| Long-term opiate treatment is known to induce a state of dependence, a phenomenon associated with the abstinence syndrome following cessation of opiate administration. Despite numerous studies, the neuronal mechanism of the opiate addiction has not been fully elucidated as yet.Neurosteroids is a new group of neurotransmitters with wide physical and pharmalogical functions. The term neurosteroids applie to those steroids that are synthesized within the central and peripheral nervous system, either de novo from cholesterol or from steroid hormone precursors. Neurosteroids including dehydroepiandresterone (DHEA), dihydroepiandresterone sulfate (DHEAS), pregnenolone (PREG), pregnenolone sulfate (PREGS), allopregnanolone (AP), deoxycorticosterone (DOC), testosterone (T), and progesterone (PROG) were named as the forth generation of neurotransmitter. It is demonstrated that neurosteroids can widely influence the function of traditional neurotransmitter receptors. Neuroactive steroids may modulate neuronal function through their concurrent influence on neuronal excitability and gene expression. This intracellular cross-talk between genomic and non-genomic steroid effects provides the molecular basis for such compounds in neuropsychopharmacology, both with regard to putative clinical effects and side effects. Neurosteroids, particularly PROG and AP can act as positive modulators at the GABAA receptor. In vivo, effects of these neurosteroids are similar to those produced by other positive modulator of GABAA receptors such as benzodiazepines and barbiturates. Many of these neurosteroids showed anticonvulsant, Myorelaxant, anesthetic and anxiolytic effects when they were administered to animals. Neurosteroid sulfate esters, such as PREGS and DHEAS, have an excitatory cellular action, since they antagonize the action of GABAA and potentiate the activation of NMDA receptor. Moreover, they can improve memory and learning, increase dopamine release in the rat nucleus accumbens, and enhance the dopaminergic response to morphine.Endogenous opioids is the term used to describe a family of peptides, which include endorphines, enkephalins, dynorphins, and endomorphine,that act like opiates in biological systems. The discovery of these endogenous substances has spearheaded the recent interest in the biological functions of numerous peptide hormonse and neurotransmitters. Indeed, over the last two decades, at least 50 peptides found in the skin, brain, and the gut have been added to the list of neurotransmitter candidates. However, the endogenous opioids have attracted more attention than some of the other bioactive peptides. As a general rule, like morphine, endogenous opioids, which are inhibitory substances, reduce neuronal firing rates and neurotransmitters release. Endogenous opioids may inhibit nerve cells by depressing presynaptic neurotransmitter release, by altering the ability of other neurotransmitters to produce postsynaptic ion conductance, or by directly interfering with the postsynaptic nerve impulse.Based on the previous results of our lab, this study was carried out to further test the effect neurosteroids on morphine-induced psychological dependence from the levels of neurotransmitters, receptors and postreceptors respectively in related brain areas such as nucleus accumbens(NA), frontal cortex (Fc), hippocampus (Hc), hypothalamus (Ht), striatum (Str) in rats. We first investigate the effect of PROG administration on the morphine-induced rewarding effect. Then, a simplified radioimmunoassay method has been established to detect the Endogenous opioids transmitters in rat Ht, NAc, Hc, Fc, and Str. By using the method of immunohistochemistry and western- blotting, we will further investigated the machenism of how PROG reverse the effects of morphine induced psychological dependence from receptors and nuclear factors. Therefore, this study will take a basis for the further study of the relationship between morphine addiction and neurosteroids. Following methods were employed in this study:1 The establishment of morphine psychological dependence rat modelsThe rat model of conditioned place preference (CPP) was used to mimic morphine psychological dependence. 40 male SD rats were designated to 4 groups randomly with 10 in each, including blank control group, morphine addiction group, progesterone group and progesterone plus morphine group. The white room was used as the drug-pairing room. In the morning, the groups of black control and progesterone had hypodermic injection of cyclodextrin and progesterone of 15mg·kg-1, 10 min later, had intraperitoneal injection of Saline 5mg·kg-1. The morphine and progesterone plus mrophine groups had hypodermic injection of cyclodextrin and progesterone of 15mg·kg-1, 10 min later, had intraperitoneal injection of morphine of 5mg·kg-1. All the rats were put into the white room for a 45-min training. And in the afternoon, all rats had hypodermic injection of cyclodextrin and intraperitoneal injection of Saline of the same dose, and were put into the black room for the same time training. After trained for 10 days, CPP test was scored during a 10-min session 24 h after the last training. Then all the rats were rapidly sacrificed by decapitation. The brain regions including NA, Fc, Hc, ST and Ht were separated and frozen until being taken other procedures.2. Determination of the endogenoud opoiod peptides in rat brainThe procedure of CPP was all the same in this study. Then the rats were rapidly sacrificed by decapitation. The brain regions including VTA, NAc, Ht, and Str were separated and frozen until the further studying.Neuropeptide radioimmunoassay established by the Department of Neorobiology of the Second Militery Medical University of Chinese PLA was applied to measure brain endogenoud opioid peptide levels. Sequential saturation sample addition was adopted. The radioactivity count per minute (cpm) of the whole reaction was 7000 per minute. The activated carbon was separated, centrifuged, and the cpm of the sedent was measured. The standard curve was made accroding to the study. Then, according to the curve, the content of brain endogenoud opioid peptide in each tube was figured out, and it was then translated into the content of endogenoud opioid peptide per miligrame of brain.3. Determination of the mu opoiod receptor in rat brainThe procedure of CPP was as the same as before. Then the rats were rapidly sacrificed by decapitation. The brain regions including VTA, NAc, Ht, and Str were separated and frozen until the further studying.Paraffin sections were