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Objective To observe the change of dopamine receptor in ventral tegmental area (VTA), nucleus accumbens (NAc) and prefrontal cortex (PFC) in morphine-dependent rats, and the regulation effect of electroacupuncture (EA). Method Thirty male SD rats were randomized into a control group, a model group and an EA group, 10 rats each. Morphine-dependent rat models were induced by morphine self-administration. In the EA group, bilateral Jiaji points (EX-B2) of T5 and L2 were selected, and the EA intervention lasted 4 d. Western blotting method was adopted to observe the change of the contents of dopamine receptor D1 and D2 in VTA, NAc and PFC of the rats. Result After the intervention, compared to the model group, the morphine intake was reduced significantly in the EA group (P<0.05); the level of D2 declined significantly in VTA of rats in the model group (P<0.05); the level of D1 increased significantly and D2 declined significantly in NAc of rats in the model group (P<0.05); the level of D1 declined significantly and D2 increased significantly in PFC of rats in the model group (P<0.05); EA produced regulation effect on the altered contents of D1 and D2 in the cerebral areas mentioned above, approaching the normal level. Conclusion EA can inhibit the hunger of addiction rats for morphine to some extent; the contents of dopamine receptors in dopamine projection pathway will take adaptive changes after morphine addiction, while EA can regulate the abnormal expressions of dopamine receptors, producing a protective effect on dopamine receptors of morphine-dependent rats.
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Objective To evaluate the role of μ-δ heterodimer in down-regulation of the expression of excitatory amino acid transporter 3 (EAAT3) in hippocampi caused by reinstatement of morphine-induced conditioned place preference (CPP) in rats.Methods Thirty-two healthy clean-grade male Sprague-Dawley rats,weighing 200-240 g,were assigned into 4 groups (n =8 each) using a random number table method:control group (group C),extinction group (group E),reinstatement group (group R) and reinstatement plus interference plasmid group (group RI).The model of morphine-induced CPP was established,and extinction of CPP was gradually induced by stopping administration.A small dose of morphine 5 mg/kg was intraperitoneally injected again to induce CPP reinstatement,and dwell time around the medicine box was recorded.μ-δ heterodimer interference plasmid 5 μl was injected into the lateral cerebral ventricle after successful establishment of CPP model in group RI.The content of glutamate (Glu) in hippocampi was measured using high-performance liquid chromatography.The EAAT3 expression in hippocampal CA1 and CA3 regions was detected using Western blot.Results Compared with group C,no significant change was found in the dwell time around the medicine box or content of Glu in hippocampi (P＞0.05),and the expression of EAAT3 in hippocampal CA1 and CA3 regions was significantly up-regulated in group E,and the dwell time around the medicine box was significantly prolonged,the content of Glu in hippocampi was increased (P＜0.05),and no significant change was found in the expression of EAAT3 in hippocampal CA1 and CA3 regions in group R (P＞0.05).Compared with group E,the dwell time around the medicine box was significantly prolonged,the content of Glu in hippocampi was increased,and the expression of EAAT3 in hippocampal CA1 and CA3 regions was down-regulated in group R (P＜0.05).Compared with group R,the dwell time around the medicine box was significantly shortened,the content of Glu in hippocampi was decreased,and the expression of EAAT3 in hippocampal CA 1 and CA3 regions was upregulated in group RI (P＜0.05).Conclusion μ-δ heterodimer is involved in down-regulation of EAAT3 expression in the hippocampus caused by reinstatement of morphine-induced CPP in rats.
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Objective:To observe the effect of reward alteration following acupuncture for morphine withdrawal rats on the behavior and neuronal discharges in the medial prefrontal cortex (mPFC). Methods:The Sprague-Dawley (SD) rats were randomly allocated into a model group, a confinement group, an electroacupuncture (EA) group, and a control group. Rats with morphine addiction were made by intraperitoneal injection of naloxone (same dose injection of saline for rats in the control group), followed by a 2-week morphine withdrawal. Acupuncture and confinement were completed during the morphine withdrawal period. Upon withdrawal, the rats received conditioned place preference (CPP) training and open field test. The multi-channel neural signal processor was used in the electrophysiological experiment to measure the neuronal discharges in different subareas of prefrontal cortex in CPP box and aversion box. Results:Rats in the model group and the confinement group spent longer period of time in CPP box than those in the EA group and the control group (allP<0.01); there was no statistically significant difference between the EA group and the control group. The total distances of movement by rats in the model group and the confinement group were longer than those in the EA group and the control group (allP<0.01). The mPFC neuronal discharge frequencies were compared between morphine preference box and aversion box. The mPFC neuronal discharge frequencies in the model group and the confinement group were higher than those in the EA group and the control group (allP<0.05); there was no statistically significant difference between the EA group and the control group. Conclusion:Acupuncture can effectively interfere with the reward alteration following morphine withdrawal, possibly because of its involvement with the mPFC neuronal discharges.
