Effect Of Urocortin2on Ventral Tegmental’ Areas Spontaneous Discharge In Morphine Dependent Rats And The Relationship With T-type Calcium Channel

ObjectiveThe purpose of this study used the method of microelectrophoresisextracellular recording to observe the effects of neuroendocrine active peptideurocortin2(Ucn2) on spontaneous discharge (SPD) and the relationship withT-type calcium channel on ventral tegmental area (VTA) of morphine dependentrats, also to explore the mechanism in the withdraw process, which provided thetheoretical basis for clinical treatment of opioid addiction.MethodsThe experiment took50healthy male Sprague-Dawley (SD) rats, randomlydivided into the model group and control group. The former which had35ratsmainly used to build the morphine addiction model by increasing doses ofintraperitoneal injection of morphine hydrochloride, the latter which had15ratsgave the normal saline in the same condition, at the end they both added thenaloxone (5mg kg-1) to induce morphine addiction withdrawal after12days.The experiment adopted the method of microelectrophoresis extracellularrecording respectively to record the spontaneous discharge activity on the VTA’sneurons about the control group and model group. First of all, depended on amultipolar glass microelectrode swam into the different concentrations of Ucn2and corticotrophin releasing factor2(CRF2) receptor blocker (astressin, AST), for researching the effects on VTA’s neuron discharge by Ucn2and theexistence of concentration dependence, whether the function of Ucn2affectedCRF2receptor in the VTA. Secondly, it added glutamate (Glu)+Ucn2, Glu+ASTto observe the influence of neurotransmitter glutamate on the VTA’s neuronsthrough Ucn2. Consequently, protein kinase A (PKA) and protein kinase Ainhibitor (H-89) and T-type calcium channel blocker (Mibefradil) were used inthis study, further to know about the effect of Ucn2on the VTA in morphinedependent rats whether by PKA and T-type calcium channel pathway.Results1. The model group were two rats died from individual differences in theprocess of experiment and the rest were successful of morphine addiction withtheir withdrawal symptoms score (17.32±2.13) points. There were three mainforms of irregular discharge, discharge and explosive discharge in the controland model group rats on VTA nuclei. The average discharge frequency of themodel group on VTA was (22.14±4.12) Hz significantly higher than the controlgroup (13.76±4.25) Hz (P<0.01). The irregular discharge and explosivedischarge significantly increased in the model group, while the control groupwas given priority to discharge, could see Irregular discharge, and accidentallysaw explosive discharge. The difference was statistically significant (P<0.05).2. Microelectrophoresised different concentrations of Ucn2on VTA’sneurons of the model group and control group, the frequency of spontaneousdischarge with the concentration of Ucn2from low to high in turn, there waslinear relationship between the two groups, comparing with the period of beforegiving drug, the difference had the statistical significance (P<0.05or P<0.01).Microelectrophoresised AST alone, the frequencies were more increased thanthe lower dosage, while microelectrophoresised the Ucn2and AST at the sametime, the result did not change.3. Microelectrophoresised Glu alone to the VTA nuclei of the model group, the frequency of neuron discharge increased significantly, then added Ucn2, theexcitability effects was inhibited by Ucn2, while used AST, it was notsuppressed.4. Microelectrophoresised Ucn2and PKA at the same time on the VTA ofthe model group, the frequency obviously decreased to the not giving drug. Itcontinued to swim into the H-89, returning to the dosing levels; whilemicroelectrophoresised Ucn2and H-89, the frequency had meaninglesslychange to compare with the not giving drug; injecting the Ucn2, PKA andmibefradil, which compared with microelectrophoresising single Ucn2and PKA,the frequency obviously decreased.ConclusionsThis experiment suggests Ucn2could inhibit the spontaneous discharge onVTA’s neurons in the morphine dependent rats, and it occurs to the dosedependent. Its inhibition may be on account of Ucn2combination with CRF2receptor, then influences the PKA signaling pathways, finally, restrains theopening of T-type calcium channel in VTA’s neuron, thus inhibits the unusuallyhigh frequency discharge on VTA in morphine dependent rats.