Amitriptyline Accelerates SERT Binding Recovery Rate in MDMA-Induced Rat Model: In Vivo 4-[18F]-ADAM PET Imaging

Background Numerous studies have conrmed that 3, 4-Methylenedioxymethamphetamine (MDMA) produces long-lasting changes to the serotonergic system and decreases the density of the serotonin reuptake transporter (SERT). However, amitriptyline (AMI) is a potent neuroprotector that can cause devastating neuropathologic injury. Use of 4-[ 18 F]-ADAM, a SERT-specic radionuclide as a molecular imaging agent, facilitates longitudinal, non-invasive assessment of SERT activity/expression post-MDMA. We used 4-[ 18 F]-ADAM PET imaging to access the longitudinal alteration of SERT binding and evaluate the synergistic neuroprotective effect of MDMA and SERT inhibition by AMI in rat model. Materials and Methods The adult male Sprague–Dawley (SD) rats are grouped into four according to drug administration (Group 1: saline, Group 2: MDMA 10mg/kg i.p., Group 3: MDMA 10mg/kg i.p. with AMI 5 mg/kg i.p., Group 4: AMI 5 mg/kg i.p.). All drugs were administrated twice daily for 4 successive days (Day 1 to Day 4). Post-drug 4-[ 18 F]-ADAM PET scans were performed on day 14, day 21 and day 28 to measure the SERT occupancy/recovery. After the last PET imaging, SERT-positive cells were measured quantitatively using immunochemical staining.