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Objective To observe expression levels of N-methyl-D-aspartate (NMDA) receptor and cholecystokinin (CCK) in the hippocampus and spinal cord in morphine withdrawal or tolerance mice treated by electroacupuncture or catgut embedding and explore the difference between the regulating effects of electroacupuncture and catgut embedding on morphine withdrawal and tolerance.Methods Fifty-six male C57BL/6J mice were randomly allocated to withdrawal control, withdrawal model, withdrawal catgut embedding and withdrawal electroacupuncture groups, and tolerance control, tolerance model, tolerance catgut embedding and tolerance electroacupuncture groups, 7 mice in each group. A model of morphine withdrawal was made by subcutaneous injection of morphine hydrochloride using 7-day increasing addiction method. The withdrawal control group was injected with an equal volume of normal saline at the same time points. In the withdrawal electroacupuncture group, electroacupuncture at bilateral points Shenshu was performed using a Han’s acupoint nerve stimulation device (HANS-200) at 15 min after an injection of morphine hydrochloride. In the withdrawal catgut embedding group, 0.5 cm chromic catgut was embedded in bilateral points Shenshu at 15 min after an injection of morphine hydrochloride. Addiction was promoted by intraperitoneal injection of naloxone 4 mg/kg at 10 o’clock on the seventh day’s morning and Withdrawal reactions were observed in the mice. The score was recorded using the Ryuta Tomoji opioid withdrawal symptoms evaluation scale. NMDA receptor and CCK contents in the hippocampus and spinal cord were measured by enzyme-linked immunosorbent assay (ELISA). A model of morphine tolerance was made by subcutaneous injection of morphine 10 mg/kg. The tolerance control group was injected with tolerance normal saline 10 ml/kg at the same time. In the tolerance catgut embedding group, catgut was embedded in point Shenshu at the first day after model making. In the tolerance electroacupuncture group, point Shenshu was given electroacupuncture at the first day after model making. After seven days of treatment, NMDA receptor and CCK contents in the hippocampus and spinal cord were measured by ELISA.Results There were statistically significant differences in hippocampal NR2B and CCK expressions between the withdrawal model and withdrawal control groups (P<0.05). There was a statistically significant difference in hippocampal NR2B expression between the withdrawal electroacupuncture and withdrawal model groups (P<0.05). There was a statistically significant difference in hippocampal CCK expression between the withdrawal catgut embedding or withdrawal electroacupuncture group and the withdrawal model group (P<0.05). There were statistically significant differences in spinal cord NR2A, NR2B and CCK expressions between the withdrawal model and withdrawal control groups (P<0.05). There were statistically significant differences in spinal cord NR2A and NR2B expressions between the withdrawal electroacupuncture and withdrawal model groups (P<0.05). There were statistically significant differences in hippocampal NR2A, NR2B and CCK expressions between the tolerance model and tolerance control groups (P<0.05). There was a statistically significant difference in hippocampal CCK expression between the tolerance catgut embedding and tolerance model groups (P<0.05). There was a statistically significant difference in hippocampal NR1 expression between the tolerance electroacupuncture group and the tolerance model or tolerance catgut embedding group (P<0.05). There was a statistically significant difference in spinal cord CCK expression between the tolerance catgut embedding or withdrawal electroacupuncture group and the tolerance model group (P<0.05).Conclusions Both catgut embedding and electroacupuncture at point Shenshu have a reducing effect on morphine tolerance and withdrawal. The therapeutic effect of electroacupuncture is better than that of catgut embedding.