used for immunohistochemical staining according to the protocol recently described by the researchers. Briefly, after treatment with 3% H2O2 and 10% normal rabbit serum for 10 and 30 minutes at 37℃, respectively, the deparaffinized and rehydrated sections were incubated with polyclonal rabbit-anti-mu opoiod receptor (1:1000) over night. Biotinylated goat anti-rabbit IgG secondary antibody and ABC complex solution were applied to the sections for 2 hours and 30 minutes at room temperature. The horseradish peroxidase reaction was detected with diaminobenzidine-H2O2 at room temperature for 5-10 minutes. Among each step, 0.01 M PBS (pH 7.4) was used to wash the sections three times with five minutes each time by swaying. Finally, sections were mounted, dehydrated, cleared, and sealed. Slides were observed under a Zeiss Axioskop2 Plus microscope linked to a digital camera from Diagnostics Instruments. Images were captured using the Spot software (Diagnostics) and analyzed with the Image Pro Plus 5.0 image analysis software, resulting in quantification of the protein levels as the mean of integrated optical density (IOD).4. Determination of the dopamine 2 receptor in rat brainBeside the deparaffinized and rehydrated sections were incubated with polyclonal rabbit-anti-D2R (1:250) over night, the other procedures were the same as section 3.5. Determination of the p-CREB levels in rat brainCPP test was used to investigate the morphine rewarding effect. The neuclear lysates were extracted and screened by using Western blot to observe the changes of p-CREB levels in NA, Fc, Hc, Ht, Str and VTA in rat brain. All frozen specimens were grinded in liquid nitrogen and homogenized in lysis buffer on ice. The lystate was centrifuged and the supernatant was prepared. Total protein concentration of the supernatant was measured by BCA Ptotein Assay Bit. 50μg of each protein sample was separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking in skimmed milk for 1 hour, the membranes were incubated with antibody over night. The membrane were then developed with peroxidase-conjugated secondary antibody. The immunoreactive proteins were visulized by enhanced chemiluminescence system. The immunoblots were quantitated using analystic software.The principal results and conclusions were as follows:1 Effects of progesterone administration on morphine-induced conditioned place preference(CPP) and its relationship to endogenous opioids in the related rat brain regionsCompared with NS control group, 5 mg·kg-1 morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg-1 progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.Compared with NS control group, the levels ofβ-EP following morphine administration were decreased in hypothalamus, hippocampus and frontal cortex(P<0.05,P<0.01 and P<0.05). Compared with morphine group, the levels ofβ-EP were increased by co-administration with 15 mg·kg-1 dose of progesterone in hypothalamus (P<0.01). Compared with NS control group, the levels of L-EK following morphine administration were decreased in hypothalamus, nucleus accumbens and frontal cortex(P<0.05, P<0.05 and P<0.01). Compared with morphine group, the levels of L-EK were increased by co-administration with 15mg·kg-1 dose of progesterone in hypothalamus, nucleus accumbens and frontal cortex (P<0.05,P<0.01 and P<0.05). Compared with NS control group, the levels of DynA following morphine administration were increased in hypothalamus and frontal cortex (P<0.05 and P<0.05). Compared with morphine group, the levels of DynA were decreased by co-administration with 15mg·kg-1 dose of progesterone in hypothalamus and frontal cortex (P<0.01 and P<0.05). 2 Effects of progesterone administration on morphine-induced conditioned place preference and its relation toμ-receptors in rat brain regionsCompared with NS control group, 5 mg·kg-1 morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg-1 progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.Compared with NS control group, the levels of mu opioid receptor following morphine administration were decreased in hypothalamus, hippocampus , striatum and frontal cortex(P<0.05,P<0.01,P<0.01 and P<0.01). Compared with morphine group, the levels of mu opioid receptor were increased by co-administration with 15 mg·kg-1 dose of progesterone in hypothalamus, striatum and frontal cortex (P<0.05,P<0.01 and P<0.01).3 Effects of progesterone administration on morphine-induced conditioned place preference and its relation to D2-receptors in rat brain regionsCompared with NS control group, 5 mg·kg-1 morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg-1 progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.Compared with NS control group, the levels of D2 receptor following morphine administration were decreased in nucleus accumbens ,striatum and ventral tegmental area (P<0.01,P<0.01 and P<0.01). Compared with morphine group, the levels of D2 receptor were increased by co-administration with 15 mg·kg-1 dose of progesterone in nucleus accumbens and striatum (P<0.05 and P<0.01).4 Effects of progesterone administration on morphine-induced conditioned place preference and its relation to p-CREB in rat brain regionsCompared with NS control group, 5 mg·kg-1 morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg-1 progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect(P<0.01).Compared with NS control group, the levels of p-CREB following morphine administration were increased in hippocampus and striatum(P<0.01 and P<0.01)but decreased in nucleus accumbens (P<0.01). Compared with morphine group, the levels of p-CREB were decreased by co-administration with 15 mg·kg-1 dose of progesterone in striatum (P<0.01) but increased in nucleus accumbens (P<0.05).In conclusion, our study indicated that chronic morphine treatment could induce the development of morphine psycological dependence and progesterone as an ectogenic neurosteroid could reverse morphine-induced rewarding effects. Durig the procedure of progesterone abolishing morpnine- induced dependence, the levels ofβ-EP, L-EK and DynA were increased or decreased in rat brain. Opioid receptors and dopamine receptors were involved in the regulation of morphine dependence by progesterone. We also found that with the changing in neurotransmitters and receptors, the levels of p-CREB were diversified in rat brain rewarding circuit. Thus, our data provide a new insight for the research of the neurobiochemical mechanism of the relationship between morphine dependence and neurosteroid.
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