Background 3, 4-Methylenedioxymethamphetamine (MDMA) is a ring-substituted that has a chemical structure similar to amphetamine. MDMA comprises of mescaline that has amphetamine stimulant having hallucinogenic effects [1]. Amphetamine is phenylethylamine derivatives. Its chemical properties comprise monoamine neurotransmitters and natural hallucinogenic compounds, such as mescaline and cathinone. Thus, the amphetamine produces the central nervous system stimulant and hallucinogenic effects [2].
MDMA affects peripheral and monoamine that plays an important neurotrophic role in the maturation of the central nervous system. Some studies conducted MDMA in vivo stimulation in brain tissue of rats, using serotonin out ow and dopamine to compare their multiple effects [3]. Some studies have pointed out that the interaction between MDMA and monoamine transporter facilitates the end of the cell synaptic neurons release serotonin, dopamine, and norepinephrine [2,4]. Thus, MDMA-induced monoamine transporter on neurons is the pharmacology and toxicity of biological targets. From the perspective of the MDMA-induced neurotoxicity, some studies argue that when MDMA is combined with the high-a nity monoamine transporter, its MDMA a nity for the serotonin transporter is much higher than other monoamine class transporters [5]. When the MDMA and serotonin transporter bind after a certain position, they transport to the presynaptic serotonin neurons. Thereby, the serotonin transporter massively releases serotonin into the synaptic cleft [6]. Presently, no clear conclusion reveals how MDMA causes a massive release of serotonin mechanisms. However, it is con rmed that MDMA can cause a massive release of serotonin in the brain after its rapid decline and produce depletion of the forebrain in rodents and primates, and this phenomenon in rodents and primates can be observed [7,8,9].
Many studies have con rmed that MDMA can cause selective damage to rodents and primate's serotonin systems. In addition, MDMA has been demonstrated to reduce serotonin levels, serotonin reuptake transporter (SERT), and the amount of serotonin synthesis tryptophan hydroxylase important to enzymes in the use of MDMA after have signi cantly reduced [10,11,12,13]. These phenomena occur because of MDMA effect on serotonin neurons injury and MDMA inhibition of the presynaptic neuron into the tryptophan hydroxylase enzyme and the disintegration of monoamine oxidase-B (MAO-B). Serotonin concentrations rise sharply after MDMA administration but quickly dropped in a few moments [14,15].
Nuclear medicine is a vital imaging technique to detect molecular serotonin transporter distribution in the central nervous system. In addition, serotonin is important in the regulation mechanism, providing important information about the diagnosis and treatment. In vivo nuclear medicine imaging of the serotonin transporter uses the quantitative technique as a research method. Positron emission tomography technology, a kind of serotonin transporter in contrast to agents [ 11 C]DASB, can have longterm use of MDMA in the human brain serotonin system. The results showed a signi cant reduction in all brain regions of serotonin transporter [16,17]. By administering 5mg/kg MDMA subcutaneously twice daily for four consecutive days, the MDMA-induced decrease in brain SERT levels, which could persist for over four years in primates [13].
As discussed above, SERT is one of the pharmacology and toxicity of MDMA biological targets. The SERT involvement in the MDMA-induced neurotoxicity mechanism has been extensively studied [18,19,20]. The neuroprotective effect of selective serotonin reuptake inhibitors (SSRIs) (i.e., uoxetine) has been studied in a rat model after MDMA intoxication [3,21]. In addition, co-administration of MDMA with SSRIs (e.g., uoxetine and citalopram) can prevent subsequent extracellular oxidative stress [22], long-term serotonin depletion and serotonin uptake site decrease, indicating that free radical production might occur following SERT activation by MDMA [23,24,25].
Amitriptyline (AMI) is one of the earliest members of the tricyclic antidepressants family. AMI functions as SERT inhibitor (ki = 1 nM), norepinephrine transport reuptake (NET) inhibitors (ki = 35 nM), and dopamine transport reuptake (DAT) inhibitor (ki = 3780 nM) [26,27]. AMI is also effective for the therapeutic of some mental disorders and treatment of neuropathic pain [28,29] AMI seems to be highly more effective than newer SSRIs [30,31]. Regarding anti-depressant actions, AMI induces dosedependent pluripotent actions of this drug [27,32]. Interestingly, several studies con rm that AMI elicits strong neurotrophic activity via a productive interaction with the brain-derived neurotrophic factor and neurotrophin tyrosine kinase receptor B (TrkB) system [28,29,33,34]. Kamińska et al., (2018) reported that chronic treatment with AMI in a unilaterally 6-hydroxydopamine lesion rat model increased dopamine levels. However, it decreased SERT and NET levels in the striatum and substantia nigra as well as improving motor dysfunction [35]. However, the in vivo interaction between SERT and AMI or the neuroprotection of AMI in MDMA-induced serotonin neurotoxicity remains unknown.
In this study, we developed a selective positron emission tomography (PET) imaging agent for the serotonin transporter, 4-[ 18 F]-ADAM, demonstrating its selectivity, speci city, and safety using rodent or primate models [36-40] and human study [41]. Furthermore, we demonstrated that uoxetine produced long-lasting protection against MDMA-induced neurotoxicity [20].
In light of these nds, this study aims to use PET 4-[ 18 F]-ADAM to assess (1) the in vivo interaction between SERT and AMI, (2) the long-term neuronal damage or recovery of SERT after MDMA administration, and (3) the evaluation of AMI against MDMA neurotoxicity in rat brain. Drug treatments and study design MDMA (purity, 98%) was obtained from the Investigation Bureau of Taiwan, and AMI was purchased from Sigma-Aldrich (St. Louis, MO). MDMA and AMI were dissolved in saline (0.9% NaCl) at a nal concentration of 10 and 5 mg/ml, respectively. Rats were treated with saline or AMI [5 mg/kg, subcutaneously (s.c.)], followed by saline or MDMA (twice a day for 4 consecutive days, 10 mg/kg, s.c.).

Animals
Rats were grouped as normal control (a saline injection, n = 6 for each time point), MDMA (saline followed by MDMA injection, n = 6 for each time point), AMI with MDMA (AMI followed by MDMA injection, n = 6 for each time point), AMI (AMI followed by saline injection, n = 6 for each time point).
The experiment was conducted using 4-[ 18 F]-ADAM PET imaging to measure SERT occupancy by amitriptyline and MDMA, as a method to gauge in vivo SERT binding of AMI and MDMA. The experimental design is schematically shown in Fig. 1. Baseline 4-[ 18 F]-ADAM PET scans were conducted when the animals were free of any drug treatment. A week after the baseline scans, 24 rats were randomly assigned to 4 treatment groups injected intravenously (i.v.) with either vehicle (saline, i.p.) or MDMA (10 mg/kg, s.c.) or MDMA (10 mg/kg) with AMI (5 mg/kg, s.c.) subcutaneously (s.c.). All drugs were administrated twice a day for 4 successive days (Day 1 to Day 4). Post-drug 4-[ 18 F]-ADAM PET scans were performed on day 14, day 21 and day 28 to measure the SERT occupancy/recovery.

