| Objective: Morphine is one of the most commonly used addictive drugs. Studies have shown that multiple brain regions, such as the prefrontal cortex, nucleus accumbens, caudate putamen, hippocampus, amygdala, substantia nigra, periaqueductal gray, ventral tegmental area and locus coeruleus, involved in morphine addiction and had different roles. Drug dependence involves a variety of neurotransmitters, neuro-modulators and neuropeptide. As the most potent endogenous anti-opioid peptides, cholecystokinin octapeptide (CCK-8) is found a broad distribution in the central system and takes part in regulation of pain, appetite, memory and other physiological processes. However, the role of this peptide during morphine dependence and withdrawal remains unclear. In this study, immunohistochemistry and in situ hybridization were used to observe the changes of CCK-8 expression in frontal cortex, nucleus accumbens, caudate putamen, hippocampus, amygdala, substantia nigra, periaqueductal gray, ventral tegmental area, locus coeruleus, and to explore CCK-8 roles in morphine dependent and withdrawal rats.Method: Thirty six healthy male Wistar rats, weighting from 200g to 220g, were randomly divided into three groups (each group 12 rats): NS control group (NS group), morphine dependent group (MOR group) and naloxone precipitated withdrawal group (NAL group). The morphine dependent model was established by injecting subcutaneously morphine. The daily dose gradually increased as following: 10, 20, 30, 40, 50mg·kg-1 twice a day, at 8:00 am and 8:00 pm, for 5 days. On the 6th day, 50mg·kg-1 morphine was given at 8:00 am, withdrawal syndrome was precipitated by intraperitoneal injection of naloxone 5mg·kg-1 after 2 hours. The animal model was evaluated by Gellert-Holtzman scoring. One hour later, the rats were given heart perfusion and the brains were quickly isolated, then we cut the brain tissue by rat Brain Slicer Matrix and the tissue was stored for future application.In our study, Image-Pro software was used to measure the optical density(mean) of CCK-8 and CCK mRNA in positive cell brain regions and the results were analyzed with The results were analyzed with SPSS 13.0 statistics software, and a level of P<0.05 was considered statistically significant.Result:1 The establishment of morphine dependent and precipitated withdrawal model in ratsOne hour after injecting naloxone, withdrawal symptoms apppered, such as teeth chattering, wet dog shakes, diarrhea, tears, salivation and jumping, body weight loss. The number of jumping times (10.92±3.99) in naloxone precipitated withdrawal rats was obviously increased compared with NS group and MOR group within 15 minutes (P<0.01). One hour later, the body weight loss in NAL group (8.37±3.53g) were remarkably increased compared with NS Group (0.98±0.24g)and MOR group (0.91±0.26g)(P<0.01). There was not different between NS Group and MOR group. The Scoring of withdrawal symptoms by Gellert-Holtzman in NAL group (37.88±11.92) were remarkably increased compared with NS Group (1.79±0.92) and MOR group (1.75±0.89) (P<0.01). There was not different between NS Group and MOR group.The data showed that the model of morphine dependent and withdrwal was successful.2 The changes of CCK-8 protein expression of related brain regions in morphine dependent and withdrawal ratsCCK-8 protein expression was observed by immunohistochemical method in each group of rats in prefrontal cortex, caudate putamen, nucleus accumbens, hippocampus, amygdala nucleus, substantia nigra, ventral tegmental area, periaqueductal gray and locus coeruleus. The results showed positive neurons were mainly present in the internal pyramidal layer in prefrontal cortex. In the substantia nigra, positive neurons mainly distributed in substantia nigra pars compacta, while pars reticulata with a few positive cells. In amygdala, positive neurons distributed in the basal amygdala. In hippocampus, positive neurons were mainly expressed in the dentate gyrus polymorphic layer, while few positive neurons were observed in CA1, CA2, and CA3 pyramidal cell layer. Some positive neurons were found in the midbrain ventral tegmental area and locus coeruleus. But in the caudate putamen, nucleus accumbens and periaqueductal gray matter, we failed to detect the positive expression of CCK-8.The changes of CCK-8 protein expression in above brain regions as following: in prefrontal cortical, substantia nigra pars compacta, ventral tegmental area, CCK-8 expression in MOR group was significantly higher than the NS group (P <0.05), decreased after withdrawal, but still higher than NS group. In dentate gyrus, amygdala nucleus, locus coeruleus, CCK-8 expression in MOR group was significantly higher than NS group (P <0.05), further increased after withdrawal (P <0.05).3 The changes of CCK mRNA expression of related brain regions in morphine dependent and withdrawal ratsCCK mRNA expression was observed by in situ hybridization in each group of rats in prefrontal cortex, caudate putamen, nucleus accumbens, hippocampus, amygdala nucleus, substantia nigra, ventral tegmental area, periaqueductal gray and locus coeruleus. The results showed that CCK mRNA positive cells are basically the same as CCK-8 protein. In prefrontal cortex, CCK mRNA positive neurons were mainly present in the internal pyramidal layer. In the substantia nigra, positive neurons mainly distributed in substantia nigra pars compacta. In amygdala, positive neurons distributed in the basal amygdala. In hippocampus, CCK mRNA positive neurons were more than CCK-8 positive neurons, and mainly expressed in dentate gyrus polymorphic layer, while few positive neurons was observed in CA1, CA2, CA3 pyramidal cell layer. Some positive neurons were found in the midbrain ventral tegmental area and locus coeruleus. But in the caudate putamen, nucleus accumbens and periaqueductal gray matter, we failed to detect the positive expression of CCK mRNA.The changes of CCK mRNA expression in above brain regions as following: in prefrontal cortical, substantia nigra pars compacta, ventral tegmental area, CCK mRNA in MOR group was significantly higher than the NS group (P <0.05), decreased after withdrawal, but still higher than NS group. In dentate gyrus, amygdala nucleus, locus coeruleus, CCK mRNA expression in MOR group was significantly higher than NS group (P <0.05), further increased after withdrawal (P <0.05).Conclusion:This study demonstrated that positive expression of CCK-8 protein and CCK mRNA were found in prefrontal cortex, amygdala, substantia nigra, ventral tegmental area, hippocampus, and locus coeruleus in rats. After morphine dependence and withdrawal, CCK-8 protein and CCK mRNA significantly increased. Howere, no positive cell was detected in the caudate putamen, nucleus accumbens, and periaqueductal gray. The findings suggested that CCK-8 involved in the development of morphine dependence and withdrawal, with significant specificity in different brain regions.
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