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Objective To investigate the changes in 20S proteasome activities in the brain and spinal cord of acute and chronic morphine-dependent mice.Metbods Male ICR mice,weighing 25-30 g,were used in the study.The experiment was performed in 2 parts.In experiment Ⅰ,16 mice were randomly divided into 5 groups (n =8 each) using a random number table:control group (group C) and acute morphine dependence group (AMD group).In experiment Ⅱ,16 mice were randomly divided into 5 groups (n =8 each) using a random number table:control group (group C) and chronic morphine dependence group (CMD group).Acute morphine dependence was induced with morphine 100 mg/kg injected subcutaneously,and the mice were sacrificed 3 h later.Chronic morphine dependence was induced by increasing doses of morphine for 4 days,the initial dose of morphine was 20 mg/kg injected subcutaneously twice a day and was increased by 10 mg/kg every day,the dose of morphine was 10 mg/kg injected subcutaneously on 5th day,and then the mice were sacrificed 1 h later.In group C,the equal volume of normal saline was given instead,and the other treatments were similar to those previously described in morphine dependence groups.After the mice were sacrificed,the hippocampus,prefrontal cortex,striatum and spinalcord were isolated for determination of 20S proteasome activity,measured as chymotrypsin-like (ChT-L),trypsin-like (T-L) and peptidylglutamyl-like hydrolyzing (PGLH) activities.Results Experiment Ⅰ Compared with C group,PGLH activity in the spinal cord and T-L activity in the striatum or prefrontal cortex were significantly weakened in group AMD.There was no significant difference in 20S proteasome activity in the hippocampus between the two groups.Experiment Ⅱ Compared with C group,ChT-L and T-L activities in the spinal cord were significantly weakened,and PGLH activity in the striatum was enhanced in CMD group.There was no significant difference in 20S proteasome activity in the prefrontal cortex and hippocampus between the two groups.Conclusion 20S proteasome activity in the spinal cord and brain is weakened in acute morphine-dependent mice,20S proteasome activity in the spinal cord is weakened,20S proteasome activity in the striatum is enhanced in chronic morphine-dependent mice,these changes have specificity in terms of position and type of activity,and the changes mentioned above may be related to development of morphine dependence in mice.
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Objective To investigate the effect of warm needling on learning and memory abilities and the calmodulin kinaseⅡ (CaMKⅡ) content of prefrontal cortex area in morphine withdrawal rats andexplore the mechanism of its action. Methods Forty clean-grade male SD rats were randomly allocated to control, model, manual needling and warm needling groups, 10rats each. A SD rat model of morphine addiction and withdrawal was made by dorsal subcutaneous injection of day-by-day incremental morphine and rapid withdrawal with Naloxone after addiction. Learning and memory abilities were tested using a Morris water maze and the CaMKⅡ content of prefrontal cortex area was measured by an immunohistochemical method in every group of rats.Results There were statistically significant differences in mean platform escape latency, the number of platform crossing and the CaMKⅡ content of PFC area between the control, manual needling or warm needling group of rats and the model group (P<0.01). There were statistically significant differences in mean platform escape latency, the number of platform crossing and the CaMKⅡ content of PFC area between the warm needling and manual needling groups (P<0.05).Conclusions Warm needling treatment can restore learning and memory abilities in morphine withdrawal rats. The mechanism of its action may be related to an increase in the CaMKⅡ content of prefrontal cortex area.
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Objective To evaluate the changes in the activity of extracellular signal-regulated kinase 5 (ERK5) in the distal cerebrospinal fluid contacting neurons (CSF-CNs) in the mid-brain of morphine dependent rats.Methods Forty-eight male adult Sprague-Dawley rats weighing 230-270 g,were randomly divided into 2 groups (n =24 each) using a random number table:control group (group A) and morphine dependence group (group B).Morphine dependence was induced by increasing doses of subautaneous morphine for 5 consecutive days.The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg everyday until 50 mg/kg on 5th dav.The equal volume of normal saline was injected subcutaneously instead of morphine in group A.On 3rd day after morphine dependence was induced,the distal CSF-CNs in the mid-brain was labeled with 30％ cholera toxin subunit B and horseradish peroxidase compound (CB-HRP) 3 μl injected in the lateral cerebral ventricle in the morning.At 4 h after the last injection of morphine,the segments in which CSF-CNs were located were removed,and CB-HRP positive neurons,phosphor-ERK5 (p-ERK5) positive neurons and CB-HRP/p-ERK5 positive neurons were counted.Results Compared with group A,the number of p-ERK5 and CB-HRP/p-ERK5 positive neurons in the mid-brain was significantly increased (P ＜ 0.05),and no significant change was found in CB-HRP positive neurons in group B (P ＞ 0.05).Conclusion The enhanced activity of ERK5 in the distal CSFCNs in the mid-brain may contribute to the development of morphine dependence in rats.
