Abnormal expression of dopamine and serotonin transporters associated with the pathophysiologic mechanism of Tourette syndrome
Correspondence Address: Source of Support: Chinese Medicine Administration Bureau of Shandong province (No: 2005064), Conflict of Interest: None DOI: 10.4103/0028-3886.68663
Source of Support: Chinese Medicine Administration Bureau of Shandong province (No: 2005064), Conflict of Interest: None
Background : Tourette syndrome (TS) is a neurobehavioral and neuropsychiatric disorder and its pathophysiology is not well understood. However, recent studies provide evidence implicating metabolic abnormalities of dopamine (DA) and serotonin (5-HT) of the basal ganglia both in TS patients and TS animal models. It is also well known that dopamine and serotonin transporters (DAT and SERT) are monoamine neurotransmitter transporters, which participate in the metabolism of DA and 5-HT, respectively. Objective : To evaluate whether expression of DAT and SERT in the striatum could lead to pathophysiological change in TS rat model. Materials and Methods : Twenty-four Wistar male rats were randomly allocated to: TS model group (n=12) and control group (n=12). The stereotypy counts were recorded during the 2-week period of inducing TS rat models. The levels of DA and 5-HT in striatum homogenate were measured by ELISA. The protein and mRNA expression of DAT and SERT in the striatum were tested respectively by Immunofluorescence, Western blot and quantitative real-time PCR. Results : ANOVA analysis indicated that the stereotypy scores were much higher in the TS model group than in the control group at different time points (P<0.01). By ELISA analysis, the DA concentration in striatum homogenate was higher in the TS model group (130.92 ± 25.60 ng/mL) than in the control group (101.00 ± 20.14 ng/mL) (P<0.01), but 5-HT concentration in striatum was found to be lower in the TS model group (59.79 ± 14.73 ng/mL) compared to the control group (77.01 ± 14.05 ng/mL) (P<0.05). Analysis of protein and mRNA levels revealed a lower expression of DAT, concomitant with a higher expression of SERT in striatum of the TS model group than in the control group. Conclusions : Lower expression in DAT, concomitant with higher expression in SERT could participate in the pathophysiology of TS.
Keywords: DAT, Pathophysiology, SERT, Tourette syndrome
Tourette syndrome (TS), a developmentally regulated neurobehavioral and neuropsychiatric disorder, is characterized by multiple motor and vocal tics that wax and wane in frequency and intensity over time during its natural course. ,, Although tics are the classical symptom characterizing TS, additional comorbidities are commonly associated with the disease, such as obsessive-compulsive behavior (OCB) and attention deficit-hyperactivity disorder (ADHD). , The prevalence of TS is 4-6 per 1000 school-aged children and adolescents , and TS is three times higher in males than in females. , Typically, the tics begin around school age, increase to a maximum severity during the preadolescent years, and then decline in frequency and severity by the beginning of adulthood. , In a small number of cases, however, the tics continue throughout adulthood. ,
Although TS is a common neuropsychiatric disorder, the definitive pathophysiological mechanism of the tics is not well understood and remains a subject of active investigation. Recent anatomical and neuroimaging studies have provided evidence for abnormal basal ganglia and dopaminergic function in TS ,, and also demonstrated metabolic abnormalities of dopamine (DA) and serotonin (5-HT) in basal ganglia. , Two kinds of monoamine neurotransmitter transporters, DAT and SERT are involved in the metabolism of neurotransmitters in order to regulate their respective levels at the neuroeffector junction; they terminate synaptic neurotransmission by active uptake of DA or 5-HT. ,,,,, Thus, both DAT and SERT can influence the metabolism of DA and 5-HT, suggesting that they may have a role in the pathophysiological mechanisms of TS. ,,
We hypothesized that both DAT and SERT participate in the pathophysiological mechanism of TS. In the present study, we induced a TS model in rats by microinfusing TS patient sera containing a quantity of antineural antibodies into the ventrolateral striatum  and subsequently investigated whether there were significant differences between TS rat models and the controls with respect to DAT and SERT expression in the striatum.
Subjects and animals
We developed experimental models of TS in Wistar rats by microinfusing with TS patient sera containing a quantity of antineural antibodies as described Xiumei Liu et al.  All experiments were approved by the Ethics Committee of the Provincial Hospital affiliated to Shandong University. Twenty-five male TS children (DSM-IV criteria; age: 8.2 ± 1.1 years), who had never received tic-suppressing medications, were recruited for the study after the full consent of both the children and their parents. Sera were separated from the blood samples drawn and tested for antineural antibodies by ELISA, this was done with the support of the Department of Neurosurgery and the clinical testing laboratory at the Provincial Hospital affiliated to Shandong University. All samples were assayed in triplicate. Finally, 12 samples having maximum concentations were used for rat striatal microinfusion for TS rat models. Each rat was microinfused with a different child's serum.
