Exp Neurobiol 2019; 28(5): 602-611
Published online October 31, 2019
© The Korean Society for Brain and Neural Sciences
So Hyeon Park1, Yoo Sung Song2, Byung Seok Moon3, Byung Chul Lee2, Hyun Soo Park1,2* and Sang Eun Kim1,2,4*
1Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 08826, 2Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seoul 03080, 3Department of Nuclear Medicine, Ewha Woman’s University Seoul Hospital, Ewha Womans University College of Medicine, Seoul 07804, 4Advanced Institutes of Convergence Technology, Suwon 16229, Korea
Correspondence to: *To whom correspondence should be addressed.
Sang Eun Kim, TEL: 82-31-787-7671, FAX: 82-31-787-4018
Hyun Soo Park, TEL: 82-31-787-2936, FAX: 82-31-787-4018
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Synaptic dopamine (DA) is mainly regulated by the presynaptic DA transporter (DAT). Single-photon emission computerized tomography (SPECT) with the DAT radiotracer [123I]FP-CIT assesses changes in synaptic DA availability when endogenous DA displaces [123I]FP-CIT or competes for DAT. Here, we investigated the effects of haloperidol (HAL) and clozapine (CLZ) on [123I]FP-CIT binding in the rat striatum and midbrain to assess the utility of [123I]FP-CIT SPECT to quantify changes in synaptic DA availability. Rats underwent [123I]FP-CIT SPECT after intraperitoneal administration of normal saline (vehicle), HAL (1 and 7 mg/kg), CLZ (10 and 54 mg/kg) and bupropion (BUP, a DAT blocker, 20 and 100 mg/kg). In the striatum and midbrain, percent differences in the nondisplaceable binding potential (BPND) of [123I]FP-CIT compared to the vehicle were calculated for the various drugs and doses. In another experiment, changes in endogenous striatal DA concentration were measured by
Synaptic dopamine (DA) availability is implicated in the pathology of various neurological and psychiatric diseases, such as Parkinson’s disease [1, 2] and schizophrenia [3, 4]. DA availability in the brain is mainly regulated by the DA transporter (DAT) , and DA receptors for its D2 and D3 subtypes expressed on the cell membrane of DA neurons (i.e., autoreceptors) also play a key role in regulating the activity of DAergic neurons and controlling DA synthesis, release, and reuptake . Under the DA hypothesis, antipsychotic drugs act mainly by regulating the DAergic systems, as evidenced by the significant association between DA receptor antagonism and improved positive and/or negative symptoms. Some of them have been proven to regulate DA release [6–8]. An antipsychotic drug haloperidol (HAL) is known to increase DA synthesis and release in the striatum and related mesolimbic structures [9–12]. The proposed mechanism of action consists of presynaptic terminal blockade by DA autoreceptors , which abolishes feedback inhibition, leading to enhanced DA synthesis or release. Clozapine (CLZ), despite acting as a DA antagonist as part of its therapeutic effect against schizophrenia symptoms, also stimulates DA release in the ventral striatum (i.e., nucleus accumbens) of rats  and in the hippocampus of schizophrenia patients . Presynaptic modulation induced by antipsychotic drugs affects the extracellular DA concentration by altering autoregulation . An increased extracellular DA concentration can lead to stimulation of DA autoreceptors, which inhibits DA release [6, 16]. DA release and metabolism in the rat striatum
The competition between endogenous transmitters and radio-labeled ligands for
We investigated differences in the nondisplaceable binding potential of [123I]FP-CIT in the rat striatum and midbrain, which compose the nigrostriatal DA system, after HAL and CLZ compared to vehicle treatment in an attempt to assess the validity of [123I]FP-CIT SPECT as a measure of synaptic DA availability induced by HAL and CLZ. The reliability of this noninvasive imaging technique was further examined for the striatum by conducting an
This study was approved by the Institutional Animal Care and Use Committee of the Seoul National University Bundang Hospital. Animals were purchased from Orient Bio Inc., Seoul, Korea. A total of 35 and 20 Sprague Dawley (SD) rats (male, 6-week-old, 260~300 g body weight) were used for [123I]FP-CIT SPECT and
HAL, CLZ and bupropion (BUP) hydrochloride were purchased from Sigma-Aldrich Korea, Yongin, Korea. HAL and CLZ were dissolved in 1% tartaric acid and 1 N HCl. A DAT blocker, BUP, the positive control drug for HAL and CLZ, was used to test whether endogenous DA displaces [123I]FP-CIT or BUP competes with [123I] FP-CIT for DAT. BUP was dissolved in NS.