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Objective To evaluate the role of spinal neuronal extracellular signal-regulated protein kinases 5/cAMP response element binding protein (ERK5/CREB) signaling pathway in withdrawal responses in morphinedependent rats.Methods Ninety-six adult male Sprague-Dawley rats in which intrathecal catheters were successfully placed,weighing 200-250 g,were randomly divided into 4 groups (n =24 each):normal control group (group A),morphine withdrawal group (group B),dimethyl sulfoxide (DMSO) + morphine withdrawal group (group C) and ERK5 inhibitor BIX02188 + morphine withdrawal group (group D).Morphine dependence (MD) was induced by increasing doses of subcutaneous morphine for 6 days.The initial dose of morphine was 10 mg/kg once a day and was increased by 10 mg/kg once a day from 2nd to 5th days until 50 mg/kg on 6th day in B,C and D groups.Morphine withdrawal response (MW) was induced by intraperitoneal naloxone 4 mg/kg at 4 h after last morphine administration in B,C and D groups.In addition,BIX02188 10 μg and 1％ DMSO 10 μl were injected intrathecally at 1 h before naloxone injection in D and C groups,respectively.MW and morphine withdrawal-induced hyperalgesia were scored.The rats were then sacrificed after hyperalgesia was scored and the spinal cord was removed for determination of CREB and phosphorylated CREB (p-CREB) expression.Results Compared with group A,MW and hyperalgesia scores were significantly increased and the expression of p-CREB was up-regulated in B,C and D groups.Compared with group B,MW and hyperalgesia scores were significantly decreased and the expression of p-CREB was down-regulated in D group,and no significant change was found in group C.Conclusion The spinal neuronal ERK5/CREB signaling pathway is involved in withdrawal responses in morphine-dependent rats.
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Objective To investigate the roles of spinal N-methyl-D-aspartic acid receptor-extracellular signal-regulated protein kinases 5 signaling pathway in naloxone-induced withdrawal response in morphine-dependent rats.Methods Ninety-six adult male SD rats weighting 230-250 g were randomly divided into 4 groups:control group,withdrawal group,DMSO group and MK801 group.Rats were subcutaneously injected with morphine.On day 6,rats were injected with naloxone (intraperitoneal) to precipitate morphine withdrawal syndromes.To identify the function of NMDAR-ERK5 signaling pathway in morphine withdrawal,morphine withdrawal-like behavior test and western blot technique were used in this research.The scores of morphine withdrawal symptom,morphine withdrawal-induced allodynia and the activation of ERK5 in spinal cord were observed after intrathecal injection of MK801.Results There was no withdrawal symptoms and withdrawal-induced allodynia in group A after intraperitoneal injection of naloxone.Compared with group A,withdrawal score (45.2±7.3),score of withdrawal-induced allodynia (14.4±3.7) of group B,withdrawal score (44.7±6.2),score of withdrawal-induced allodynia (13.2±2.7) of group C and withdrawal score (28.3±1.6),score of withdrawal-induced allodynia (5.9± 1.1) of group D were significantly increased (P＜ 0.05).Compared with group C,the total withdrawal score (28.3 ± 1.6),score of withdrawal-induced allodynia (5.9± 1.1) of group D were significantly decreased (P＜0.05).Compared with group A,the expression of spinal p-ERK5 of group B (12848±621) and group C (12579±396) were significantly increased (P＜0.05).Compared with group C,the expression of spinal p-ERK5 of group D (5 123±546) was significantly decreased (P＜0.05).Condusion The signaling pathway of spinal N-methyl-D-aspartic acid receptor-extracellular signal-regulated protein kinases 5 contributes to naloxone-induced withdrawal response in morphine-dependent rats.
