Background
The crucial role of the central serotoninergic system has been increasingly recognized in the field of addiction, as the reduction of serotonin transporters (SERTs) or upregulated serotonin levels contribute to the pathological mechanism and behavioral changes induced by drug abuse [
1,
2]. The majority of pathological mechanism studies have focused on the effects of drug addiction in the striatum and amygdala, which are deeply involved in the development of addiction behaviors. Exposure to psychostimulants, as well as the withdrawal phase, is associated with gene-specific changes in SERTs in serotonergic terminals [
3,
4].
The SERTs recycle serotonin to regulate its concentration in a gap, or synapse, and thus are the targets for selective serotonin reuptake inhibitors (SSRIs) [
4]. While SERTs are mainly located on serotonergic terminals and cell bodies in the brainstem nuclei [
5], they are also heterogeneously distributed in rat and human brains [
6]. In rats, high densities of immunoreactivity were observed within the caudate, putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nuclei, olfactory nuclei, and hippocampus [
7]. In general, the binding of autoradiographic [
3H]citalopram to its binding sites in SERTs in the human brain was highest in the limbic cortices, followed by the brainstem, striatum, pallidum, isocortex, and thalamus [
8].
Neuroimaging using either positron emission tomography (PET) or single-photon emission computed tomography (SPECT) coupled with appropriate radiopharmaceuticals provides a noninvasive and functional means to evaluate drug effects on SERT distribution in the human brain. Imaging neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA), an analog of methamphetamine (METH) that is known as ecstasy, has been extensively studied in nonhuman primates and humans using either [
123I]β-CIT SPECT [
9] or [
11C]McN5652 and [
11C]-DASB PET [
10]. However, [
123I]β-CIT and [
11C]McN5652 are nonspecific for SERTs with only moderate signal contrast in human studies [
9]. [
11C]DASB is suitable for probing SERTs with PET; however, the short half-life of carbon-11 (~ 12 min) and the requirement of an on-site cyclotron hindered the possibility for routine practice in our laboratory. Because
18F has a half-life of 110 min, it can be produced off-site and transported to medical facilities for imaging use, and many
18F-labeled ligands have been developed for SERT PET [
11].
Our group developed (
N,
N-dimethyl-2-(2-amino-4-[
18F]-fluorophenylthio) benzylamine), termed as 4-[
18F]ADAM, and demonstrated that, compared to others, 4-[
18F]ADAM had suitable characteristics for imaging SERTs because of its high target-to-nontarget ratio, little in vivo de-fluorination, easy preparation, and acceptable radiochemical yield (~ 15%). Studies of toxicity and radiation dosimetry carried out in rats and rhesus macaques also suggested that 4-[
18F]ADAM is safe [
12]. The 4-[
18F]ADAM PET and autoradiography for imaging SERTs have been validated in 5,7-dihydroxy tryptamine-lesioned and p-chloroamphetamine-induced, 5-hydroxy tryptamine (5-HT) depletion and paroxetine SSRI-treated rat models [
12,
13] to evaluate the degrees of MDMA neurotoxicity and therapeutic response. We previously reported the neuroprotective effect of dextromethorphan against MDMA-induced neurotoxicity [
14].
However, to date, few imaging studies have observed the in vivo changes in central SERTs in METH cases [
15] but have focused on the alteration of dopamine or dopamine transport (DAT). Studies suggested that repeated high-dose treatment with METH in rats caused a decrease in central dopamine (DA) levels [
16] and DAT binding [
17]. METH causes significant depletion of serotonin [
18], reduced tryptophan hydroxylase (TPH) activity [
19], and decreased SERT binding [
20] in rats.
Our group assessed the side effects of recreational drugs such as ketamine, cocaine, and METH on dopamine neurons in the peripheral organs using PET imaging and quantitative whole-body autoradiography with [
18F]FDOPA, an analog of
l-dihydroxyphenylalanine (L-DOPA). We demonstrated that the dose-dependent effect of acute administration (single injection) of these three recreational drugs and the inhibitory effects of the [
18F]FDOPA accumulation (or the ability to raise dopamine) in the striatum or other tissues varied [
21].
Repeated high-dose administrations of METH in monkeys also cause persistent decreases of dopamine and serotonin levels in the brain [
22]. Studies in postmortem humans partially confirmed that METH abusers showed significantly reduced dopamine, TH, and DAT levels in the striatum and nucleus accumbens and decreased SERT levels [
23]. Yamamoto and Zhu concluded that free radicals and oxidative stress, excitotoxicity, hyperthermia, neuroinflammatory responses, mitochondrial dysfunction, and endoplasmic reticulum stress might be responsible for the METH-induced neuronal fiber degeneration and apoptosis [
24].
In a previous study, we demonstrated that lower 4-[
18F]ADAM PET binding was associated with reduced SERT immunoreactivity by 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a rat model [
25]. This result was in agreement with the study that reported by [
123I]ADAM/SPECT imaging that SERT levels were decreased in monkey brains following 6-OHDA injections into the medial forebrain bundle [
26]. Those studies determined that 4-[
18F]ADAM PET could be used to detect serotonergic neuron loss or dysfunction of SERTs.
Based on the well-established experiences of SERT and DA imaging in animal models of substance abuse and addiction including MDMA, ketamine, cocaine, and METH, we modeled typical human METH exposure using acute administration of several repeated doses (5 or 10 mg/kg, s.c. four times, with each injection 1 h apart) in rats. The aims of this study were (1) to further characterize the long-lasting effects of METH by examining the brain region vulnerability and ligand binding to SERTs by 4-[18F]ADAM PET and (2) to determine the association of SERT availability/activity with the levels of TH, the rate-limiting enzyme for dopamine synthesis, and thus the maintenance of dopamine levels, used as a surrogate biomarker in METH-induced neurotoxicity.
