| Tourette syndrome(TS)is a common mental disorder of neurodevelopmental disease,characterized by multiple motor tics and vocal tics,etc.Although both of the genetic and environmental factors are involved,the exact etiology of TS is unknown.Numerous studies have found that TS is diagnosed much higher in males than in females;the androgen level is higher in TS children than in healthy children;and the treatment with flutamide which is an antagonist of androgen receptor,has remarkably alleviated symptoms in patients with TS.These findings imply that androgen is likely to be involved in the development of TS.Neurotransmitters play pivotal roles in the regulation of cerebral function activities.The balance among different neurotransmitters in the nerve circuits ensures the normal brain activities.A variety of studies have indicated that TS patients have increased brain dopamine,dopamine transporter and dopamine receptors D1,D2 in frontal cortex.Serotonin and its metabolite 5-hydroxy indole acetic acid(5-HIAA)in the cerebrospinal fluid are reduced in TS patients in comparison with control subjects.Motor tics are negatively correlated with serotonin content in cerebrospinal fluid.TS comorbidity of obsessive-compulsive disorder(OCD)manifests increased dopamine receptors D2 in ventral striatum and serotonin transporter in midbrain and caudate putamen.Above lines of evidence suggest that TS patients have dysfunctional dopaminergic and serotonergic nervous system in the brain.Finasteride is a type II 5α-reductase inhibitor which prevents conversion of testosterone to the more active dihydrotestosterone that reduces the binding of dihydrotestosterone to receptors and therefore suppresses the effect of androgen.Recent pre-clinical studies have shown that treatment with finasteride for adult TS patients could significantly improve tics.One of the contributing factors for the improvement of tics is the finasteride induced decreas in the activity of androgen;the other one is that finasteride modulates brain dopaminergic neurologic activities and therefore regulates motor behaviors of tic symptoms in TS.Finasteride has become the potential therapeutic medication in the treatment of various diseases associated with dopaminergic hyperactivity.Based on above research findings,we hypothesize that inhibiting the production of dihydrotestosterone during early development and adolescence may induce alterations of neurotransmitters and correlate behaviors.To this end,the current study is 1)to investigate the effects of administration of testosterone propionate(TP)during early development on neurochemicals alterations and detect androgen-associated neurotransmitters changes in the brain;2)to study the effects of inhibition of androgen conversion by finasteride during early development and mid adolescence on behavioral changes and to explore whether the behavioral changes are correlated to TP regulated neurotransmitters alterations.These studies would provide neurobiological basis of androgen disturbance associated neurodevelopmental diseases.Part 1 Administration of testosterone propionate during earlydevelopment induced brain neurochemicals alterationsObjectives: To determine the effects of testosterone propionate administration during early development on neurochemicals alterations and to detect androgen-associated neurotransmitters changes.Methods: Twtenty-nine postnatal day 7 male Wistar rats were divided into Control group and TP group.Rats in TP group received testosterone propionate(TP)injection subcutaneously,0.05 mg per rat,once per day for 14 consecutive days.Rats in Control group received same volume of sesame oil injection subcutaneously.Rats were euthanized and decapitated on postnatal day 21(weaning,early adolescence)and 49(late adolescence)respectively.Frontal cortex,caudate putamen(CPu),nucleus accumbens(Acb)and hippocampus(Hip)were separated and collected.The content of dopamine(DA),dihydroxy phenyl acetic acid(DOPAC),homovanillic acid(HVA),serotonin(5-HT),and 5-hydroxy indole acetic acid(5-HIAA),norepinephrine(NE),γ-aminobutyric acid(γ-GABA),histamine(His),glutamate(Glu)were determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results:Effects of testosterone propionate administration during early development on neurochemicals alterations1 Frontal cortex1.1 Postnatal day 21 ratsIn comparison to Control group,there was no significant change for all neurochemicals in TP group.1.2 Postnatal day 49 ratsIn comparison to Control group,serotonin and 5-HIAA levels were significantly increased by 25%(P<0.01)and 26%(P<0.05)respectively in TP group,and there was no other significant neurochemical change.2 Caudate putamen2.1 Postnatal day 21 ratsIn comparison to Control group,dopamine level was significantly increased by 