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Objective To investigate the roles of spinal extracellular signal-regulated protein kinases 5 (ERK5) on morphine withdrawal in rats.Methods Ninety-six male and adult SD rats weighting 230-250 g were randomly divided into saline-naloxone-DMSO group (group A),saline-naloxone-BIX02188 group (group B),morphine-naloxone-DMSO group (group C) and morphine-naloxone-BIX02188 group (group D).To set up morphine dependent model,rats were subcutaneously injected with morphine in the increasing dosage method.On day 6,4 h after the injection of morphine,rats were injected with naloxone (intraperitoneal) to precipitate morphine withdrawal syndrome.The scores of morphine withdrawal symptom and morphine withdrawal-induced allodynia were observed after intrathecal injection of ERK5 inhibitor BIX02188.Result There were not withdrawal symptoms and withdrawal-induced allodynia in group A and B after intraperitoneal injection of naloxone.Compared with group A,teeth chatting (7.5± 1.1),wet dog shacks (4.6± 0.7),jump (5.3± 0.7),abnormal position (8.9± 1.9),diarrhea (7.1 ± 1.6),salivation (2.8±0.6),weight loss (7.9±0.9),total withdrawal score (44.8±5.9),score of withdrawalinduced allodynia (14.6±2.4) of group C and teeth chatting (3.1±0.5),wet dog shacks (1.5±0.4),jump (2.2± 0.5),abnormal position (7.9± 1.6),diarrhea (1.8±0.5),salivation (2.8±0.9),weight loss (3.7±0.6),total withdrawal score (23.1± 1.3) and score of withdrawal-induced allodynia (3.5± 1.1) of group D were significantly increased (P＜0.05).Compared with group C,teeth chatting (3.1±0.5),wet dog shacks (1.5±0.4),jump (2.2±0.5),diarrhea (1.8±0.5),weigbt loss (3.7±0.6) and total withdirawal score (23.1±1.3),score of withdrawal-induced allodynia (3.5±1.1) of group D were significantly decreased (P＜0.05).But there was not significant change in abnoral position (7.9±1.6) and salivation (2.8±0.9).Conclusion Inhibition of the activation of spinal cord ERK5 can significantly alleviate withdrawal symptoms of morphine dependent rats by intrathecal injection BIX02188.
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Objective To evaluate the role of extracellular signal-regulated protein kinase 5 (ERK5) in the spinal cord in withdrawal responses in morphine-dependent rats.Methods Ninety-six adult male SpragueDawley rats in which intrathecal catheters were successfully placed,weighing 200-250 g,were randomly divided into 4 groups (n =24 each) using a random number table:normal saline group (group A),withdrawal group (group B),dimethyl sulfoxide (DMSO) group (group C) and ERK5 inhibitor BIX02188 group (group D).Morphine dependence (MD) was induced by increasing doses of subcutaneous morphine for 6 days.The initial dose of morphine was 10 mg/kg once a day and was increased by 10 mg/kg once a day from the 2nd to 5th days until 50 mg/kg on the 6th day in B,C and D groups.Morphine withdrawal response (MW) was induced by intraperitoneal naloxone 4 mg/kg at 4 h after last morphine administration in B,C and D groups.In addition,BIX02188 and 1％ DMSO 10 μl were injected intrathecally at 1 h before naloxone injection in D and C groups,respectively.MW and morphine withdrawal-induced hypemlgesia were scored.The rats were then sacrificed after hyperalgesia was scored and the spinal cord was removed for determination of ERK5 and phosphorylated ERK5 (p-ERK5) expression.Results Compared with group A,MW and hyperalgesia scores were significantly increased and the expression of pERK5 was up-regulated in B,C and D groups (P ＜ 0.05).Compared with group B,MW and hyperalgesia scores were significantly decreased and the expression of p-ERK5 was down-regulated in D group (P ＜ 0.05),and no significant change was found in group C (P ＞ 0.05).Conclusion ERK5 in the spinal cord is involved in withdrawal responses in morphine-dependent rats.
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Objective To observe the protein expression of grow thassociated protein-43 (GAP-43) in mid-brain ventral tegmental area in morphine withdrawal rats at different time, and to evaluate the effect of GAP-43 on morphine withdrawal memory. Methods Rat models of morphine dependent 1 week, 2 weeks and 4 weeks were established by morphine hydrochloride intraperitoneal injection with increasing doses to establish natural withdrawal. The protein expression of GAP-43 in midbrain ventral tegmental area was observed by im munohistochemical staining and the results were analyzed by Im age-Pro Plus 5.1 im-age analysis system . Results With prolongation of dependent time, the expression of GAP-43 was de-creased then increased in midbrain ventral tegmental area . Conclusion GAP-43 could play arole in morphine withdrawal memory in midbrain ventral tegmental area.
