Therapeutic Effects of Quetiapine and 5-HT1A Receptor Agonism on Hyperactivity in Dopamine-Deficient Mice

Some diseases that are associated with dopamine deficiency are accompanied by psychiatric symptoms, including Parkinson’s disease. However, the mechanism by which this occurs has not been clarified. Previous studies found that dopamine-deficient (DD) mice exhibited hyperactivity in a novel environment. This hyperactivity is improved by clozapine and donepezil, which are used to treat psychiatric symptoms associated with dopamine deficiency (PSDD). We considered that DD mice could be used to study PSDD. In the present study, we sought to identify the pharmacological mechanism of PSDD. We conducted locomotor activity tests by administering quetiapine and drugs that have specific actions on serotonin (5-hydroxytryptamine [5-HT]) receptors and muscarinic receptors. Changes in neuronal activity that were induced by drug administration in DD mice were evaluated by examining Fos immunoreactivity. Quetiapine suppressed hyperactivity in DD mice while the 5-HT1A receptor antagonist WAY100635 inhibited this effect. The number of Fos-positive neurons in the median raphe nucleus increased in DD mice that exhibited hyperactivity and was decreased by treatment with quetiapine and 5-HT1A receptor agonists. In conclusion, hyperactivity in DD mice was ameliorated by quetiapine, likely through 5-HT1A receptor activation. These findings suggest that 5-HT1A receptors may play a role in PSDD, and 5-HT1A receptor-targeting drugs may help improve PSDD.


Introduction
Dopamine is a neurotransmitter that plays a very important role in motor control, motivation, reward, and cognitive function [1]. When dopamine levels in the brain decrease, basal ganglia circuits, including the striatum, become imbalanced, resulting in parkinsonism, characterized by bradykinesia, rigidity, resting tremor, and postural instability [2,3]. Such drugs as metoclopramide and cerebrovascular disorders, tumors, and inflammatory and infectious processes that involve areas of the nigrostriatal pathway can decrease dopamine in the brain and cause parkinsonism [4]. Dopamine levels also decrease in Parkinson's disease (PD) through the degeneration of dopaminergic neurons in the substantia nigra, the cause of which has not yet been clarified [5].
Some diseases that are associated with dopamine deficiency are accompanied by psychiatric symptoms [6]. Manganese has been reported to accumulate in dopamine neurons in the ventral tegmental area and substantia nigra [7] and is known to cause secondary parkinsonism and psychiatric symptoms, such as hallucinations [8]. Hallucinations have also been reported in patients with pathologically proven vascular parkinsonism [9]. Cases of brain exhibited hyperlocomotion. These results are consistent with our previous study [16]. We found that quetiapine suppressed locomotor activity in both WT and DD mice. The other drugs, including pimavanserin, tandospirone, paroxetine, and trihexyphenidyl, did not significantly affect locomotor activity in either WT or DD mice. The time course of locomotor activity in saline-and 20 mg/kg quetiapine-treated mice indicated that quetiapine significantly suppressed locomotor activity in both WT and DD mice beginning 20 min after administration until the end of the test. Data for quetiapine doses of 10, 40, and 80 mg/kg are included in the Supplementary Materials (Figures S1a,b-S3a,b). The transient increase in activity in WT mice just prior to drug administration may be attributable to the fact that the mice may have detected that the researchers had entered the laboratory and administered the drugs to the other mice. The increase in activity immediately after drug administration may be attributable to the pain of the injection. Conversely, in DD mice, excitation was transiently suppressed immediately after drug administration (Figure 2a,b).   exhibited hyperlocomotion. These results are consistent with our previous study [16]. We found that quetiapine suppressed locomotor activity in both WT and DD mice. The other drugs, including pimavanserin, tandospirone, paroxetine, and trihexyphenidyl, did not significantly affect locomotor activity in either WT or DD mice. The time course of locomotor activity in saline-and 20 mg/kg quetiapine-treated mice indicated that quetiapine significantly suppressed locomotor activity in both WT and DD mice beginning 20 min after administration until the end of the test. Data for quetiapine doses of 10, 40, and 80 mg/kg are included in the Supplementary Materials ( Figures S1a,b-S3a,b). The transient increase in activity in WT mice just prior to drug administration may be attributable to the fact that the mice may have detected that the researchers had entered the laboratory and administered the drugs to the other mice. The increase in activity immediately after drug administration may be attributable to the pain of the injection. Conversely, in DD mice, excitation was transiently suppressed immediately after drug administration (Figure 2a,b).

Figure 1.
Behavioral changes related to PSDD induced by drug administration. Total locomotor activity increased (3 h after drug administration-3 h before drug administration). ** p < 0.01, compared with saline administration (Scheffé test). The graph shows the number of animals per group. The data are expressed as mean ± SEM. WT, wildtype mice; DD, dopamine-deficient mice.  The graphs show the number of animals per group. The data are expressed as mean ± SEM. WT, wildtype mice; DD, dopamine-deficient mice.