22%(P<0.05)in TP group,and there was no other significant neurochemical alteration.2.2 Postnatal day 49 ratsIn comparison to Control group,dopamine and HVA levels were significantly elevated by 20%(P<0.05)and 27%(P<0.05)respectively in TP group,and there was no other significant neurochemical alteration.3 Nucleus accumbens3.1 Postnatal day 21 ratsIn comparison to Control group,dopamine and serotonin levels were significantly elevated by 65%(P<0.01)and 23%(P<0.05)respectively in TP group,and there was no other significant neurochemical alteration.3.2 Postnatal day 49 ratsIn comparison to Control group,dopamine and serotonin levels were significantly increased by 22%(P<0.05)and 23%(P<0.01)respectively in TP group,and there was no other significant neurochemical change.4 Hippocampus4.1 Postantal day 21 ratsIn comparison to Control group,there was no significant change for all neurochemicals in TP group.4.2 Postnatal day 49 ratsIn comparison to Control group,there was no significant change for all neurochemicals in TP group.Summary: Administration of testosterone propionate during early development increased the contents of DA and 5-HT and their metabolites in both early and late adolescent male rats,which indicated that the activities of dopaminergic and serotonergic systems were up-regulated.Part 2 Treatment of finasteride during early development had no effect onneurobehaviors and neurochemicals in the brainObjective: To observe the effects of finasteride treatment during early development on alterations of behaviors and dopaminergic and serotonergic nervous systems.Methods: Seventy postnatal day 7 male Wistar rats were divided into Control group,Fin-3 group,Fin-25 group and Fin-50 group.Rats in Fin-3 group,Fin-25 group and Fin-50 group received subcutaneous injection of finasteride according to 3 mg/kg,25 mg/kg and 50 mg/kg,once per day for 14 consecutive days.Rats in Control group received same volume of sesame oil injection subcutaneously.Behavior tests were conducted by the postnatal day 21 or 49.Behavior tests included open field test,sucrose preference test,novelty-suppressed feeding test and forced swimming test.Rats were euthanized and decapitated after the end of behavior tests.Frontal cortex,caudate putamen(CPu),nucleus accumbens(Acb),hippocampus(Hip),substantia nigra(SN),ventral tegmental area(VTA)and dorsal raphe nucleus(DR)were separated and collected.The content of dopamine(DA),dihydroxy phenyl acetic acid(DOPAC),homovanillic acid(HVA),serotonin(5-HT),and 5-hydroxy indole acetic acid(5-HIAA),norepinephrine(NE),γ-aminobutyric acid(γ-GABA),histamine(His),glutamate(Glu)were determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Tyrosine hydroxylase(TH)and tryptophan hydroxylase(TPH)were assessed and detected by Real-Time qRT-PCR and western blot.Results:1 Effects of finasteride on neurobehaviorsBoth postnatal day 21 rats and postnatal day 49 ratsIn comparison to Control group,there was no significant change for open field test,sucrose preference test,novelty-suppressed feeding test and forced swimming test in Fin-3 group,Fin-25 group and Fin-50 group.2 Effects of finasteride on neurochemicals in the brain2.1 Frontal cortexBoth postnatal day 21 rats and postnatal day 49 ratsIn comparison to Control group,there was no significant change for all neurochemicals in Fin-3 group,Fin-25 group and Fin-50 group.2.2 Caudate putamenBoth postnatal day 21 rats and postnatal day 49 ratsIn comparison to Control group,there was no significant change for all neurochemicals in Fin-3 group,Fin-25 group and Fin-50 group.2.3 Nucleus accumbens2.3.1 Postnatal day 21 ratsIn comparison to Control group,HVA level was significantly decreased by 28%(P<0.05)in Fin-3 group;5-HIAA level was significantly reduced by 19%(P<0.05)and 17%(P< 0.05)in Fin-25 group and Fin-50 group respectively.There was no other significant neurochemical change in comparison with Control group.2.3.2 Postnatal day 49 ratsIn comparison to Control group,there was no significant change for all neurochemicals in Fin-3 group,Fin-25 group and Fin-50 group.2.4 Hippocampus4.1 Postnatal day 21 ratsIn comparison to Control group,there was no significant change for all neurochemicals in Fin-3 group,Fin-25 group and Fin-50 group.4.2 Postnatal day 49 ratsIn comparison to Control group,dopamine level was significantly reduced by 28%(P<0.05)and 25%(P<0.05)in Fin-25 group and Fin-50 group respectively.There was no significant change for other neurochemicals.3 Effects of finasteride on mRNA expressions of TH and TPH in the brainBoth postnatal day 21 rats and 49 rats,in comparison to Control group,there was no significant change for mRNA expression levels of TH and TPH in substantia nigra,ventral tegmental area and dorsal raphe nucleus in Fin-3 group,Fin-25 group and Fin-50 group.4 