The Effect Of Intra-accumbens Injecting Protein Kinase A Inhibitor On Morphine-produced Conditioned Place Preference And The Phosphorylation Levels Of CREB In Specific Brain Regions In Rats

Objectives: Neural system will produce adaptive change after repeated drug use. The mesolimbic dopamine system, which efference to nuecleus accumbens, has been implicated in the reward-related learning and the second messenger pathways play critical role in the learning. It has been demonstrated in many studies that the cyclic adenosine 3',5'-monophosphate (cAMP) cascade is involved in dopamine stimulants reward-related learning. Although the phosphorylation levels of protein kinase A and its target cAMP response element binding protein (CREB) are also up-regulated after chronic morphine treatment, the study on the effect of cAMP-PKA cascade in the morphine reward-related learning is still scare. The present study is to explore the effect of the Rp-cAMPS, a PKA inhibitor on rat's conditioned place preference (CPP) developed by intra-accumbens morphine injection to try to understand the role of PKA in acumbens in the morphine reward related learning. Method : Forty-eight male Sprague-Dawley (SD) rats were randomly assigned to saline, morphine+saline, morphine+Rp-cAMPS and Rp-cAMPS groups. Injections were made in a volume of 0.5μl of saline. Dose for morphine was 15nmol per side. Dose for Rp-cAMPS was 500ng. The training and measurement of CPP adapted approaching procedure and the natural non-preference side was taken as drug-paired side. Drugs were injected at 8 AM on day 1, 3, 5 and at 8 PM on day 2, 4, then the rats were put in the drug-paired side. Saline was infused at 8 PM on day 1, 3, 5 and at 8 AM on day 2, 4, then the rats were put in the non-drug-paired side. After recording the CPP changes, the rats were scarified and perfused. Verification of injection sites was observed under microscope. Data were analyzed using one ANOVA, followed by LSD and paired t test. Results: ⑴There were significant difference on the time change in the drug paired side among four groups(F(3, 20)=23.26, P<0.01). The time in drug-paired side in morphine+saline, and morphine+Rp-cAMPS groups are significantly longer than that in the saline group. ⑵There was no difference in the time change in the drug paired side between morphine+saline group and morphine+Rp-cAMPS group (P>0.05); ⑶ There was no difference in the time change in the drug-paired side in saline group and Rp-cAMPS+saline group (t=0.58,P>0.05, t=0.52,P>0.05), so the rats in the two groups didn't develop CPP; there was significant difference in the time change drug-paired side in the morphine+saline group and morphine+Rp-cAMPS group (t=5.06, P<0.01,t=5.66, P<0.01),so the rats in the two groups developed CPP。 Conclusion:⑴Nuclueus accumbens is an important structure in the morphine reward-related learning because the rats developed CPP after intra-accumbens infusion of morphine. ⑵ PKA inhibitor Rp-cAMPS didn't block the morphine induced CPP, meaning the PKA pathway is not involved in the morphine reward-related learning. Key Words: Morphine, Nuclues Accumbens, Protein Kinase A, Conditioned Place Preference Part II The Effect of Intra-accumbens Injecting Protein Kinase A Inhibitor on the Phosphorylation Levels of CREB in Specific Brain Regions in Morphine Treated Rats Objectives: Repeated psychoactive substance exposure always produces neural-plasticity changes, and the second messenger pathway is responsible for the changes. After repeated opiates exposure, all the components of cAMP cascade change accordingly. On the basis of results of part I that morphine-produced CPP was not blocked by the PKA inhibitor, Rp-cAMPS, the phosphorylation levels of CREB in the nuclues accumbens (NAc) and locus ceruleus (LC) after chronic morphine treatment and PKA inhibitor perfusion were observed in order to further explore the role of PKA in NAc on the morphine produced CPP.Method: SD rats of each group in the part I were perfuse transcardially after CPP test. The brains were removed and cut coronally. The sections including NAc and LC were selected. The phosphorylation levels of CREB were determined by immunohistochemistry and Motic analyze system. The gray intensity of each group analyzed using one-way ANOVA, followed by LSD or Tamhane's T2. Results: ⑴There were significant difference in the gray intensity in the NAc and LC among the four groups (P<0.01), so there were significant difference among the four groups; ⑵The gray intensity were lower in NAc in the morphine+saline and morphine+Rp-cAMPS groups than the saline group (P<0.05, P<0.01), so the levels of the p-CREB in the two groups were up-regulated; ⑶ The gray intensity were lower in LC in the morphine+saline and morphine+Rp-cAMPS groups than the saline group (P<0.01) , so the levels of the p-CREB in the two groups were up-regulated; ⑷There were no difference in the gray intensity between Rp-cAMPS+saline group and saline group (P>0.05), so the levels of the p-CREB in the group was not changed. Conclusion: ⑴ The phosphorylation levels of CREB were up-regulated after intra-accumbens morphine injections; ⑵ Rp-cAMPS didn't block the up-regulation of phosphorylation levels of CREB in NAc and this up-regulation might be independent to the PKA cascade in NAc; ⑶ The up-regulation of p-CREB level in LC might be a adaptiveresponse to the increase of external opiate.