The Effects Of Testosterone Propionate On Open Field Behaviors In The Early Development Of Male Rats And The Participation Of Midbrain DA Neurons In Open Field Behaviors Change

Tourette Syndrome(TS) is a common children’s behavior disordersyndrome in clinic. It is a comprehensive syndrome characterized byinvoluntary muscles twitch on the face, limbs and trunk with abnormallaryngeal pronunciation, thus dirty language. This disease occurred mosty inboys, and incidence ratio of men and women is3-9:1, while in parts of ourcountry it is even as high as10.6:1. Patients with this disease are accompaniedby at least one behavior or mood disorders, about a third of the patients withOCD. Many studies demonstrated that TS has obvious course characteristics,characterized by childhood-onset, illness is aggravating in adolescence andmotor symptoms gradually improve as an adult, even disappear. It has beenreported that urine of TS children with severe symptoms contains high level oftestosterone. Patients can be with hyperandrogenism performance.Antiandrogen treatment can significantly alleviate TS symptoms in adultpatients. These results showed the incidence may be related to androgen levelsdisorder in children.Recent clinical data suggested that dysfunction of dopamine (DA)nervous system is closely related to the TS. By Genin neural imagingtechnology, TS children (6to12years of age) can be detected with newstriatal dopamine transporter (DAT) signal enhancement, and TS adolescentpatients (12to17years of age) can be detected with abnormal DOPAdecarboxylase activity enhancement in caudate nucleus. Clinical data showedthat amphetamine can enhance striatum dopamine release in putamen andveutro of TS patients, and dopamine receptor antagonist has inhibited twitchsymptoms of TS children. These results indicated that TS may have closerelationship with brain dopamine nervous system. Most of testosterone in men is produced by leydig cells. Testosterone isan important form of androgen in the body, and has the characteristics offat-soluble. It can be used as a male steroid hormones (AAS), and can throughthe blood brain barrier and act on central nervous system. Researches showedthat abuse of AAS will lead to hyperactivity, lack of impulse control andemotional instability. In addition, the animal experiments also found thattestosterone can lead to the increase of motor behavior and excessivegrooming of normal adult male rats. The above experiments proved that AAShas affected the behavior changes in male rats.DA neurons in the brain are mainly concentrated in the midbrainsubstantial nigra compacta (SNc) and ventral tegmental area (VTA), etc. Thesubstantia nigra-striatum and VTA-cortical dopaminergic neurons regulatemovement, cognition and thinking ability respectively. Whether the change ofandrogen levels are related to the abnormal behavior disorders in the TSchildren development or the function of the brain dopaminergic nerve system,it is not very clear.Therefore, the study chose male Wistar rats as experimental object,respectively subcutaneous injection of testosterone propionate (testosteronepropionate, TP group), androgen receptor inhibitor flutamide (flutamide, Flugroup), and flutamide+testosterone propionate (Flu+TP group) to establishthe experimental animal model. The open field experiment, LC-MS/MS,western blot and RT-PCR technique are used to observe behaviors of theWistar rats and the related parameters of the midbrain DA neurons in3weeks(youth),7weeks of age (adolescence) and6months of age (adult). The aimsof the study are to explore the possible mechanisms in androgen acting ondopamine nervous system of rats in development, and it is expected thatprovide certain experiment basis for exploring the pathogenesis of TS.Part1Effects of TP on male rats open field behaviors in the earlydevelopmental stageObjective To study the effects of chronic administration of TP onmotor-related behaviors of male rats in the three developmental stages. Methods Using open field test to observe rat behavior parameters