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Objective To evaluate the role of extracelluar signal-regulated kinase (ERK)-cyclic AMP response element binding protein (CREB) signaling pathway in the spinal cord in naloxone-induced withdrawal response in morphine-dependent rats.Methods Fifty male adult SD rats,aged 2 months,weighing 200-250 g,in which intrathecal catheters were successfully implanted without complications,were randomly divided into 5 groups (n =10 each):group control (group C); group morphine dependence (group MD); group morphine withdrawal (group MW); group U0126 (ERK signaling pathway blocker); group dimethyl sulfoxide (DMSO,solvent for U0126).Morphine dependence was induced by increasing doses of subcutaneous morphine for 6 days.The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg twice every other day until 50 mg/kg on 6th day in groups MD,MW,U0126 and DMSO.Morphine withdrawal response was induced by intraperitoneal naloxone 4 mg/kg at 4 h after last morphine administration in groups MW,U0126 and DMSO.U0126 150μg (in DMSO 10 μl) and DMSO 10 μl were administered intrathecally at 30 min before naloxone administration in groups U0126 and DMSO respectively.Morphine withdrawal response (0=no withdrawal response,3 =severe response)and touch evoked agitation (0 =no agitation,2 =severe agitation) were observed and scored during 1 h after naloxone administration.The animals were then sacrificed and the spinal cord was removed for determination of the expression of phosphorylated ERK (p-ERK) and phosphorylated CREB (p-CREB) by immuno-histochemistry and Western blot.Results Morphine withdrawal significantly up-regulated the p-ERK and p-CREB expression in group MW compared with group C ( P ＜ 0.05).Withdrawal response score and touch evoked agitation score were significantly increased in groups MW,U0126 and DMSO as compared with group MD ( P ＜ 0.05).U0126 pretreatment significantly attenuated naloxone-induced increase in withdrawal response score and touch evoked agitation score and down-regulated p-ERK and p-CREB expression in group U0126 as compared with group MW ( P ＜ 0.05).Conclusion ERK-CREB signaling pathway in the spinal cord is involved in morphine withdrawal response in morphine-dependent rats.
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ObjectiveTo investigate the role of NO and extracellular signal-regulated kinase (ERK) signaling pathways in the spinal cord in naloxone-induced withdrawal response in morphine-dependent rats.Methods Ninety male adult SD rats weighing 200-250 g in which IT catheters were successfully implanted without complication were randomly divided into 9 groups (n ＝ 10 each):group control (group C); group morphine dependence (group MD); group morphine withdrawal (group MW); group N(G)-nitro-L-arginine methyl ester (eNOS inhibitor) (L-NAME group) ; group 7-nitroindazole (nNOS inhibitor) (group 7-Ni) ; group aminoguanidine(iNOS inhibitor) (group AG); group U0126 (ERK signaling pathway blocker); group cremophor (solvent for 7-Ni) and group DMSO (solvent for U0126).Morphine dependence was induced by increasing doses of subcutaneous morphine for 6 days.The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg twice every other day until 50 mg/kg on the 6th day in groups MD,MW,L-NAME,7-Ni,AG,U0126,cremophor and DMSO.Morphine withdrawal response was induced by intraperitoneal (IP) naloxone 4 mg/kg at 4 h after last morphine administration in groups MW,L-NAME,7-Ni,AG,U0126,cremophor and DMSO.L-NAME 400 μg,7-Ni 400 μ g,AG 400μg,U0126 150 μg,cremopher 10 μl and DMSO 10 μl were administered IT at 30 min before naloxone administration in groups L-NAME,7-Ni,AG,U0126,cremophor and DMSO respectively.Morphine withdrawal response (0 ＝ no withdrawal response,3 ＝ severe response) and touch evoked agitation (0 ＝ no agitation,2 ＝ severe agitation) were observed and scored during 1 h after naloxone administration.The animals were then sacrificed and the spinal cord was removed for determination of the expression of iNOS,nNOS and phosphor-ERK (p-ERK) by immunohisto-chemistry and Western blot.ResultsMorphine withdrawal significantly increased withdrawal response score and touch evoked agitation score in group MW as comparedwith group MD.L-NAME,7-Ni,AG and U0126 pretreatment significantly attenuated naloxone-induced increase in withdrawal response score and touch evoked agitation score in groups L-NAME,7-Ni,AG and U0126 as compared with group MW.Morphine withdrawal significantly up-regulated the nNOS and iNOS expression in group MW compared with groups C and MD.L-NAME,7-Ni and AG pretreatment significantly down-regulated p-ERK expression in groups L-NAME,7-Ni and AG as compared with group MW.ConclusionThe interaction between NO and ERK signaling pathways may be involved in morphine withdrawal response in morphine-dependent rats.
