| Objective: To observe the expression changes of the N-methyl-D-aspartate receptor NR1 subunit(NR1) in the morphine induced Conditioned place Preference(CPP) rats midbrain ventral tegmental area-the nucleus accumbens-prefrontal cortex(VTA-NAc-PFC) neural circuits and morphine tolerance cells,and was to reveal inducing functions of NR1’s spiritual dependent on the tissues and cells. This research was also focused on observing the CPP effects and expression changes in neural circuits and NAc after the treatment with levo-tetrahydropalmatine(l-THP) and add new experimental evidence for applications of l-THP treat opioid dependence.Methods:1. Changes of the expression of NR1 and of morphine dependent rats’ VTA-NAc-PFC neural circuits. The SD rats were selected, and randomly divided into normal saline(NS) group and morphine in model group, Dose escalation method(starting doses of morphine from d1 10 mg/kg to 100 mg/kg d10) has been taken respectively during the injections of saline solution and morphine on the back of the neck subcutaneous. After the rear injection of 20 min they were put into black and white boxes(white box was with morphine paired) for further training with continuous injection of 10 d. After the last 48 h after training for CPP test, this research confirms that the CPP rats model was successful, and two groups of rats were killed soon, and the VTA, NAc,PFC various brain regions were immediately taken out, respectively,by using PCR and Western blot to detecte the expression of NR1.2. The expression changes of NR1 in various brain regions of VTA-NAc-PFC nerve loop in the regression phase of morphine dependent rats. Based on the successfully established the morphine CPP model of rats, on 28 days’ watching process of saline controlling group and the morphine-model group, the CPP detection was taken and the morphine CPP effect was found disappeared. Immediately the two groups of rats were killed, VTA, NAc and PFC areas of the brain were respectively taken to detect changes in the expression of the NR1 by means of PCR and Western blot analysis.3. NR1 expression changes in rat morphine tolerance rats cortex culture neuron After culture the rat cortex neuron, two groups were set, the physiological saline control group and morphine group, respectively with physiological saline and 10μm/l morphine,48 h after collecting cells respectively, and extracted protein, using western blot to detect the expression of NR1 protein in cells.4. The morphine CPP effects in rats of the l-THP treatment and the NR1 expression in the NAc areastablished morphine CPP model of rats, the qualified 60 healthy male SD rats were selected, and randomly divided into 6 groups: NS control group, morphine CPP group, NS treatment group, the low, middle and high l-THP-dose treatment group. Upon the confirmation of the successful building of the model of CPP, the NS group and model group rats were immediately killed and the experimental materials were taken immediately; in addition to the NS treatment group the remaining 4 groups of rats were lavaged with NS,other l-THP group of rats were given low, medium and high dose, respectively of l-THP 0.94 mg/kg, 1.88 mg/kg and 3.76 mg/kg lavage treatment. After 6 days of continuous treatment they were immediately killed and the detection of CPP was taken again. The experimental materials were as well in the same way of method(1). Also by using PCR and Western blot to detect the expression of NR1 in the VTA-NAc-PFC neural circuits of various brain regions.Results:1. The change of NR1 expression in morphine dependent rats VTA-NAc-PFC neural circuits in various brain regions.After the last 48 h training, and the CPP test, the durational time of the staying of the controlled group of rats in the CPP white box normal saline(NS): 393 ± 81 s before training, after training for the 416 ± 58 s. No significant difference was found before and after the training. CPP model group rats in the retention period of white box; Training before and after: 408 ± 93 s and 528 ± 81 s, after training rats significantly extendedresidence time in the white box(P < 0.01). And the CPP model group was compared with NS control group, the residence time in the white box model group rats was significantly longer(P < 0.01). Morphine was compared with NS control group, For NAc CPP group the NR1 m RNA expression had been increased significantly with more significant statistics(P < 0.05), while the VTA and PFC NR1 m RNA levels between the two groups were also increased, but with no statistical significance(P > 0.05). For NAc CPP group the NR1 protein expression had been increased significantly(P < 0.01), while the VTA and PFC NR1 protein levels between the two groups were also increased,but with no statistical significance(P > 0.05).2. The expression changes of NR1 in various brain regions of VTA-NAc-PFC nerve loopin the regression phase of morphine dependent ratsOn 28 days’ natural recess of the successfully established morphine CPP in the rats, the CPP detection was taken: the durational time of the staying of the rats in the CPP white box(NS control group of rats: 410 ± 69 s; morphine-induced CPP group of rats: 396 ± 117 s). No significant changes of durational time of the staying of the two groups of rats. And the expression levels of NR1 m RNA and protein in each brain region, the VTA, NAc, and PFC of the two groups of rats had no significant difference(P > 0.05).3. NR1 expression changes in nerve cortex cells with morphine toleranceIn 48 hours after the addition of morphine to neurons, compared with the saline-controlling group, the protein expression level of NR1 in the experimental group was significantly increased(P < 0.01)..4. The effect upon NR1 expression in neural circuits of the NAc and the CPP effects of treatment of l-THP.After the last 48 h training, the staying time of NS controlled group before and after training in the white box, was respectively 383 ± 62 s and 401 ± 76 s CPP was 407 ± 83 s and 528 ± 40 s. For NS treatment group: 386 ± 75 s before training, 527 ± 55 s after training, after six days treatment: 524 ± 79 s, l-THP treatment group, low dose(0.94 mg/kg) was 396 ± 81 s before training, after training was 523 ± 32 s; after six days treatment: 519 ± 47 s, the medium dose(1.88 mg/kg) 389 ± 95 s before training, after training:528 ± 42 s, after six days treatment: 386 ± 76 s, high dose(3.76 mg/kg) 399 ± 65 s before training and after training was 536 ± 67 s. after 6 days treatment: 386 ± 88 s. The CPP test, compared with NS control group, the CPP model group, the NS therapy, the l-THP low,medium and high dose therapy, the during time of 5 groups of rats was obviously increased in the white box(P < 0.01); After 6 days treatment with the different l-THP, compared with NS group, the duration time of group of rats treated with l-THP 1.88 mg/kg, 3.76 mg/kg in the white box was obviously reduced(P < 0.01). Compared with NS group, NR1 protein content was significantly decreased(P < 0.05) in the brain region(NAc) of rats treated with l-THP 1.88 mg/kg and 3.76 mg/kg.Conclusion:1. The NR1 m RNA and protein have obvious expressional increases in the NAc brain region of the morphine-induced rats CPP phase. But the NR1 m RNA and protein have recovered to NS control group level in the regression phase of morphine. This may play an important role in the shaping process and maintain morphine dependence, and the expression changes of NR1 protein was significantly increased in the culture neuron with morphine, most possibly was one of the forming mechanism of morphine psychological dependence.2. The abnormal expression of NR1 protein caused by the reversing function of l-THP in NAc brain regions, may be another mechanism of pharmacological effects which should accelerate the fading process of morphine CPP effects in the experimented bodies. |
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