in3weeks,7weeks and6months of age; Measuring the rat body weight, gonadorgans, pituitary weight. Radioimmunoassay (RIA) was used to detecttestosterone, follicle-stimulating hormone and luteinizing hormoneconcentration in serum after administration of TP.Results1At3weeks and7weeks, compared with the control group, TP groupimmobile-sniffing behavior, exploratory behavior, locomotor behavior andgrooming behavior were increased significantly. However, compared with thecontrol group, Flu+TP group open field behavior parameters showed noobvious change but the amount of Walking reduction at3weeks and7weeksand number of grooming increasing in7weeks. Compared with the TP group,TP group immobile sniffing, locomotor behavior, exploration behavior andgrooming behavior significantly reduced. The thigmotaxic behavior has noobvious change in TP or Flu group at3weeks,7weeks and6months. At6months, each behavior had no obvious change in all groups of rats.2There were no significant differences between groups of rats weigh at3weeks,7weeks and6months.3Effect of early TP administration on seminal vesicle, testis and pituitaryweight.At3weeks, compared with the control group, average vesicle weight inTP group increases by1028%（P<0.01），average testis weight decreases by24%（P<0.05）, average testis weight in Flu group decreases by16%（P<0.05）,average seminal vesicle weight in Flu+TP group has increases by201%(P<0.01), average testis weight decreases by60%(P <0.01).Weight of averageseminal vesicle and testis weight in Flu+TP group compared with TPdecreases by73%(P <0.01) and47%(P <0.01). Average seminal vesicleweight in Flu+TP group Compared with Flu group has increases by256%(P<0.01), average testis weight has decreases by62%(P <0.01). Pituitary weightof each group has no obvious change (P>0.05)At7weeks, compared with the control group，average testis weight decreases25%（P<0.01）in TP group，average testis weight in Flu group hasno obvious difference, average testis weight in Flu+TP group has decreasedby62%(P <0.01).Average weight of testis in Flu+T P group compared withTP reduce49%(P <0.01).Average weight of testis in Flu+TP groupcompared with Flu group decreases58%(P <0.01). Average pituitary weightof each group has no obvious change (P>0.05)At6months, each above organ has no obvious change in all groups（P>0.05）.4Effect of Early TP administration on serum T, LH and FSH in threedevelopmental stagesAt3weeks, compared with the control group， concentration of serumTin TP group increases4400%（P<0.01）, serum T in Flu+TP group increases4229%（P<0.01）. Serum T in Flu+TP group compared with Flu increases3996%（P<0.01）. serum T, LH and FSH in each group has no obvious change（P>0.05）.At7weeks, compared with the control group，concentration of serum Tin TP group decreases53%（P<0.01）, serum T in Flu+TP group decreases88%（P<0.01）. Serum T in Flu+TP group compared with TP decreases74%（P<0.01）.Serum T in Flu+TP group compared with Flu decreases88%（P<0.01）. serum T, LH and FSH in each group has no obvious change（P>0.05）.At6months, concentration of serum T, LH and FSH has no obviouschange in all groups（P>0.05）Conclusions:1Subcutaneous administration of TP lead rats to remain at a high level ofblood testosterone.2Early administration of TP on pup rats leads to open field behaviorparameters increasing significantly in childhood and puberty period,suggesting that high levels of androgens in the early development time maychange the brain neural signal transmission. Part2Testosterone propionate promotes the expression of DA,DOPAC and HVA in the midbrain dopaminergic neurons in the earlydevelopment stageObjective To observe the effect of TP on DA neurotransmitters and theirmetabolic products in the substantia nigr-caudate putamen and the ventraltegmental area-the nucleus accumbens neurons, discussed the relationshipbetween early behavior changes by administration of TP and dopaminergicnerve signal transmissionMethods Using LC-MS/MS to observe the expressions of DA, DOPACand HVA on rat corpus striatum and nucleus accumbens