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Objective To investigate changes of synaptic interface structural in the hippocampus Cal in high and low conditioned place preference(CPP) rats after chronic morphine treatment.Methods The male SD rats were randomly distributed to experiment group 130 cases(intraperitoneal injected morphine twice a day for ten days in an ascending dosage schedule) and control group 30 cases(injected saline of the same volume at same time).The rats in experiment group were re-classified into high preference group(HP),middle group and low preference group(LP) according to the numerical value of the CPP.The middle group was rejected.The rats in HP and LP were scarified at the time of 3h,3d and 14d after the last injection.The hippocampus Cal were removed and prepared for electron microscope specimen.The synaptie interface structure parameter were analyzed by image processing technique.Results ①No significant difference of pretest scores staying at the non-preference chambet existed among the three groups(F=0.78,P=0.47).However,the test scores of the CPP minus the time stayed at pretest natural preference in the high group was significantly higher than that of the low group(P=0.00).②At the 3h and 3d,the PSD of the high group((15.20±-3.65)nm) was significantly lower than low group((17.63±6.61)nm,P<0.01);the synaptic cleft of high group((5.77±2.08)nm) was significantly higher than low group ((4.92±1.65)nm,P<0.05).At the 14d,the PSD of the high group((16.22±4.93)nm) was significantly lower than low group((18.42±3.78)nm,P<0.01).Conclusion In hippocampal Cal area the synaptic cleft in the HP group was higher than that of LP group,the post-synaptic density in the HP group was lower than that of LP group.These changes may be the synaptic basic of the different susceptibility.
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Objective To investigate the effect of NR2B antisense oligonucleotide on naloxone-induced withdrawal responses in morphine-dependent rats. Methods Famale SD rats weighing 230-270 g were anesthetized with intraperitoneal pentobarhital 60 mg/kg. Intrathecal (IT)catheter was placed at L3,4 interspace.Thirty-two rats in which FT catheter was successfully placed were randomly divided into 4 groups ( n = 8 each) : group C control; group MD morphine dependence; group AO NR2B antisense oligonucleotide (aNR2B) and group SO NR2B sense oligonucleotide (sNR2B) . In group MD, AO, SO chronic morphine dependence was induced by increasing doses of subcutaneous morphine for 6 days. The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg twice every other day and reached 50 mg/kg on the 6th day. In group AO and SO IT aNR2B or sNR2B 15 nmol was administered simultaneously with subcutaneous morphine. Morphine withdrawal responses was induced by IT naloxone 4 mg/kg and scored based on the responses (0 = normal; higher scores signify severer responses) . The weight loss was calculated.The expression of NR1, NR2A and NR2B mRNA in hippocampus was determined by RT-PCR. Results The morphine withdrawal syndrome and weight loss were significantly incresed in group MD, AO and SO, while NR2B mRNA expression in hippocampus was up-regulated in group MD and SO compared with group C. The morphine withdrawal syndrome and weight loss were significantly decreased, NR2A mRNA expression in hippocampus was up-regulated and NR2B mRNA expression was down-regulated in group AO compared with group MD. There was no significant difference in NR1 mRNA expression between the 4 groups . Conclusion NR2B antisense oligonucleotide can suppress morphine withdrawal responses through the regulation of NMDA receptor level and construction in hippocampus.
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Objective: To study the impact of different abstinence periods on locomotor activity and contextual fear conditioning in morphine-dependent mice. Methods: Morphine hydrochloride was administrated (sc) to mice at a gradually increasing dose for 7 d to establish morphine-dependent model. After the last injection, animals were divided into MW1 d (Morphine Withdrawal for 1 d), MW7 d, MW21 d, SW1 d (Saline Withdrawal for 1 d), SW7 d and SW21 d groups randomly; the spontaneous activities of mice, including the numbers of crossing and rearing, grooming times, the number of center entries and time spent in center area, and the time spent in peripheral area, were observed in the open field box after different periods of abstinence. After the observation, the mice were trained using eight unconditioned stimulus (0.5 s; 0.5 mA foot shock) and contextual fear conditioning was tested 24 h later. Results: The times of crossing and center entries decreased in MW1 d and MW7 d mice (W1 d: P<0.001, P<0.001; W7 d: P<0.001, P<0.05); the time of rearing in MW1 d group was also decreased significantly (P<0.001); mice in MW1 d group had more grooming behaviors (P<0.05). The periods spent in the center and peripheral areas were not significantly different between all the 3 morphine-dependent groups and their corresponding saline withdrawal groups. In addition, a history of morphine injection selectively impaired the contextual fear conditioning in mice of MW1 d group (P <0.05). Conclusion: During the early period of morphine abstinence, mice have a decreased spontaneous activity and an impaired contextual fear conditioning.