Twenty-four Wistar male rats weighing 180-220 g each were randomly allocated to the TS model group (n=12) and control group (n=12). Rats in the TS model group were deeply anesthetized with chloral hydrate (400 mg/kg intraperitoneally) and placed in a stereotaxic apparatus (Stoelting, USA) with the incisor bar set at 3.5 mm below the interaural line. Then, using an aseptic surgical technique, the skull was exposed and holes were drilled, through which 28-gauge guide cannulae were passed and implanted into both the striatum. Rats were allowed to recover for 1 week and, during this time, an osmotic mini-pump (Alzet Corp., Palo Alto, CA) filled with PBS was connected to each cannula by a polyethylene tube loaded with 50 μL of undiluted TS sera under sterile conditions. Sera were microinfused at a rate of 0.5 μL/hour for 72 hours. After 72 hours, the pumps were removed, the wound was closed with surgical suture, and the rats were returned to their home cages. Simultaneously, the other 12 healthy male Wistar rats in the control group were microinfused with normal saline (0.9%), using the same method as described above.
In this study, rats were monitored for 2 weeks (Nikon L1, Japan) from the day of removal of the osmotic pumps. The behaviors of the rats were video- and audio-taped in natural light for 30 minutes once a day during monitoring period. The stereotypy counts were recorded by the method described by Liu et al.  The recorded stereotypy counts included bites (teeth touching, chewing the cage and wood chips), taffy pulling (raising of the forepaw to the mouth and face), self-gnawing, licking not associated with grooming, grooming, head shaking, paw shaking, rearing, and episodic utterances. Two weeks after microinfusion, analysis of the recorded stereotypy counts confirmed that the models were successful. During the entire 2-week period, all rats were housed in a natural day-night cycle at a room temperature of 21-23°C, with free access to food and tap water.
At the end of the experiment, all rats were sacrificed under anesthesia and the striatal tissue was extracted from the brain by the method described by Hida et al.  After the animals were decapitated, the brains were removed and 6-mm-thick coronal slices were made. The boundaries of the striatum were determined according to the rat anatomy. During the course of the experiment, three different rats per group (TS model and control groups) were randomly harvested in every single experiment.
Contents of DA and 5-HT in striatum by ELISA
The levels of DA and 5-HT in the striatum were measured by ELISA according to the manufacturer's (USCN, Wuhan, China) instructions. A 30-mg portion of the homogenate was diluted in 300 μL normal saline (0.9%) for detection. We assayed DA and 5-HT in supernatant fluid using the ELISA kit (USCN, Wuhan, China) after centrifugation of homogenized tissue for 10 minutes at 20000 g at 4°C. Dispensed antigen standards and samples were added to each well of 96-well plates precoated with primary antibodies. After adding biotin conjugate reagent and enzyme conjugate reagent into each well, the plates were incubated at 37°C for 60 minutes. Then the plates were rinsed 5 times with distilled water. Within 30 minutes of the chromogenic reaction, the absorbance was measured at 450 nm using a microtiter plate reader.
Three samples were randomly picked from each group. The frozen striatal tissue, the mid part of the entire striatum was cut into 20-μm slices in a coronal plane, with the interslice interval being about 10 μm. The frozen slice was washed thrice in PBS (pH 7.4) for 8 minutes each. Afterwards, it was incubated with blocking solution (consisting of 10% goat serum in PBS) at room temperature for 30 minutes. Endogenous peroxide activity was quenched with 0.3% hydrogen peroxide solution. Next, the paraffin sections were incubated overnight at 4°C with primary antibodies of rabbit polyclonal DAT (dilution 1:500; Chemicon, USA) or SERT (dilution 1:5000; Abcam, England). The following day, the paraffin sections were washed thrice in PBS (pH 7.4) for 8 minutes each and were then incubated with goat anti-rabbit secondary antibody (dilution 1:100; Zhongshan, Beijing, China) in darkness for 2 hours at room temperature. We then washed the paraffin sections thrice in PBS (pH 7.4) in darkness for 5 minutes each. Images were captured using fluorescent microscopy (IX71, Olympus, Japan).