In the [123I]FP-CIT SPECT study, the animals were divided into four groups defined by different drugs (vehicle (n=5), HAL (n=10), CLZ (n=10), BUP (n=10) treatment groups). While the vehicle-treated group had no dose conditions, the others had low and high dose conditions for the drug treatment. The low and high doses of HAL, CLZ and BUP were 1 and 7 mg/kg body weight, 10 and 54 mg/kg body weight, and 20 and 100 mg/kg body weight, respectively (n=5 per drug and dose condition). The doses were selected based on previous studies showing that low and high doses induce ~25% increases in synaptic DA availability and greater than 80% DA receptor occupancy by drugs in the striatum, respectively [24–27].
In another experiment, changes in endogenous DA concentration in the striatum were monitored by
In both experiments, drugs were injected intraperitoneally (
The [123I]FP-CIT SPECT/CT study was performed on a dedicated small-animal SPECT/CT system (NanoSPECT/CT, Mediso Inc., Budapest, Hungary). Helical small-animal SPECT scans were performed using a 4-head γ-camera outfitted with multipinhole collimators (1.4-mm-diameter pinholes) designed for rats. [123I] FP-CIT was injected at a dose (mean±SD) of 39.5±7.2 MBq 1 h after drug treatment; 2 h later (once [123I]FP-CIT had reached equilibrium in the striatum), SPECT/CT data were acquired from the animals for 30 min under 2% isoflurane anesthesia. After the scan, the SPECT data were reconstructed using iterative three-dimensional ordered subset expectation maximization with the single-slice rebinning method. CT-based attenuation correction was performed, as were scatter and random correction. The reconstructed images were 176×176×136 pixels with a voxel size of 0.6×0.6×0.6 mm (x, y, z). PMOD software (PMOD Technologies LLC., Geneva, Switzerland) was used for processing and analysis of SPECT and CT images. Images were spatially normalized to standard stereotaxic space with the predefined magnetic resonance imaging (MRI) rat brain template. The striatum, midbrain and cerebellum were defined using automated anatomical labeling embedded in PMOD software . Synaptic DA availability in the striatum and midbrain was quantitatively assessed in terms of the nondisplaceable binding potential (BPND) of [123I]FP-CIT, which is proportional to the density of available binding sites (i.e., DAT). The cerebellum (which is known as a DAT-poor region or nondisplaceable binding site of [123I]FP-CIT) was set as the reference region for estimating BPND according to the following equation: BPND=(
Extracellular DA concentrations in the striatum of freely moving rats were directly measured by
Data were analyzed using GraphPad Prism (version 7.0, Graph-Pad Software Inc., La Jolla, CA, USA). The statistical significance of differences in mean BPND between drug and dose conditions was tested by two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test between dose conditions for each drug.
In the vehicle-treated group, the mean [123I]FP-CIT BPND was 1.64±0.13 and 1.69±0.32 in the striatum and midbrain, respectively; these values are comparable to the results of previous studies [24, 30]. BUP dose-dependently occupied DAT to a considerable degree, as evidenced by decreases in [123I]FP-CIT BPND of −16.50% (20 mg/kg) and −56.29% (100 mg/kg) in the striatum and −31.57% (20 mg/kg) and −53.08% (100 mg/kg) in the midbrain, implying that [123I]FP-CIT SPECT is a reliable and sensitive technique for measuring drug-induced changes in DAT activity and can conceptually allow the assessment of changes in synaptic DA availability
Treatment with HAL and CLZ markedly altered synaptic DA availability compared to the vehicle, as evidenced by changes in [123I]FP-CIT BPND in both the striatum and the midbrain. Intriguingly, the level of changes in [123I]FP-CIT BPND treatment varied across drugs, doses, and regions (Fig. 1 and Table 1). Compared to the vehicle, HAL decreased [123I]FP-CIT BPND in the striatum (−25.29% and −2.27% for 1 and 7 mg/kg, respectively) and to a greater degree in the midbrain (−58.74% and −49.64% for 1 and 7 mg/kg, respectively), whereas the CLZ-treated group showed an increase in the striatum (18.85% and 38.64% for 10 and 54 mg/kg, respectively) but a decrease in the midbrain (−38.60% and −40.38% for 10 and 54 mg/kg, respectively).
The changes in extracellular striatal DA concentrations by HAL (1 and 7 mg/kg) and CLZ (10 and 54 mg/kg) treatment were evaluated by
The relationships between percentage differences in [123I]FP-CIT BPND and time-averaged percentage changes from baseline extracellular DA concentration after treatment with varying doses of HAL and CLZ are depicted in Fig. 3 (A and B for HAL and CLZ, respectively). An inverse relationship between percentage differences in [123I]FP-CIT BPND and time-averaged % percentage change from baseline extracellular DA concentration (lower [123I] FP-CIT BPND, greater synaptic DA availability) appeared only in the HAL-treated group.