Radiopharmaceutical
The 4-[ 18 F]-ADAM was synthesized in an automated synthesizer as described previously. Brie y, Puri cation with high-performance liquid chromatography (HPLC) produced the desired compound with a radiochemical yield (EOS) of ~ 3%, in a synthesis period of 120 min. The radiochemical yield of 4-[ 18 F]-ADAM increased to ~ 15% when using a different precursor and synthesized manually [37]. The chemical and radiochemical purities were > 95%, and the speci c activity was > 3 Ci/µmol (111 GBq/µmol).

Image data acquisition and analyses
Imaging was performed according to a previous report [36] with minor modi cations. Rats were anesthetized by passive inhalation of iso urane/oxygen mixture (5% iso urane for induction and 1% for maintenance). After 60 minutes of administration of 4-[ 18 F]-ADAM (14.8-18.5 MBq; 0.4-0.5 mCi) via tail vein, the static PET images were acquired for 30 min using a Concorde R4 Microsystem (Knoxville, TN, USA), which produced 63 image slices over a 7.89-cm axial eld of view, with a slice thickness of approximately 1.25 mm. All images were reconstructed with the Ordered Subset Expectation Maximum (OSEM) algorithm, producing a 128 × 128-pixel image matrix, 16 subsets, four iterations, and a Gaussian lter. Then, images were reconstructed by the Fourier rebinning algorithm and two-dimensional ltered back-projection, applying a ramp lter cutoff using the Nyquist frequency. The reconstructed images were analyzed with PMOD (PMOD Technologies, Switzerland) to measure standardized uptake value (SUV) in various brain regions. Volumes of interest of the striatum, auditory cortex, cingulate cortex, visual cortex, hippocampus anterodorsal, hippocampus posterior, hypothalamus, thalamus, and Cerebellum were drawn manually on the reconstructed PET images, using a MRI-based rat brain atlas with PMOD (PMOD Technologies, Switzerland). The regional radioactivity concentrations (KBq/mL) of 4-[ 18 F]-ADAM PET were estimated from the maximum pixel values within each ROI and expressed as SUV.
The nal data were expressed as speci c uptake ratios (SURs), expressed as (SUV target region - A semiquantitative assessment of the protein of interest expression was carried out based on the number of cells that showed nuclear expression of each SERT marker over 5 non-overlapping microscopic elds (at ×100 microscope objective magni cation) according to: 0 = absent, less than 5% immunopositive neurons seen, 1 = rare, 10-20% immunopositive neurons per eld, 2 = mild, 20-40% mildly or moderately positive neurons per eld, 3 = moderate, 40-60% moderately or strongly positive neurons per eld, 4 = strong, more than 80% strongly positive neurons per eld.
A percentage score for each case was calculated as: Actual rating x 100/maximal score (i.e., a rating value of 4).