Protein analysis by Western blotting
Three samples per group of striatal homogenate were chosen. About 50 mg homogenate was diluted in 500 μL normal saline (0.9%), and the debris was then removed by centrifugation for 10 minutes at 20000 g at 4°C. The protein concentrations of supernatant were determined using BCA-100 Protein Quantitative Analysis Kit (Shenneng, Shanghai, China). Equal amounts of protein (20 μg) were subjected to electrophoresis on 10% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes by electroblotting. The membranes were first incubated in blocking solution (5% skim milk) for 1 hour at room temperature and then incubated overnight at 4°C with the primary antibodies: anti-DAT (1:500 dilution, Chemicon, USA) or anti-SERT (1:100-dilution, Abcam, England). After being washed with TBST (10 mM Tris-HCl, 0.15 M NaCl, 8 mM sodium azide, 0.05% Tween-20; pH 8.0) three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000-dilution, Zhongshan, China) for 1 hour and then washed with TBST three times again. Finally, protein bands were visualized with the enhanced chemiluminescence (ECL) (Chemicon, USA) detection system. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected with anti-GAPDH antibodies. The relative quantification of protein expression was done using Alphalmager IS-2200 (Nature Gene, USA).
mRNA isolation and quantitative real-time polymerase chain reaction (PCR) analysis
Three specimens in each group were randomly selected and analyzed by quantitative real-time PCR. Afterwards, 30 mg of even homogenate per sample was tested. Total RNA was extracted from striatal homogenate using Trizol (Invitrogen, CA) reagent according to the manufacturer's protocol. First-strand cDNA was generated using a commercial Takara RT kit (Takara, Dalian, China) and amplified by real-time PCR using a QuantiTest SYBR Green kit (Takara, Japan) and ABI Prism 7500 real-time PCR instrument and software (Applied Biosystems, CA). Primer sequences used in the PCR are shown in [Table 1]. All quantifications were performed with rat GAPDH as an internal standard. The reverse transcription reaction was performed at 37°C for 15 minutes, 85°C for 5 seconds, and 4°C for 20 minutes. The real-time PCR was performed for 40 cycles at 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The relative quantification of mRNA expression was analyzed by 2-Delta Delta C (T) method,  and the results were expressed as extent of change with respect to control values.
Every experiment was repeated three times for each sample. All data in the text and tables were expressed as the mean ± standard deviation. Statistical significance of differences between groups was evaluated by one-way ANOVA. The relationship between the stereotypy counts and the levels of DA, 5-HT, DAT, and SERT of prepared tissue in the striatum of TS rats was analyzed by calculating the Pearson correlation coefficients. A value of P<.05 was considered as statistically significant.
TS models and assessment of stereotypic behavior
Twelve TS children with maximum antineural antibodies in the sera participated in the study. These patients' sera were used to successfully generate 12 TS model in rats. Another group of 12 rats were treated with saline (0.9%) and these rats formed the controls.
All rats in both the groups were monitored for 2 weeks, with 30 minutes of observation per day after removal of the osmotic pumps. The stereotypy counts are shown in [Figure 1] at time points of 2, 4, 6, 8, 12, and 14 days after intrastriatal microinfusion. The stereotypy scores were significantly much more in the TS model group than in the control group at different time points (P<.01).
Contents of DA and 5-HT in striatum by ELISA
The DA content in the striatum homogenate [Figure 2], was higher in the TS model group (130.92 ± 25.60 ng/mL) than in the control group (101.00 ± 20.14 ng/mL) (*P<.01). However, a lower 5-HT content in striatum was found in the TS model group (59.79 ± 14.73 ng/mL) compared to the control group (77.01 ± 14.05 ng/mL) (**P<.05).
Weaker and thinner fluorescence was seen in the DAT-model group than that in the DAT-control group, but stronger and denser fluorescence was noted in the SERT-model group than that in SERT-control group[Figure 3].
Protein analysis by Western blotting
Western blotting [Figure 4]a and b also demonstrated that there was significantly lower protein expression of DAT in the striatum of the TS model group (0.67 ± 0.08) than in the control group (0.97 ± 0.15) and higher protein expression of SERT in the TS model group (0.82 ± 0.15) compared to the control group (0.50 ± 0.10). Statistically significant differences were confirmed between groups from the above data (*P<.001, **P<.05).
mRNA isolation and quantitative real-time PCR analysis
Real-time PCR results were analyzed by the 2-Delta Delta C (T) method.  As shown in [Figure 5], the mRNA expression of DAT is much weaker in the striatum of the TS model group (0.99 ± 0.17) than in the control group (1.46 ± 0.23). In contrast, the mRNA expression of SERT was higher in the TS model group (0.95 ± 0.14) than in the control group (0.57 ± 0.15). There were statistically significant differences between the TS and control groups (*P<.001, **P<.05).
Correlative analysis of stereotyped behavior and the levels of DA, DAT, 5-HT, and SERT
The results showed that increase in stereotypy counts in rats correlated with higher total DA content and lower expression of DAT in rat striatum (P<.001) as well as lower levels of 5-HT and higher expression of SERT.
