced high levels of plasma biochemical parameters. Furthermore, acetylcholinesterase activity was positively correlated with both LDH (P=0.000) activity and GSH (P=0.001) level and negatively correlated with MDA (P=0.009) level in the brain. Conclusion: Results suggest that NAC could effectively ameliorate the DZN-induced oxidative stress and cholinergic hyperactivity in various tissues especially in the brain, through free radicals scavenging and GSH synthesis. Prophylactic approach exerted a stronger protective effect compared to a therapeutic treatment.

scholarly journals Involvement of N-methyl-D-aspartate receptors and nitric oxide in the anticonvulsant effects of dantrolene against pentylenetetrazole-induced seizures in mice

2017 ◽  
Vol 04 (02) ◽  
pp. 112-118
Author(s):  
Akbarzadeh Samad ◽  
Heidary Fatemeh ◽  
Keshavarz Mojtaba
Keyword(s):  
Nitric Oxide ◽  
Nmda Receptors ◽  
Clonic Seizure ◽  
Negative Control ◽  
No Synthase ◽  
Tonic Clonic Seizure ◽  
Control Group ◽  
No Donor ◽  
Clonic Seizures ◽  
Anticonvulsant Effects

Abstract Objective N-methyl-d-aspartate (NMDA) receptors and nitric oxide (NO) have important roles in the pathology and treatment of pentylenetetrazole (PTZ)-induced seizures. We aimed to show the involvement of these two systems in the anticonvulsant effects of dantrolene against PTZ-induced seizures. Methods The male albino Swiss strain of mice (N = 56) randomly allocated to the seven separate groups and treated with dantrolene (40 mg/kg), dantrolene (40 mg/kg) + L-arginine (100 mg/kg, a NO donor), dantrolene (40 mg/kg) + N-Nitroarginine methyl ester (L-NAME) (100 mg/kg, a NO synthase inhibitor), dantrolene (40 mg/kg) + NMDA (50 mg/kg), dantrolene (40 mg/kg) + MK801 (1 mg/kg, a selective NMDA antagonist), Diazepam (5 mg/kg, the positive control) and saline (the negative control). Seizures were induced by intraperitonial injection of PTZ (90 mg/kg). The onsets of clonic and tonic-clonic seizures, as well as the death of animals, were recorded. Results Dantrolene significantly increased the onset of clonic, tonic-clonic seizures and death of animals challenged with PTZ. The onset of tonic-clonic seizure in animals treated with dantrolene alone and dantrolene + L-NAME was higher than the control group. In contrast, the onset of tonic-clonic seizure in the animals treated with dantrolene + L-arginine was significantly lower than the dantrolene-treated group. The onset of clonic and tonic-clonic seizures in animals treated with dantrolene + MK801 were significantly higher than the control and dantrolene + NMDA groups. Conclusion Dantrolene protected animals against PTZ-induced seizures and mortality. The inhibition of NO synthase and NMDA receptors may contribute to the dantrolene anticonvulsant effects on the PTZ-induced seizure.

scholarly journals Effect of Artesunate Treatment on Some Brain Biomolecules and its Behavioral Implication

1970 ◽  
Vol 4 (2) ◽  
pp. 44-50
Author(s):  
MB Ekong ◽  
AO Igri ◽  
TB Ekanem
Keyword(s):  
Therapeutic Dose ◽  
Wistar Rats ◽  
Tap Water ◽  
Control Group ◽  
Anthropometric Parameters ◽  
Whole Brain ◽  
Long Duration ◽  
Male Wistar Rats ◽  
Group A ◽  
The Brain

Background: Artesunate (AS) is an artemisinin antimalarial drug used as a single drug or in combination with other antimalarials. Objective: This study was to find its effect on some brain biomolecules and behavioural activities in Wistar rats. Methods: Forty adult male Wistar rats weighing between 150- 180g were divided into four groups of A, B, C and D with 10 animals each. Group A served as the control that received tap water, while groups B, C and D served as the experimental groups that received 2.85mg/kg (therapeutic dose-TD) and 5.71mg/kg (high pharmacologic dose-HPD) of AS per day for 3 days, and 2.85mg/kg (long duration therapeutic dose -LDTD) of AS per day for six days respectively. Half of the dose was administered twelve hourly (twice a day), and twelve hours after the last treatments, behaviour test using the ‘open field maze' was carried out. Immediately after, the animals were sacrificed with chloroform anaesthesia and the whole brain removed and weighed. Whole brain homogenates were used to determine brain total protein (TP), triacylglycerol (TAG) and cholesterol (CH).Data were analyzed statistically by ANOVA and Tukey-Kramer Multiple Comparative Test as applicable. Results: There were no difference (p<0.05) between the experimental groups and the control group in the anthropometric parameters and behavioural activities. In the brain biomolecules concentration, TP was lower in concentration in the HPD group, TAG was lower in concentration in the LDTD group, while the HPD and LDTD groups had lower CH concentration compared to the control. In all the parameters studied no difference was found between the TD group and the control. Conclusion: AS at recommended dose may not affect some behaviour and brain biomolecule concentration, unlike when taken in excess of dose and or time. Even at these doses/time there may have been no behavioural manifestation. Key words: Artesunate, Brain, Anthropometry, Behavior, Biomolecules, Rats DOI: 10.3329/jbsp.v4i2.4171 J Bangladesh Soc Physiol. 2009 Dec;4(2): 44-50  

scholarly journals The Decrease In The Anxiolytic Effect of Morphine After Repeated Use of The Drug In Rats is Associated With Inflammatory Cytokine Signaling Pathways In The Prefrontal Cortex

2021 ◽  
Author(s):  
Shamseddin Ahmadi ◽  
Shiva Mohammadi Talvar ◽  
Kayvan Masoudi ◽  
Mohammad Zobeiri
Keyword(s):  
Gene Expression ◽  
Prefrontal Cortex ◽  
Mrna Level ◽  
Anxiolytic Effect ◽  
Cytokine Signaling ◽  
Control Group ◽  
Repeated Injection ◽  
Mrna Levels ◽  
Male Wistar Rats ◽  
Repeated Use

Abstract We aim to examine anxiety-like behaviors and expression of specific genes complicated in neuroinflammation in the prefrontal cortex (PFC) after repeated use of morphine. A group of male Wistar rats received injections of morphine (10 mg/kg) twice a day for eight days while a control group received saline (1 ml/kg) instead of morphine. On days 1 and 8, anxiety-like behaviors were evaluated using a light/dark box test. On day 8, opioid dependence was confirmed by measuring the behavioral expression of morphine withdrawal precipitated with naloxone. Expression of neuroinflammation genes were also evaluated at mRNA levels in the PFC on day 8. The results revealed that morphine induced anxiolytic-like effects on day 1, which significantly decreased after the repeated injection of the drug on day 8. The results also revealed that repeated morphine injection significantly increased the mRNA level of Il1, Tnfα, and Il6 but decreased Il1r and Tnfr while increased Il6r in the PFC. The gene expression results also revealed a significant decrease in Tlr1 but not in Tlr4 in the PFC of morphine-dependent rats. Although Erk1 expression had no significant alteration but p38 increased and Jnk3 decreased significantly in the PFC in morphine-dependent rats. Creb and Nfkb significantly increased but Fos expression decreased. Let-7c, mir-133b, and mir-365 also significantly increased in the PFC in morphine-dependent rats. We conclude that the alteration in neuroinflammatory pathways at gene expression level in the PFC may party underlie neuroadaptive changes leading to the decrease in anxiolytic effect of morphine in dependent rats.

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