Materials and methods
Animals and METH treatment
The animal study was performed according to the protocol approved by the Animal Care and Use Committee of the National Defense Medical Center Taipei, Taiwan (IACUC10-093). Male Sprague-Dawley (SD) adult rats (3 months old) weighing 250–300 g were housed in the animal center of the National Defense Medical Center at a constant temperature and a controlled 12/12-h light/dark cycle (light from 7:00 AM to 7:00 PM). Male rats were used to avoid the cyclic hormonal changes in female rats that are associated with the estrus cycle and could confound the results. METH was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 5 mg/mL saline (0.9% NaCl).
On the day of METH administration, rats were housed individually with restricted water and food. Rats received four administrations of METH (5 or 10 mg/kg, s.c.) at 1-h intervals or an equal volume of 0.9% saline [
27]. After METH administration, two rats were housed per cage and had free access to water, food, and sawdust shavings.
According to previous studies [
28], 30 days is adequate for the body to clear METH; however, the striatal DA levels are depleted to about 50% in METH-treated animals. Thus, the PET imaging of in vivo SERT availability/activity was carried out 30 days after the administration of METH to investigate the 30-day long-lasting depletion of serotonin as well as damage to striatal serotonergic nerve terminals. One week after the PET imaging study, the rats were sacrificed and tissues assessed by immunohistochemistry for TH levels.
Synthesis of 4-[18F]ADAM
The 4-[
18F]ADAM was synthesized in an automated synthesizer as described previously [
29]. All the 4-[
18F]ADAM formulations used in this study were prepared in our PET cGMP laboratory, which is inspected regularly by the Council of Atomic Energy and the Department of Health, Taiwan. The radiochemical purity was > 95%, and the specific activity was 0.6 Ci/μmol or 22.2 Gbq/μmol (EOB). All 4-[
18F]ADAM was prepared in the PET-Cyclotron Laboratory of the National Defense Medical Center.
4-[18F]ADAM micro-PET imaging
Imaging protocols and acquisition for the METH-treated and vehicle groups (controls) were performed as described by Ma et al. [
30]. Briefly, the rats were gas anesthetized (2% isoflurane with 98% oxygen mixture) and injected with 4-[
18F]ADAM (14.8–18.5 MBq; 0.4–0.5 mCi) via the tail vein. PET imaging was performed 60–90 min after the administration of 4-[
18F]ADAM.
The static PET images were acquired for 30 min on a small animal micro-PET R4 scanner (Concorde MicroSystems, Knoxville, TN, USA). The energy window was 350–650 keV, and the timing window was 6 ns. Images were then reconstructed by the Fourier rebinning algorithm and two-dimensional filtered back projection with a ramp filter with a cutoff using Nyquist frequency. The regional radioactivity concentration (KBq/cc) of 4-[
18F]ADAM was estimated from the maximum pixel values within volumes of interest (VOI). The radioactivity concentration (KBq/cc, μCi/cc) was converted to percent injected dose per gram (%ID/g), and the mean and standard deviation values of radiotracer accumulation in various tissues were calculated. Specific uptake ratios (SURs) were expressed as (target region-cerebellum)/cerebellum [
13].
Data were analyzed with ASIPro VM6.3.3.1 software (Concorde MicroSystem, Knoxville, TN, USA) or PMOD 3.7 software (PMOD Technologies Ltd., Zurich, Switzerland).
Immunohistochemistry
Rats were anesthetized by intraperitoneal injection of 7% chloral hydrate (5 mL/kg). Their thoracic cavities were opened followed by an incision in the right auricle. Perfusion was performed via the ascending aorta with 300 mL of 0.9% normal saline followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The rat brains were quickly removed and immersed in the same fixative for 2 h.
Then, the brain was stored overnight in a solution of 0.1 M PBS with 30% sucrose at 4 °C. The sagittal sections (30 μm) were cut using a cryostat (Leica CM 3050; Leica Microsystems Nussloch, GmbH, Nussloch, Germany). Afterwards, the brain sections were washed with PBS and incubated in 1% H2O2 in PBS for 30 min. Then, the sections were washed extensively with PBS and incubated to reduce background in blocking solution (1% normal goat serum in 0.1 M PBS plus 1% Triton X-100) for 1 h. Next, the sections were incubated overnight at 4 °C with rabbit anti-tyrosine hydroxylase (anti-TH) antibody (1:2000; Millipore Corporation, Bedford, MA, USA). Following the overnight incubation, the sections were washed and incubated with goat anti-rabbit biotinylated IgG (1:250; Vector Laboratories, Burlingame, CA, USA) for 1 h and with avidin-biotin complex (1:200; Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) for 1 h. After these incubations, the sections were washed with PBS and exposed to 0.05% 3′3-diaminobenzidine (DAB, dissolved in 0.1% H2O2 in 0.05 M Tris buffer, pH 7.6) for 5 min. Finally, the sections were washed three times with distilled water and mounted on gelatin-coated glass slides.
IHC images were captured using an optic imaging system, Nikon OPTIPHOT-2 × 10 and MICROPHOT-FXA × 200–400 (Nikon Instruments Inc., Melville, NY, USA). The optical density of dopaminergic fiber images was analyzed and quantified with Image-Pro Plus v. 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The selected images were then converted into an 8-bit grayscale. The optical density was measured with the region of interest (ROI) and calibrated with a control region (corpus callosum). The ratio of ROI-to-control region was calculated and averaged for each animal.
Statistics
Data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with post-hoc Bonferroni tests were used for statistical evaluations. A p < 0.05 was considered statistically significant. Statistical analyses of data were performed using GraphPad Prism 4 (GraphPad, La Jolla, CA, USA).
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