Result
SERT recovery shows region-speci c and time-dependent Figure 2A is the illustration of the location of the brain regions was used to estimate the SERT binding of 4-[ 18 F]-ADAM. The 3D 4-[ 18 F]-ADAM PET images in the rat brain are shown in Fig. 2B-C. Brain uptake of 4-[ 18 F]-ADAM in all regions was signi cantly lower in rats pretreated with MDMA than in control rats from day 14 to day 28 (second row). However, the uptake in the control groups was similar in each imaging data (top row). In the baseline, the hypothalamus showed the highest 4-[ 18 F]-ADAM uptake followed by the thalamus, striatum, hippocampus posterior, motor cortex, cingulate cortex, hippocampus anterodorsal, auditory cortex, and visual cortex (Fig. 3 black line). In all brain regions, the SURs in the MDMA group signi cantly decreased up to day 28 (Fig. 3 red line). Detailed results were summarized in Table 1.
After normalized to the baseline value, we calculated the SERT recovery rate in each time point. Figure 4 showed that the recovery rate at the control group remained relatively at (black line); whereas the MDMA group appeared at its lowest recovery rate at day 14 (64.34% ± 2.05%) and slightly increased at day 21 (71.11% ± 1.96%) to day 28 (70.70% ± 3.96%) (red line). According to the SERT recovery rate, from day 14 to day 28, brain regions of the MDMA group averagely divided into three classi cations: Low recovery rate (< 50%)-thalamus, Mid recovery rate (< 65%)-hypothalamus, hippocampus anterodorsal and Hippocampus Antero Dorsal, and High recovery rate (< 80%)-cingulate cortex, motor cortex, auditory cortex, striatum, and visual cortex. Detailed results were summarized in Table 2.

Amitriptyline Accelerated SERT Level recovery after MDMA induction
In all brain regions, co-administration of AMI with MDMA resulted in higher 4-[ 18 F]-ADAM uptake compared to the MDMA group ( Fig. 2B-C). At day 14, the SURs of 7 of 9 regions showed signi cant difference in the two groups (p < 0.05 ~ p < 0.005). AMI dramatically increased 4-[ 18 F]-ADAM uptake in all brain regions (Fig. 3  Amitriptyline did not affect normal brain Since AMI was a non-selective SERT inhibitor, we further tested whether it altered normal brain SERT levels. The results showed that pre-treatment with AMI alone slightly decreased 4-[ 18 F]-ADAM uptake in all brain regions. However, no signi cant effect is noted regarding the curves of SURs or recovery rate of the AMI group, showing a similar pattern with the controls (Fig. 2B-C, Fig. 3-4 green line). Detailed results were summarized in Tables 1 and 2. AMI markedly increased the serotonergic ber density after MDMA-induction The results of SERT immunohistochemical localization were obtained to compare in vivo PET images. A dense meshwork of bers and high densities of SERT labeling was found in all brain regions in the control group (Fig. 5 left panel). Widespread heterogeneous distribution of SERT immunoreactivity was observed in the striatum, frontal cortex, and dorsal raphe (midbrain). A high dense of SERT-expressing bers were found in the hypothalamus and thalamus in control animals (Fig. 5 left panel). In contrast to the control group, MDMA treatment reduced densities of SERT immunoactivity in all brain regions (Fig. 5  second panel). However, co-administration of MDMA with AMI restored the SERT-expressing immunoactivity (Fig. 5 middle panel). Similar to PET results, all brain regions of the AMI group showed a slight reduction of SERT immunoreactivity when compared with the controls. However, no signi cant difference is observed (Fig. 5 right panel).