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Objective To investigate the effect of penehyclidine hydrochloride (PHCD) on the reinstatement of conditioned place preference (CPP) in morphine dependent rats. Methods Forty male adult SD rats weighing 180-220 g were randomly divided into 5 groups (n = 8 each): group control (group C); group morphine (group M) and 3 PHCD groups (group P1-3 ). Morphine 10 mg/kg was injected subcutaneously once a day for 8 days to induce morphine CPP. The rats were then subjected to extinction of CPP for 10 days with normal saline (NS) instead of morphine. After the extinction, the rats were put into the drug-paired side of the box. A single priming dose of morphine 4 mg/kg was injected to reinstate the morphine CPP. In group P1-3 the rats received PHCD 0.5, 1.0 and 1.5 mg/kg intraperitoneally 30 min prior to priming dose of morphine, whereas in group C and M the rats received NS. The second day the rats underwent CPP test. Results Compared with group M, the time spent in the drug-paired side (grey area) was significantly shortened in group P1-3 (P ＜ 0.05 or 0.01 ).Compared with group P1 ,no significant change in the time spent in the drug-paired side (grey area) was found in group P2(P ＞ 0.05), but the time spent in the drug-paired side (grey area) was significantly shortened in group P3 ( P ＜ 0.05). Conclusion PHCD could significantly inhibit the reinstatement of CPP induced by priming dose of morphine in morphine dependent rats and it is related to the dose.
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Objective To evaluate the effect of intrathecal (IT) NR2B antisense oligonucleotide (aNR2B) on cognitive function in morphine-dependent rats.Methods Male SD rats weighing 230-270 g were used in this study. The animals were anesthetized with intraperitoneal pentobarbital 60 mg/kg.IT catheter was placed at L3-4 interspace according to the technique described by Yang. Thirty rats in which IT catheter was successfully placed without any complication were randomly divided into 3 groups(n=10 each):control group (group C), morphine dependence group (group MD) and group aNR2B.Morphine dependence was induced in group MD and aNR2B by increasing doses of morphine for 6 days. The initial dose of morphine was 10mg/kg injected subcutaneously (SC) twice a day and was increased by 10 mg/kg.every other day.The final dose was 50mg/kg. Then morphine 30 mg/kg was administered SC once a day for 4 weeks. aNR2B 15 nmol was administered IT at 30 min before SC morphine every day in group aNR2B.In control group normal saline was administered instead of morphine. Morris water maze was used to assess the cognitive function at 0 (T0, baseline),1 and 3 weeks of morphine administration (T1,T2).The escape latency and the number of times the animals crossing the plateform were recorded. The animals were killed after the test and the hippocampus was isolated for determination of choline acetytransferase(ChAT)expression.Results There was no significant difference in the baseline escape latency and the baseline number of times the animals crossing the plateform at T0 among the 3 groups. The escape latency was significantly prolonged and the number of times the animals crossing the plateform decreased at T1 and T2 as compared with the baseline at T0 in group MD.The ChAT expression was significantly down-regulated in group MD as compared with control group. IT aNR2B significantly ameliorated cognitive dysfunction at T1 and T2 and increased ChAT expression in group aNR2B compared with group MD.Conclusion IT NR2B antisense oligonucleotide can attenuate cognitive dysfunction through up-regulation of ChAT expression in hippocampus in morphine-dependent rats.
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Objective To evaluate the changes in the expression of cytochrome cholesterol side chain cleavage enzyme (P450scc) in the brain of morphine-dependent rats. Methods Twenty-four male SD rats aged 4-8 months weighing 180-200 g were randomly divided into 3 groups (n = 8 each): group Ⅰ normal saline (group NS), group Ⅱ morphine dependence (group MD) and group Ⅲ morphine withdrawal (group MW). In group MD and MW, the rats were given intraperitoneally increasing doses of morphine starting from 5 mg/kg to 10, 15, 20, 30, 40 and 50 mg/kg twice a day for 7 days. In group NS, the rats were given equal volume of normal saline instead of morphine. The rats were decapitated 1 h after last injection in group NS and MD. In group MW, naloxone 2 mg/kg was given 1 h after last injection, and then the animals were decapitated 30 min after withdrawal symptoms were observed. The brains were immediately removed and the frontal cortex, hippocampus, striatum and thalamus were separated. The expression of P450see was determined by Western blot. Results The expression of P450scc in the frontal cortex, hippocampus and striatum was significantly decreased in group MD and MW compared with group NS (P<0.05). Conclusion The down-regulation of P450scc expression might be involved in the development of morphine dependence, but it is not involved in the morphine withdrawal.