| Dopamine in the central nervous system is closely correlated with neuropsychiatric disorders, which is mainly localized in nigro-striatum system. Dopamine transporter (DAT) is the membrane protein located in axon terminals of DAenergic neurons. It reuptakes DA specificly from synaptic cleft to the presynaptic nerve terminals and terminates DA action on the postsynaptic neurons. DAT is an important index to reflect the founctional status of DA system and has been widely used in ligand neuroimaging technique to detect the malfunction of the dopamine system in related diseases.Many studies showed that the attention-deficit hyperactivity disorder (ADHD) mainly afflict gradeschool children. ADHD children behaved inattention, hyperactivity and so on. With obvious gender difference, more boys are diagnosed with ADHD than girls, suggesting that the disturbance of androgen level may be one possible factor for the ADHD. Neuroimaging technique showed that there is the altered signals of DAT binding in the brain of ADHD children. Disturbed androgen level maybe caused or induced ADHD onset.Recent studies found that anabolic– androgenic steroids (AAS) can affect the brain reward system, which is related to dopamine sytem. Ligand neuroimaging technique detected the alteredβ-CIT binding signal in adult rats brain following the administration of adrogen. ?-CIT is a commonly-used ligand detecting DAT in neuroimaging. However, besides DAT, it can also bind 5-HT transporter and noradrenaline transporter. Studies showed that the overlapping distribution of three transporters was found in most brain areas. Therefore, ligand neuroimaging technique using ?-CIT is difficult to analyze the true change of DAT in brain.Objective: The open field test, immuocytochemistry and western blot were used in this study to observe the changes of the locomotion activity and the dopamine system in TP rats, to discuss the influence of early testosterone propianate treatment on the expression of DAT and the behavior of rat, to probe into the relationship between testosterone propianate and ADHD and to expect to provide some experiment evidence for the neurobiology meachanism of ADHD.Methods:Pups used for experiment were randomly divided into experimental and control groups. animals in the experimental group were given a subcutaneous injection of 500μg testosterone propionate from postnatal day 7(PND7) to PND21; and those in the control group were given an injection of 0.05 ml vehicle(sesame oil) alone. Open field test were observed on postnatal days 45,46 and 47 three consecutive days, immuocytochemistry and westernblot was used to analyze the alteration of the expression of DAT in rats brain.Results: 1. open field test: Among the observed index,The results indicated that the TP rats showed significant differences in both Horizontal activity and total path length in 8minute and 15 minute group according to the Veh rats(p<0.05); the vertical locomtion had no significant difference once in the 8 minute group(p>0.05), but there was a incerased trend,and all had statistical difference in 15 minute group(p<0.05);the numbers of grooming of rat was significantly increased in 15minute group(p<0.05),and had no significant difference once in the 8 minute group too(p>0.05), but there was a incerased trend. In addition,during the growth,the weight of rats in these group had no significantly difference,but the TP rats had a more complicated approaching line than the Veh rats in the open field test,and had a more sensitive vigilance to the surrounding environment. 2. The experimental results of immunohistochemical staining:①The results of DAT immunohistochemical staining: The highest density of DAT(Rat-anti-DAT) was mainly found in the substantia nigra and striatum. The early testosterone treatment can enhance the expression of DAT in substantia nigra and striatum of rats. The gray scale mean value are 101.41±4.72(TP) and 111.01±12.14(Veh) in substantia nigra, 102.61±5.73(TP) and 113.68±12.08(Veh) in striatum, indicating that the gray scale mean value of DAT in substantia nigra and striatum of the experimental are significently lower than that of control(P<0.01), and decreased 9.47% and 10.79% respectively.②The results of TH immunohistochemical staining: No significant change of the count of substantia nigra TH-immunoreactive neurons was observed in two groups in SN(P>0.05). The gray scale mean value of TH are significently lower than that of control(P<0.01), decreased 13.46% and 14.15% respectively in substantia nigra and striatum. 3. Western-Blot:The results showed that the ratio of IOD were higher in the TP group than in the Veh.(1)Western-Blot shows the ratio of IOD of TH/β-actin in Veh are: substantia nigra 1.4604±0.2174, striatum 2.6931±0.2523;those in TP are: substantia nigra 1.9263±0.1647, striatum 4.2186±0.3987. all the two groups are of significant statistical importance (P<0.05). (2)Western-Blot shows the ratio of IOD of DAT/β-actin in Veh are(Rat-anti-DAT): substantia nigra 0.7044±0.0734(80kDa) and 0.4179±0.041(50kDa), striatum 4.2416±0.5436(80kDa) and 1.0976±0.3659(50kDa);those in TP are: substantia nigra 0.9081±0.0199(80kDa) and 0.5374±0.0567(50kDa), striatum 5.4952±0.6285(80kDa) and 1.5117±0.1134(50kDa). all the dates are of significant statistical importance (P<0.05). (3) Western-Blot shows the ratio of IOD of DAT/β-actin in Veh are(Rb-anti-DAT): substantia nigra 1.0297±0.4702(80kDa) and 0.4377±0.1389(50kDa), striatum 0.5227±0.3035(80kDa) and 0.2148±0.0898(50kDa);those in TP are: substantia nigra 2.5606±0.8808(80kDa) and 0.7371±0.1302(50kDa), striatum 1.1914±0.232(80kDa) and 0.4352±0.0938(50kDa). all the dates are of significant statistical importance (P<0.05), increased 31.9% and 56.7% in substantia nigra and striatum respectively.Conclusion: 1. Early testosterone propionate treatment altered the locomotion activity of adolescent rats, which showed the increase of horizontal activity, vertical locomtion, total path length and numbers of grooming. 2. Early testosterone propionate treatment increased the dopamine transporter content in Substantia Nigra and caudate-putamen of rats. 3. Early testosterone propionate treatment increased the tyrosine hydroxylase content in Substantia Nigra and caudate-putamen of rats. 4. The altered locomotion activity and DAT in early testosterone-treated rats implied the disturbed androgen level can influence the rat's locomotion activity via effecting the dopamine system, which may be related to the ADHD. |
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