Study On The Interaction Between Dopaminergic And Serotonergic System In An Animal Model Of Tourette Syndrome

IntroductionTourette syndrome (TS) is a neurological disorder in humans characterized by persistent motor and phonic tics and occurs in patients before the age of 18 years. Tics range in severity and are coupled with sensory urges. The disorder affects approximately 0. 1~1% of the population and males are affected 3~4 times more than females. In addition, behavioral disorders such as obsessive compulsive disorder and attention deficit hy-peractivity disorder are sometimes linked with TS.The etiology of TS is unknown, however, a genetic component and abnormalities of neurotransmitter systems are suggested. Twin and family studies indicate that TS has strong genetic determinants. Furthermore, an imbalance among neurotransmitter systems including the dopa-mines, 5 —HT and NE may contribute to the symptoms of the disorder. It is also postulated that a developmental abnormality in cortico striato thalamo cortical circuits is responsible for tic symptoms. The neurochemical hypotheses tend to be based on extrapolations from clinical trials evaluating the response to specific medications;from neurochemical assays on a few postmortem brain tissues;and PET/SPECT studies. Most neuro-transmitters involved in cortico—striato—thalamo—cortical circuitry including the dopaminergic (DA), glutamatergic, GABAergic, serotoner-gic, cholinergic, noradrenergic, and opioid systems have all been implicated. Which, if any, of these proposals represents the primary pathologi-cal factor remains to be definitively determined. Even though most people support that the DA system has a significant role, because many transmitters systems are interrelated in the production of complex actions, it is indeed possible that imbalances exist within several transmitter systems.Pharmacological treatment of TS utilizes neuroleptics such as halo-peridol, risperidone, and whose actions include blockade of dopamine receptors. Motor and phonic tics decreased or eliminated in about 80% patients after treatment with Haloperidol. Although these agents have shown efficacy in treating symptoms of TS, severe side effects such as drowsiness, muscle rigidity, and movement disorders may limit their use and long—term effectiveness.It has been proposed that shakes or twitches of the head in mice or shakes in the shoulders in rats following administration of the selective serotonin 5 —HT2A/2C agonist 1 — (2,5 — dimethoxy—4 — iodophenyl) — 2 — aminopropane (DOI) can serve as an animal model of tics seen in TS. In this study, the rats model of Tourette syndrome were established by peritoneal injection of DOI, the levels of dopamine and 5—HT in the rat brain of DOI—induced head twitch response were measured, the level of dopamine transporter in the presynaptic membrane and the DA receptors in the postsynaptic membrane of dopaminergic nerve were observed. The interrelation of dopaminergic and serotonergic system was revealed in TS, and the neurochemical pathogenesis of TS was studied.Materials and Methods1 AnimalsMale Wistar rats weighing 180—220 g were provided by experimental animal department of China Medical University.2 Male Wistar rats were housed separately with free access to food and water. The rats model of Tourette syndrome were established by peritoneal injection of DOI 2 mg/kg, and rat of control group were administered the same dose of physiologic saline.3 Measurement of Brain MonoaminesThe rat were decapitate and their brains were rapidly removed. The brain tissue was placed in 100 fi\ of 5% perchloric acid, homogenized with an ultrasonic cell disrupter and centrifuged at 20000 g for 20 min at 4°C in a microcentrifuge. 20 jzl of tissue sample supernatants were injected into high—performance liquid chromatography with an electrochemical detector. As a standard, 10 ng dopamine, HVA, 5 —HT and 5 —HI A A were also treated using the same procedure.4 Distribution and density of DAT in rat brain were evaluated 4.1 Preparation of 99mTc-TRODAT-lEach vial of TROD AT-1 was added with 1. 0 ml (37—1 110 MBq) of 99mTc pertechnetate. The reaction mixture was heated for 30 minutes at 100°C and then cooled to room temperature. The radiochemical purity was higher than 90%.4. 2 Biodistribution in ratsTen rats per group were used for each biodistribution study. 0. 3 ml of a saline solution containing 5. 55 MBq of Tc—TRODAT—1 was injected into the vena caudalis. The rats were sacrificed at the indicated time. Organs of interest were removed and weighed, and the radioactivity was counted using automatic gamma counter. The percentage dose per organ was decay corrected and calculated by a comparison of the tissue counts of 1 % of the initial dose measured at the same time.4. 3 Distribution and density of DAT in rat brain were evaluated by autoradiographyFive rats per group were injected intravenously with 666 ~740 MBq 99mTc—TRODAT—1. At 60min post injection, the animals were sacrificed , the brains were rapidly removed, and put in the freezing microtome 30 min, consecutive 20 fim coronal sections were cut on a freezing microtome. The section were put in 4% paraformaldehyde fixation for 10 min, smeared nuclear emulsion, and wraped with black paper, exposed in the —80*C refrigerator. After exposition 24 h, the sections were take out developing, fixing, and HE staining. The silver granules in striatum wereobserved under light microscope.5 D1R mRNA and D2R mRNA expression in the striatum and prefrontal lobeRats were decapitated 24 h after the last drug dose, and their brains were rapidly removed and placed on ice. The striatum and prefrontal lobe dissected, weighted and stored at — 80 °C till RNA isolation. RNA was i-solated from brain tissues according to the instruction with RNA out. The isolated total RNA was quantified by determining absorbance in spec-trophotometer at 260 nm. The reverse transcription reaction was performed for 30 min at 62°C. The amplification of Dl mRNA was carried out for 30 cycles with 40 s denaturation at 94 °C , 40 s annealing at 56. 5°C and 1 min extension at 72"C. The last extension step at 72°C was prolonged to 7 min. The amplification of D2 mRNA was carried out for 30 cycles with 40 s denaturation at 94 °C, 40 s annealing at 54 °C and 1 min extension at 72 °C. The last extension step at 72 °C was prolonged to 7 min. The 10 /Jtl portions of the amplified PCR products were subjected to electrophoresis on 2% agarose for small fragments stained with ethidium bromide in 1% Tris - acetate/EDTACTAE) electrophoresis buffer and analyzed by densitometry. The mRNA levels were determined by measuring the mean optical density (OD) values of outer RT—PCR products u-sing imaging analysis system. The results were normalized with the expression of the |3—actin gene. The experiments were repeated three times for each rat and type of brain tissues. The average values of relative optical density were subjected to statistical analysis.6 Statistical treatment The significance of differences between the different treatment groups was assessed using t test and analysis of variance (ANOVA), followed by Tuke/s post hoc test. The level of p<0. 05 was considered as the limit for statistical significence.Results 1 DOI—induced head twitches in miceThe HTR is a very distinctive behavior that rarely occurs spontaneously and usually cannot be mistaken for spontaneous behaviors such as head shakes (lateral movements of the head from side to side) or head jerks (up and down jerks).2 The levels of DA, HVA, 5-HT and 5-HIAA in the striatum of rat brainThe levels of DA and HVA in rats with DOI —induced HTR were significantly lower than those in the controls (P<0. 05). The levels of 5 — HT and 5 —HIAA in rats with DOI—induced HTR were significantly lower than those in the controls (P