Structural and functional neuroimaging studies have indicated that the basal ganglia and cortico-striato-thalamo-cortical circuits may be the neuroanatomical sites involved in TS. ,,, It is widely believed that abnormalities of DA and 5-HT neurotransmission play a vital role in the pathophysiology of TS. ,, Both DAT and SERT are important monoamine neurotransmitter transporters that are essential regulators of monoaminergic neuronal function.  The efficiency of monoaminergic neurotransmission is controlled by efficient reuptake of neurotransmitters out of the synaptic cleft by monoamine transporters. ,, Therefore, we hypothesized that abnormalities of DA and 5-HT neurotransmission in TS may be associated with abnormal expression of dopamine and serotonin transporters. To the best of our knowledge, no other study has observed different concentrations of DAT and SERT between TS models and controls. Thus, our finds may shed light on the pathophysiologic mechanism of TS.
In this study, we chose to use the TS rat model described by Xiumei Liu et al. because the method has good clinical relevance and high success rate. , Our data showed that higher DA content, but lower levels of 5-HT, existed in striatum homogenate of the TS model group compared with the control group. Prior studies have also demonstrated increased DA content ,, and also decreased 5-HT content ,,, in TS as well as in impulsive aggression and obsessive-compulsive disorder, both of which are features of TS. In comparison to the control group, the TS model group showed lower protein and mRNA of DAT but higher SERT expression in the striatum.
Obviously, the decreasing DAT expression in the striatum of the TS model could lead to decrease of DA uptake from the synaptic cleft and thus the accumulation of DA in the synaptic cleft of dopaminergic neurons. In other words, the increments of DA content in the synapse maybe the result of the decrement of DAT. Also, the accumulation of DA in the synapse may indicate hyperactivity of DA in TS. Meanwhile, several studies have demonstrated that blockage of DA receptors, especially DRD2, by some receptor antagonists could reduce the tic frequency and severity. , More importantly, the correlation analysis revealed that there was a close correlation between the stereotypy counts and higher total DA content or lower expression of DAT in the striatum of TS rat models. The result also revealed that abnormal expression of dopamine transporters may be involved in the pathophysiologic mechanism of TS. The increase in the DA content in our study may be the result of the increase in the DA content in the synaptic cleft of dopaminergic neurons in the TS model; the correlation analysis also supports this conclusion. However, our study was not designed to differentiate between intra- and extracellular DA content. Consequently, hyperactivity of DA involved in the pathophysiology of TS may also be associated with the decrease of DAT in striatum tissue in the TS model. However, the positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging of striatal DAT binding was either normal ,, or increased ,,, in TS patients, which is in contrast to our results. Our conclusions may be in disagreement with those studies due to the different empirical approaches adopted (for example, measurement of total protein or mRNA vs PET or SPECT imaging assays) or because of interspecies differences between human and rats.
Meanwhile, the experimental results have also revealed that more stereotypy counts correlated with lower levels of 5-HT and higher expression of SERT in rat striatum. SERT is involved in the metabolism of 5-HT by removing extracellular 5-HT and recycling it back into the neuron and also plays a key role in shaping neurotransmission. Since SERT can reuptake 5-HT from the synaptic cleft and regulate the concentration of 5-HT in the synaptic cleft, low content of 5-HT may be associated with the high SERT expression in the striatum of the TS model in our study. Although the relationship between the total 5-HT content and synaptic 5-HT content could not be determined in our study, our data indicates that the stereotypy counts were associated the levels of 5-HT in the TS model. The result indicated that the increment of SERT in striatum tissue may also be involved in the pathophysiology of TS. Also, PET has demonstrated that the SERT binding potential in the midbrain and caudate/putamen is significantly increased in TS patients.  Consequently, higher SERT expression in the striatum is also likely to be associated with the pathophysiology of TS.
Our study has some limitations: the sample size was small, there are interspecies differences between human and rats, and we did not differentiate between intra- and extracellular neurotransmitter content in rat brain. Our data merely suggests that the low expression of DAT and high expression of SERT could influence the increased DA content and decreased 5-HT level seen in the striatum of TS rat models, which may be one of the possible pathophysiological mechanisms of TS. Our findings also indicate that regulating some neurotransmitter levels and using blockers or agonists to alter transporter function in the striatum may provide potential therapeutic targets for the treatment of TS.
First and foremost, we have to express our heartfelt thanks to our sponsor, Chinese Medicine Administration Bureau of Shandong province (No: 2005064). Without its full support, this research would have been impossible. Our thanks also go to all the lab technicians in the Science Center of Shandong Provincial Hospital for the excellent technical assistance.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]