in3weeks,7weeksand6months.ResultsThe changes of DA and metabolite in corpus striatum, nucleusaccumbens.At3weeks, compared with control group, the expression of DA,DOPAC and HVA in corpus striatum are increased, the expression of DA,DOPAC in nucleus accumbens in TP group are increased（P<0.01）, there is nosignificant change on DOPAC+HVA/DA, DOPAC/DA and HVA/DA. Theexpression of DA in corpus striatum and nucleus accumbens in Flu group aredecreased, the specific value of DOPAC+HVA/DA and DOPAC/DA incorpus striatum are increased（P<0.01）, the specific value of corpus striatumHVA/DA is increased by40%. The expression of DA in corpus striatum inFlu+TP group is reduced by43%, the specific value of DOPAC+HVA/DAand DOPAC/DA are increased（P<0.01）, there is no significant change oncorpus striatum. Compared with TP group, the expression of DA and DOPACin Flu+TP group is obviously decline. There is no obvious decreased onnucleus accumbens HVA.At7weeks, compared with control group, the expression of DA,DOPAC and HVA in corpus striatum and nucleus accumbens in TP group areobviously increased（P<0.01）, there is no significant change on the specificvalue of DOPAC+HVA/DA,DOPAC/DA and HVA/DA. The expression ofcorpus striatum HVA in Flu group are increased by25%, there is no change on all targets of nucleus accumbens; there is no obvious change on all targetsof corpus striatum in Flu+TP group. Compared with TP group. There is noobvious change on corpus striatum in Flu+TP group, the expression ofnucleus accumbens DA is reduced by26%.At6months, there is no obvious change on DA neurotransmitter and itsmetabolite of male rats in each group.Conclusion The early TP treatment on neonatal rats can promote themidbrain DA, DOPAC and HVA expression in childhood and adolescenceperiods, while administration of flutamide inhibited the effect of exogenousTP.Part3Testosterone propionate early treatment increase theexpression of TH, DAT, MAOA and MAOB and COMT in midbraindopaminergic neurons of the male ratsObjective To observe the effect of TP administration on pup rats on therelevant activities indicators of the midbrain dopaminergic neurons. Then weexplore in depth the relationship between these effects and how TP canpromote DA, DOPAC and HVA expressionMethods Detecting the expressions of nigra TH, DAT protein, TH, DATof male rats, MAO, MAO and COMT mRNA after treating by testosteronepropionate by western blot and realtime RT-PCR.Results The effect of TP’s earlier treatment on TH and DAT in SN,CPu, VTA, Acb.1At3weeks and7weeks, compared with control group, the expressionsof TH， DAT in substantial nigra, ventral tegmental area and nucleusaccumbens in TP group are increased. Compared with TP group, theexpressions of TH, DAT in substantial nigra, ventral tegmental area andnucleus accumbens in Flu+TP group are obviously reduced.At6months, there is no obviously change on the expressions ofsubstantial nigra, ventral tegmental area and nucleus accumbens TH, DAT ineach group.2The effect of TP’s earlier treatment on TH, DAT, MAOA, MAOB, COMT gene in SN, VTA.At3weeks and7weeks, compared with control group, the expressionsof TH mRNA, DAT mRNA, MAOA mRNA, MAOB mRNA and COMTmRNA in substantial nigra, ventral tegmental area are obviously increased inTP group. Compared with TP group, the expressions of TH mRNA,DATmRNA, MAOA mRNA, MAOB mRNA and COMT mRNA insubstantial nigra, ventral tegmental area are obviously reduced in Flu+TPgroup.At6months, there is no obviously change on the expressions ofsubstantial nigra, ventral tegmental area TH mRNA, DATmRNA, MAOAmRNA, MAOB mRNA and COMT mRNA in each group.Conclusion Early TP administration on pup rats leads to change on TH,DAT and its mRNA and MAOA and MAOB, COMT mRNA expression levelin childhood and adolescence midbrain DA neurons, which performance theenhancement effect of TP. Flu inhibited the effect of exogenous TP, whichsuggested that the midbrain neural signal transmission of DA changesassociated with TP regulation of neurotransmitter DA metabolic enzymes andthe expression of DA transporter.