Under the investigational assumption that if endogenous DA displaces radioligands or competes with them for presynaptic binding sites, radioligand binding to the DAT could be affected, we investigated differences in the [123I]FP-CIT BPND in the rat striatum and midbrain, which compose the nigrostriatal DA system, after HAL and CLZ treatments that increased synaptic DA availability. The reliability of this noninvasive imaging technique was further examined for the striatum by using an
Our results showed that [123I]FP-CIT SPECT allows the detection of apparent increases in synaptic DA concentration induced by low (1 mg/kg
The [123I]FP-CIT SPECT and
Although it is possible that there were experimental errors and unknown contamination factors that could have led to these results, the magnitude of error in the measured values (BPND and DA concentration) was quite acceptable. The reliability of increased striatal [123I]FP-CIT BPND could be validated from decreases in the midbrain of the same subject on CLZ treatment. In the midbrain, non-dose-dependent decreases in [123I]FP-CIT binding after treatment were consistently shown among drugs (although the DAT blocker BUP occupied DAT dose-dependently). Regardless of dose, the %difference was greater for HAL (−49.64~58.74% difference) than CLZ (−38.60 to −40.38% difference) in accordingly with pharmacologic characteristics in DA regulation of HAL and CLZ, implying their own typicality. That CLZ affects increasing binding affinity to DAT is unlikely, but it is not impossible, as some ligands occasionally act in this way. For example, an antiepileptic drug, tiagabine, which binds to the central benzodiazepine receptor, affects the increased binding affinity of radiolabeled ligand ([18F] flumazenil) to the central benzodiazepine receptor, known as the “GABA shift” . On the other hand, the interaction between CLZ and [123I]FP-CIT could also be considered. The potential effect of CLZ on the group of antipsychotic drugs was examined; for instance, only CLZ induced a decrease in the protein kinase C level , but the effect of decreased protein kinase C level and interaction between [123I]FP-CIT and DAT is unexpected.
In addition, this study examined changes in synaptic DA availability after acute administration of HAL and CLZ in an attempt to resolve the previously reported controversy [12, 13] through an analogous approach to the experimental paradigm and to determine whether [123I]FP-CIT SPECT can be used to assess changes in endogenous DA concentration based on alterations in [123I]FP-CIT BPND to DAT as it is displaced by endogenous DA. Importantly, we partly overcame the experimental limitations of the earlier studies by performing
DAT imaging is increasingly used for diagnostics and drug development for neurological and psychiatric diseases as well as for precision medicine in cases of complicated medication status—for instance, a Parkinson’s disease patient receiving antipsychotic drugs. Consequently, there is a need for further experimental evidence supporting the utility of [123I]FP-CIT SPECT for quantitation of acute changes in available DA. Recently, there has been growing interest in assessing DAT binding in schizophrenia patients. However, the results obtained to date on striatal DAT binding in schizophrenia subjects have been inconsistent, with reports of elevated [37, 38], reduced [22, 38, 39], or unaltered [23, 40–44] DAT binding. Interestingly, unaltered , decreased [22, 39], or increased  DAT binding has also been observed in medicated patients. These results are difficult to interpret because factors such as illness duration and phase–which could vary between patients and investigations—are likely to affect the regulation of pre- and postsynaptic binding sites. Moreover, our findings suggest that antipsychotic drugs themselves may confound presynaptic binding data. Schizophrenia patients who are not responding to antipsychotic drug treatment can have a high percentage of occupied D2 receptors without any relief of symptoms . In light of the present findings, it is conceivable that presynaptic autoreceptor or transporter function may be dysregulated in this subgroup of schizophrenia patients. In routine clinical studies as well as scientific studies, patients are frequently on medication and sometimes even take drugs of abuse . Moreover, in preclinical studies, animals are anesthetized for their scans. Prescribed drugs, drugs of abuse, and anesthetics may influence the visual interpretation and/or quantification of [123I]FP-CIT SPECT scans.
The present study has several limitations. We used different methods to measure alterations in the DAergic neurotransmission system induced by treatment with the antipsychotic drugs HAL and CLZ. The two techniques employed have pros and cons with respect to their ability to measure alterations in the DAergic neurotransmission system, and they were used under fundamentally different experimental conditions (anesthetized animals vs. awake animals). Nonetheless, we felt that the utility of [123I]FP-CIT SPECT compared to the
In conclusion, this study demonstrates that [123I]FP-CIT SPECT may be a useful preclinical technique for detecting increases in synaptic DA availability induced by HAL treatment in both the midbrain and the striatum, with results comparable to those obtained by
This study was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C-0947), and by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning, Republic of Korea (NRF-2018R1D1A1B07047994, 2016R1D1A1A02937028).
Table 1. Differences in [123I]FP-CIT BPND.
|BPND||% Difference||p||BPND||% Difference||p|
Values are the mean±SEM. VEH, vehicle; BUP, bupropion; HAL, haloperidol; CLZ, clozapine..