Discussions
Using a selective SERT PET radiotracer, we monitored a long-term SERT occupancy/recovery in vivo and evaluated the AMI neuroprotection after MDMA induction. Our results showed that acute and repeated administration of MDMA signi cantly induced SERT reduction levels in all regions at day 14 compared to the controls, which were supported by previous studies, revealing that the effect of MDMA on SERT binding was a robust nding in rodents [13,20,42,43,44]. Those reports indicate that the effect of MDMA on SERT binding is a robust nding in rodent.
Regarding the long-term effects of MDMA exposure, we further investigated the effect of the duration of ecstasy abstinence on the SERT binding by examining the reversibility of the in vivo SERT binding during the period of abstinence from MDMA administration. We found that neurotoxicity induced by MDMA in rat brain was regional-speci c, re ecting the varied SURs or progression of the self-recovery rate of SERT. In the study period (28 days), we found the regions, such as the thalamus, hypothalamus, hippocampus anterodorsal, and hippocampus anterodorsal (low or mid-self-recovery rate), had relatively slower selfrecovery progression compared to cingulate cortex, motor cortex, auditory cortex, striatum, and visual cortex (high self-recovery rate) ( Table 2 and Fig. 4). The results also indicated that the SERT self-recovery in rat brain after MDMA-induction was time-dependent and returned to 70.7% ± 3.96% of baseline values at day 28. The regions of low or mid-self-recovery rate were the most affected region by MDMA [4,45].
In contrast, MacGregor et al., (2003) reported a clear loss of SERT binding sites in rats 3 months after administering the high-dose MDMA regime (4 × 5 mg/kg over 4 h in 2 consecutive days) [12], This agrees with numerous previous studies that show a SERT loss in the cingulate cortex, hippocampus, entorhinal cortex, medial hypothalamic area, and the medial and lateral thalamic nuclei of rats, following MDMA administration in a rodent model [46,47]. Lew et al., (1996) reported that a progressive recovery of SERT binding was noted from 2 to 52 weeks following MDMA exposure [48]. Moreover, Li et al. (2010) reported < 50% SERT recovery rate using in vivo 4-[ 18 F]-ADAMPET in the midbrain, thalamus, hypothalamus, caudate-putamen, hippocampus, and frontal cortex at day 31 after MDMA administration [20].
The difference in recovery time-course between the present study and previous reports described above could be (1) methodological issues that affected the accuracy of the quantitative measurement. For example, the reports published before 2000 used quantitative autoradiographic to analyze tissue slices or high-performance liquid chromatography (HPLC) for homogeneous tissue. However, the present study used PET imaging. (2) The analysis techniques, i.e. the accuracy of PET and MRI-based atlases registration or the correction of partial volume effect could underestimate the SERT binding in small volumes.
In a primate study, Scheffel et al. (1998) showed that SERT binding increased from 40 days to 9 months after MDMA administration in the pons, midbrain, and hypothalamus. However, it decreased in cortical regions [49]. Ma et al., (2016) reported that the SERT recovery rate was on average of ~ 66.6% and ~ 68.6% after MDMA administration in the striatum, thalamus, and midbrain at 24 and 54 months, respectively [13].
In human studies, some studies examined the reversibility of the SERT binding during abstinence from MDMA administration. Several reports demonstrated no difference in SERT binding between former ecstasy users and drug-naive controls after 1 year of abstinence [50,51,52].
Some preclinical or clinical studies con rmed a recovery in SERT binding after MDMA administration [40].
However, the question raised is the correlation between recovery of SERT binding and the function of SERT neurons. In the rat model, Andó et al. reported that 6 months after administering high-dose (15 or 30mg/kg, i.p), MDMA-induced damage of serotonergic axons showed recovery in most brain areas. However, SSRIs reduce serotonergic functions. Anxiety and aggression remain altered [53]. Li et al., (2010) suggested that when the SERT recovery rate reached ~ 35.2% compared to the controls, the density of serotonergic bers and cell bodies decreased at day 31 after MDMA treatment (10 mg/kg, i.p) [20].
In the human study, several studies reported that after one year of abstinence, ex-MDMA users showed de cits in the Rey Auditory Verbal Learning Test similar to current MDMA users although SERT binding was similar to control level [50]. A review of empirical research (2013) supported those cognitive impairments following MDMA administration, which could result in a long-term cognitive effect, such as retrospective memory, prospective memory, higher cognition, problem-solving, and social intelligence. It can also result to sleep architecture, sleep apnoea, complex vision, pain, neurohormones, and psychiatric status [54]. Golding et al., (2007) reported that light ecstasy users showed a small signi cant cognitive impairment. However, no such impairment was detected among ex-users absent from the drug for at least 6 months [55]. Thus, neuroimaging studies show reduced serotonin transporter levels across the cerebral cortex, associated with neurocognitive impairments. SERT recovery positively correlates with the duration of MDMA abstinence. However, it is unclear whether the cause is associated with the SERT neurons recovery or other causes. Future longitudinal studies are recommended to investigate the serotonin level in blood or cerebrospinal uid [45] or behavior tests.
The present results demonstrated that co-administration of MDMA with AMI rapidly blocked MDMAinduced serotonin release and MDMA neurotoxicity, restored globally, and largely accelerated SERT levels at day 14. After day 14, the progression of SERT recovery rate increased slowly at a rate of approximately 3% per 7 days and reached ~ 70% of baseline at day 28. Among all regions, those regions with low or midself-recovery rates had weaker responses to AMI when compared to regions with high recovery rates. Li et al., (2010) reported that co-administration of MDMA with SSRI, uoxetine, restored SERT binding rate to ~ 79.6 % of the control level at day 31 post-MDMA [20]. Compared to uoxetine in the current results, AMI showed a 84.38% ± 2.05% of recovery rate at day 28. AMI had a higher neuroprotective effect because of its antiapoptotic effect that prevented PC12 cell death caused by hydrogen peroxide [56]. Another reason could be that MDMA can cause hyponatremia that induces seizures [57], resulting in an anoxic brain. However, AMI can protect primary cultured hippocampal neurons and in vivo hippocampal neurons from oxygen-glucose deprivation-induced apoptosis [34]. Moreover, AMI showed signi cant improvement in long-and short-term memory and increased neurogenesis and neurosynaptic marker proteins in an AD mouse model [32]. However, MDMA can in uence a long impact on cognitive impairments.
Cytochrome P450 2D6 is the main enzyme involved in MDMA metabolism [58], and the AMI is a potent inhibitor of the Cytochrome P450 2D6 enzyme [59], which may inhibit MDMA metabolism and increase the MDMA toxicity.
In contrast to the expensive, risk-overt, and time-consuming nature of de novo drug development, a more effective approach is to apply the well-tolerated, therapeutics in new pharmacogenomic settings. Seeking effective treatments in Food and Drug Administration-approved drugs has become a promising drug discovery route for neuroprotection by MDMA.
Our ndings in immunochemical staining con rmed the PET study, revealing that at day 28 post-MDMA, the density of serotonergic bers and cell bodies decreased in the MDMA group. On the contrary, coadministration of MDMA with AMI showed improvement in structural damage of serotonin neurons (Fig. 5-6). After being measured by RT-PCR, our results supported a decrease in SERT gene expression in the striatum, parietal cortex, and hippocampus after MDMA treatment [60]. The results were consistent with several studies that reported dramatic decreases in SERT binding following various MDMA dosing regimens and post-administration [61]. This previous study also showed the effect of MDMA on SERT depletion region-speci c. For example, areas such as the striatum, raphe nuclei seem to affect stronger than other areas such as the hypothalamus. In the long term, the evidence suggests that SERT gene expression is negatively regulated by MDMA exposure [62], leading to reductions in SERT binding and immunoreactive ber density in the absence of physical damage.