Effects of finasteride on protein expressions of TH and TPH in the brainBoth postnatal day 21 rats and 49 rats,in comparison to Control group,there was no significant change for protein expression levels of TH and TPH in caudate putamen,nucleus accumbens,substantia nigra,ventral tegmental area and dorsal raphe nucleus in Fin-3 group,Fin-25 group and Fin-50 group.Summary: There was no significant alteration by the treatment of finasteride during early development in spontaneous motor,exploratory and depressive behaviors in both early and late adolescent male rats.Apart from several changes in dopaminergic and serotonergic systems in few brain areas,there was no other metabolite alteration.Part 3 Treatment of finasteride to adolescent male rats reducedneurobehaviors,activities of dopaminergic and serotonergicnervous systems in adolescent male rat brainObjective: To observe the effects of finasteride treatment to adolescent male rats on alterations of spontaneous motor,exploratory,depressive behaviors,dopaminergic and serotoniergic nervous systems.Methods: Twenty-eight postnatal day 35 male Wistar rats were divided into Control group,Fin-3 group,Fin-25 group and Fin-50 group.Rats in Fin-3 group,Fin-25 group and Fin-50 group received subcutaneous injection of finasteride according to 3 mg/kg,25 mg/kg and 50 mg/kg,once per day for 14 consecutive days.Rats in Control group received same volume of sesame oil injection subcutaneously.Behavior tests were conducted by the postnatal day 49.Behavior tests included sucrose preference test and open field test.Rats were euthanized and decapitated after the end of behavior tests.Frontal cortex,caudate putamen(CPu),nucleus accumbens(Acb),hippocampus(Hip),substantia nigra(SN),ventral tegmental area(VTA)and dorsal raphe nucleus(DR)were separated and collected.The content of dopamine(DA),dihydroxy phenyl acetic acid(DOPAC),homovanillic acid(HVA),serotonin(5-HT),and 5-hydroxy indole acetic acid(5-HIAA)were determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Tyrosine hydroxylase(TH)and tryptophan hydroxylase(TPH)expression levels were assessed and detected by Real-Time qRT-PCR and western blot.Results:1 Behavior1.1 Sucrose preference behaviorIn comparison to Control group,the percentage of sucrose intake was significantly reduced by 36%(P<0.01)and 39%(P<0.01)in Fin-25 group and Fin-50 groups respectively.In comparison to Fin-3 group,the percentage of sucrose intake was significantly reduced by 35%(P<0.01)and 38%(P<0.01)in Fin-25 group and Fin-50 groups respectively.1.2 Open field behavior1.2.1 Exploratory behaviorIn comparison to Control group,the numbers of walking,sniffing and climbing were significantly decreased by 31%(P<0.01)and 58%(P<0.01),38%(P<0.01)and 64%(P<0.01),42%(P<0.05)and 68%(P<0.01)in Fin-25 group and Fin-50 group respectively;In comparison to Control group,the number of rearing was significantly reduced by 63%(P<0.01)in Fin-50 group;There was no significant change for both Fin-3 group and Fin-25 group in comparison to Control group.1.2.2 Spontaneous motor behaviorIn comparison to Control group,the numbers of vertical activity and horizontal activity were significantly decreased by 40%(P<0.05)and 66%(P<0.01),34%(P<0.01)and 56%(P<0.01)in Fin-25 group and Fin-50 group respectively;In comparison to Control group,the total path length was significantly decreased by 13%(P<0.05),33%(P<0.01)and 54%(P<0.01)in Fin-3 group,Fin-25 group and Fin-50 group respectively.2 Dopaminergic and serotonergic nervous systems2.1 Dopamine,serotonin and their metabolitesFrontal cortex: In comparison to Control group,dopamine level was significantly reduced by 35%(P<0.01)and 59%(P<0.01)in Fin-25 and Fin-50 group respectively;serotonin level was significantly decreased by 58%(P<0.01)in Fin-50 group;5-HIAA level was significantly reduced by 28%(P<0.05)and 53%(P<0.01)in Fin-25 group and Fin-50 group respectively.Caudate putamen: In comparison to Control group,dopamine level was significantly reduced by 35%(P<0.05)and 55%(P<0.01)in Fin-25 group and Fin-50 group respectively;DOPAC level was significantly decreased by 23%(P<0.05)and 45%(P< 0.01)in Fin-25 group and Fin-50 group respectively;HVA level was significantly reduced by 23%(P<0.05)and 51%(P<0.01)in Fin-25 and Fin-50 group respectively;serotonin level was significantly decreased by 27%(P<0.05),37%(P<0.01)and 64%(P<0.01)in Fin-3 group,Fin-25 group and Fin-50 group respectively;5-HIAA level was significantly reduced by 26%(P<0.01)and 49%(P<0.01)in Fin-25 group and Fin-50 group respectively.Nucleus accumbens: In comparison to Control group,dopamine level was significantly reduced by 34%(P<0.05)and 57%(P<0.01)in Fin-25 group and Fin-50 group respectively;DOPAC level was significantly decreased by 23%(P<0.05)and 38%(P<0.01)in Fin-25 group and