Study On The Roles Of Calcium And Calmodulin In The Mechanism Of Morphine Addiction

Part one Changes of Ca2+ in Primary cultured hippocampalneurons with acute or chronic morphine treatment[Abstract] Objective: To investigate the change of Ca²⁺ in primary cultured hippocampal neurons with acute or chronic morphine treament by using immunofluorscence and laser scanning confocal microscope(LSCM) techniques,try to find possible neurobiological mechanisms and appropriate treatments for morphine addiction. Methods: The hippocampi were dissected from newbron Sprague-Dawley rats. Primary hippocampal neuronal cultures of 7 days in vitro were used and were divided randomly into 7 Groups(n=6 plate): acute morphine treatment group, acute abstinent group, chronic morphine treatment group, chronic abstinent group, pretreatment with antagonist of opiate receptors group , pretreatment with blocker of calcium tunnel group and control group. Impacts of morphine on Ca²⁺ in primary cultured hippocampal neurons were detected by using laser scanning confocal microscope(LSCM) after marking free [Ca²⁺]i with a new type fluorence probe Fluo-4. Results: We found that [Ca²⁺]i levels increased significantly both in acute and chronic treatment groups compared with the control group. Withdrawl with naloxone did not block the increase,but caused intense increase. Selective antagonist of δ 2 opiate receptor- Naltrindole blocked the increase,while selective antagonist of u opiate receptor- CTOP did not. Pretreatment with verapamil could not inhibit the increase of [Ca2+]i in hippocampal neurons .but specific inhibitor of calcium pump in Endoplasmic reticulum -TG could. Conclusion: Ca²⁺ in primary cultured hippocampal neurons increase with acute or chronic morphine stimulation , which may be associated with release of calcium pool sensitive to IP3mediated by 8 2 opiate receptor.Part two Changes of CaM protein in Primary culturedhippocampal neurons with acute or chronic morphine treatment[Abstract] Objective: To investigate the change of CaM protein in primary cultured hippocampal neurons with acute or chronic morphine treament by using immunofluorscence and laser scanning confocal microscope(LSCM) techniques. Methods: The hippocampi were dissected from newbron Sprague-Dawley rats. Primary hippocampal neuronal cultures of 7 days in vitro were used and were divided randomly into 5 Groups(n=6 plate): acute morphine treatment group, acute abstinent group, chronic morphine treatment group, chronic abstinent group and control group. Control group was treated with saline.The CaM protein levels were detected with LSCM imaging techniques. Result:We found that (1) In the acute morphine treatment group and acute abstinent group , CaM protein levels did not change compared with control group.;(2) In the chronic morphine treatment group and chronic abstinent group, CaM protein levels increased significantly compared with the control group , and the latter increased more than the former although there were no signigicant differences between the two groups. Conclusion: Chronic,repeated use of morphine may cause changes of cellular signal transduction ,which could be responsible for significant increase of CaM protein levels in primary cultured hippocampal neurons.Part three The different changes of CaM proteins in brain regions of acute or chronic morphine dependent and withdrawl ratsl.Immunohistochemistry[Abstract] Objective: To investigate the changes of CaM proteins in five brain regions of morphine addiction and withdrawl rats with the technique of immunohistochemistry. Methods:Thirty six adult male Sprague-Dawley rats were divided randomly into six groups(n=6):acute morphine dependent group,acute abstinent group,acute control group,chronic morphine dependent group,chronic abstinent group and chronic control group. Dependent groups and abstinent group were administered with morphine by intraperitoneal injection till morphine dependent models were founded.The rats in abstinent groups were induced withdrawal sydromes with naloxone 5mg/kg for 30min.The rats in control group were injected with Saline.All rats were sacrificed by decapitation.The coronal sections of discrete brain regions (hypothalamus,nucleus accumbens,prefrontal cortex,locus coeruleus, hippocampus)were cut.The relative concentrations of CaM protein were detected by the technique of immunohistochemistry. Results: l)In acute morphine dependent group and acute abstinent group, CaM protein decreased significantly in locus coeruleus compared with the acute control group (p<0.01),but nor of the other four brainregions(p>0.05);2)In chronic morphine dependent group and chronic abstinent group, CaM protein increased significantly in locus coeruleus -. hippocampus > cortex and locus coeruleus(p<0.05) compared with the chronic control group. But CaM protein decrease significantly in nucleus accumbens compared with the chronic control group (p>0.05). Conclusion: The morphine-induced differential changes in CaM protein levels may reflect AC-cAMP changes in gene expression and the result in changes of CaM protein may affect signal transductionpathways in morphine dependent animals. Maybe that is the molecular mechanism of oipioid tolerance and dependence.2.Western-blotting[Abstract] Objective: To investigate the different changes of CaM proteins in three brain regions of acute or chronic morphine addiction and withdrawl rats with the technique of western-blotting . Methods:Thirty six adult male Sprague-Dawley rats were divided randomly into six groups(n=6):acute morphine dependent group,acute abstinent group,acute control group,chronic morphine dependent group,chronic abstinent group and chronic control group. Dependent groups and abstinent group were administered with morphine by intraperitoneal injection till morphine dependent models were founded.The rats in abstinent groups were induced withdrawal sydromes with naloxone 5mg/kg for 30min.The rats in control group were injected with Saline. All rats were sacrificed by decapitation.The brain region of nucleus accumbens, prefrontal cortex and hippocampus were separated .The relative concentrations of CaM protein were detected by the technique of western-blotting. Results:l)In acute morphine dependent group and acute abstinent group, CaM protein didn't change significantly in prefrontal cortex, nucleus accumbens and hippocampus compared with control group(p>0.05);2)In chronic morphine dependent group and chronic abstinent group, CaM protein increased significantly in cortex and hippocampus (p<0.0\ ,p<0.05) more than the chronic control group. But CaM protein decrease significantly in nucleus accumbens compared with the chronic control group (p>0.05). Conclusion: The acute morphine dependence did not change the CaM level,which may beassociated with a long-term biological effect of mophine dependence. While the chronic morphine dependence changes the CaM level diversely because of the function difference in the course of morphine dependence formation,which exhibits diversity of different brain regions response to morphine-induced CaM expression level.