Conclusions
Based on the longitudinal in vivo 4-[ 18 F]-ADAM PET, the present study found a clear loss of SERT binding sites in rats after the low-dose MDMA regime. We demonstrated that SERT recovery was positively correlated to the MDMA abstinence duration, implying that the lower SERT densities in MDMA-induced rats re ected neurotoxic effects, which varied by region-speci c and reversible. Current data also supported that AMI might have neuroprotective effects that globally accelerated the recovery rate of SERT besides its anti-depressive effects. Future studies should verify the neuroprotective effects of AMI in neuronal cells.

Consent for publication
Not applicable.

Availability of data and material
The authors con rm that the data supporting the ndings of this study are available within the article.

Con icts of Interests
The authors have no nancial or competing interests to declare.       Comparison of recovery rate, based on graphical analyses of 4-[18F]-ADAM binding before and after drug administration. Among four groups, the MDMA group (red line) appeared the lowest recovery rate at day 14, slightly increased at day 21, and recovered to ~70% of baseline value at day 28. AMI with MDMA (blue line) signi cantly accelerated the recovery rate from day 14 and slowly increased up to day 28 when compared with the MDMA group. The control and AMI alone (green line) groups showed no difference in curve tendency. Data are mean ± SD. Dark-eld photomicrograph of SERT immunoreactivity in different brain regions after drug treatment. Compared to the control, SERT immunoreactivity was signi cantly lower in the MDMA group, slightly lower in AMI with the MDMA group, and almost equal in the AMI alone group. Scale bar, 100 µm. Figure 6