Fin-50 group respectively;HVA level was significantly reduced by 50%(P<0.01)in Fin-50 group;serotonin level was significantly decreased by 25%(P<0.01)and 48%(P<0.01)in Fin-25 group and Fin-50 group respectively;5-HIAA level was significantly reduced by 52%(P<0.01)in Fin-50 group.Hippocampus: In comparison to Control group,dopamine level was significantly reduced by 34%(P<0.05)and 63%(P<0.01)in Fin-25 group and Fin-50 group respectively;serotonin level was significantly decreased by 26%(P<0.05)and 50%(P< 0.01)in Fin-25 group and Fin-50 group respectively;5-HIAA level was significantly reduced by 62%(P<0.01)and 72%(P<0.01)in Fin-25 and Fin-50 group respectively.2.2 mRNA expressions of TH and TPHSubstantia nigra: In comparison to Control group,TH mRNA expression level was significantly reduced by 33%(P<0.01)and 37%(P<0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-3 group,TH mRNA expression level was significantly decreased by 27%(P<0.01)and 31%(P<0.01)in Fin-25 group and Fin-50 group respectively.Ventral tegmental area: In comparison to Control group,TH mRNA expression level was significantly reduced by 35%(P<0.01)and 42%(P<0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-3 group,TH mRNA expression level was significantly decreased by 32%(P<0.01)and 39%(P<0.01)in Fin-25 group and Fin-50 group respectively.Dorsal raphe nucleus: In comparison to Control group,TPH mRNA expression level was significantly reduced by 20%(P<0.05)and 37%(P<0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-3 group,TPH mRNA expression level was significantly decreased by 28%(P<0.01)in Fin-50 group.2.3 Protein expressions of TH and TPHCaudate putamen: In comparison to Control group,TH protein expression level was significantly reduced by 32%(P<0.01),66%(P<0.01)and 84%(P < 0.01)in Fin-3 group,Fin-25 group and Fin-50 group respectively.In comparison to Fin-3 group,TH protein expression level was significantly decreased by 49%(P<0.01)and 76%(P<0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-25 group,TH protein expression level was significantly decreased by 52%(P<0.01)in Fin-50 group.Nucleus accumbes: In comparison to Control group,TH protein expression level was significantly reduced by 65%(P<0.01)in Fin-50 group.In comparison to Fin-3 group,TH protein expression level was significantly decreased by 60%(P<0.01)in Fin-50 group.In comparison to Fin-25 group,TH protein expression level was significantly decreased by 49%(P<0.01)in Fin-50 group.Substantia nigra: In comparison to Control group,TH protein expression level was significantly reduced by 40%(P<0.01),57%(P<0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-3 group,TH protein expression level was significantly decreased by 40%(P<0.01)and 57%(P<0.01)in Fin-25 group and Fin-50 group respectively.Ventral tegmental area: In comparison to Control group,TH protein expression level was significantly reduced by 45%(P<0.01),64%(P<0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-3 group,TH protein expression level was significantly decreased by 32%(P<0.01)and 56%(P < 0.01)in Fin-25 group and Fin-50 group respectively.In comparison to Fin-25 group,TH protein expression level was significantly decreased by 34%(P<0.05)in Fin-50 group.Dorsal raphe nucleus: In comparison to Control group,TPH protein expression level was significantly reduced by 48%(P<0.01)in Fin-50 group.In comparison to Fin-3 group,TPH protein expression level was significantly decreased by 48%(P<0.01)in Fin-50 group.In comparison to Fin-25 group,TPH protein expression level was significantly decreased by 32%(P<0.05)in Fin-50 group.Summary: Treatment of finasteride to adolescent male rats induced deficits in spontaneous motor,exploratory and depressive behaviors,reduced the contents of DA and 5-HT and their metabolites,and decreased mRNA and protein expression levels of DA and 5-HT snythetases TH and TPH in the brain,which suggested the reduction in the activities of dopaminergic and serotonergic systems.Conclusions:1 The administration of testosterone propionate during early development increased activities of dopaminergic and serotonergic nervous systems mainly in late adolescent male rats.2 Treatment of finasteride to adolescent male rats induced deficits in spontaneous motor,exploratory and depressive behaviors,reduced the contents of DA and 5-HT and their metabolites,and decreased mRNA and protein expression levels of DA and 5-HT snythetases TH and TPH in the brain,which suggested the reduction in the activities of dopaminergic and serotonergic systems.3 Above findings suggested that finasteride-induced behavioral alterations are likely to be correlated with its regulation in dopaminergic and serotonergic nervous systems in adolescent male rats. |
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