5-HT 1A Receptor Antagonist Partially Inhibited the Effect of Quetiapine
In a previous study, clozapine was shown to be effective against hyperactivity in DD mice, and the mechanism was considered to involve an increase in acetylcholine [16]. Quetiapine is a Multi-Acting Receptor-Targeted Antipsychotic (MARTA) like clozapine, but it is known to have minimal actions on acetylcholine receptors. To further investigate the mechanism of action, we administered the 5-HT 1A receptor antagonist WAY100635 (1 or 0.2 mg/kg) and anticholinergic drug scopolamine (0.1 mg/kg) 30 min before 20 mg/kg quetiapine administration to examine whether these receptors mediate the locomotorsuppressive effect of quetiapine. The effects of quetiapine were partially blocked by 1 mg/kg WAY100635 (Figure 3a). When 0.2 mg/kg WAY100635 was administered, the effect was not as clear ( Figure S4). Scopolamine did not significantly block the effects of quetiapine (Figure 3b). In WT mice, the effects of quetiapine were not blocked by WAY100635 or scopolamine ( Figure S5a,b). The effects of quetiapine are presumed to be partially mediated by the 5-HT receptor system.

5-HT1A Receptor Antagonist Partially Inhibited the Effect of Quetiapine
In a previous study, clozapine was shown to be effective against hyperactivity in DD mice, and the mechanism was considered to involve an increase in acetylcholine [16]. Quetiapine is a Multi-Acting Receptor-Targeted Antipsychotic (MARTA) like clozapine, but it is known to have minimal actions on acetylcholine receptors. To further investigate the mechanism of action, we administered the 5-HT1A receptor antagonist WAY100635 (1 or 0.2 mg/kg) and anticholinergic drug scopolamine (0.1 mg/kg) 30 min before 20 mg/kg quetiapine administration to examine whether these receptors mediate the locomotorsuppressive effect of quetiapine. The effects of quetiapine were partially blocked by 1 mg/kg WAY100635 (Figure 3a). When 0.2 mg/kg WAY100635 was administered, the effect was not as clear ( Figure S4). Scopolamine did not significantly block the effects of quetiapine ( Figure 3b). In WT mice, the effects of quetiapine were not blocked by WAY100635 or scopolamine ( Figure S5a,b). The effects of quetiapine are presumed to be partially mediated by the 5-HT receptor system.

5-HT1A Receptor Agonist Ameliorated Hyperactivity in DD Mice
The effects of quetiapine were partially mediated by 5-HT1A receptors, suggesting that 5-HT1A receptor function might be involved in hyperactivity in DD mice. Quetiapine also targets 5-HT2A receptors. Thus, receptor subtype-specific drugs were administered to determine which specific receptor subtypes may be involved in hyperactivity in DD mice. 8-OH-DPAT (5-HT1A receptor agonist) and EMD281014 (5-HT2A receptor antagonist) were administered in WT and DD mice. 8-OH-DPAT was more effective than EMD281014 in suppressing hyperactivity (Figure 4a

5-HT 1A Receptor Agonist Ameliorated Hyperactivity in DD Mice
The effects of quetiapine were partially mediated by 5-HT 1A receptors, suggesting that 5-HT 1A receptor function might be involved in hyperactivity in DD mice. Quetiapine also targets 5-HT 2A receptors. Thus, receptor subtype-specific drugs were administered to determine which specific receptor subtypes may be involved in hyperactivity in DD mice. 8-OH-DPAT (5-HT 1A receptor agonist) and EMD281014 (5-HT 2A receptor antagonist) were administered in WT and DD mice. 8-OH-DPAT was more effective than EMD281014 in suppressing hyperactivity (Figure 4a

Number of Fos-Positive Cells was Reduced by Quetiapine and 8-OH-DPAT in the MRN
We performed immunohistochemical staining using an anti-c-fos antibody to examine changes in activated brain regions that are related to the 5-HT system when hyperactivity was suppressed by quetiapine or 8-OH-DPAT. Fos-positive cells were counted in a 400 μm × 100 μm area in the MRN and 200 μm × 200 μm area in the rostral linear nucleus (RLi). Hyperactive DD mice exhibited an increase in Fos-positive cells in the MRN, which was reduced by quetiapine and reduced further by 8-OH-DPAT ( Figure 5). In contrast, few Fos-positive cells were found in the RLi ( Figure 6). No Fos-positive cells were detected in the dorsal raphe nucleus (data not shown). (a)

Number of Fos-Positive Cells was Reduced by Quetiapine and 8-OH-DPAT in the MRN
We performed immunohistochemical staining using an anti-c-fos antibody to examine changes in activated brain regions that are related to the 5-HT system when hyperac

Number of Fos-Positive Cells was Reduced by Quetiapine and 8-OH-DPAT in the MRN
We performed immunohistochemical staining using an anti-c-fos antibody to examine changes in activated brain regions that are related to the 5-HT system when hyperac

Discussion
In the present study, we found that quetiapine, a drug that is used to treat psycho symptoms of PD [13], effectively suppressed hyperactivity in DD mice.
Although quetiapine is a MARTA, it does not target muscarinic receptors. As e pected, we confirmed that scopolamine did not block the effects of quetiapine. Therefo hyperactivity should be inhibited through other molecular targets beyond muscarinic r ceptors. Among multiple receptors that are targeted by quetiapine, the present study f cused on 5-HT receptors. WAY100635 partially inhibited the effects of quetiapine, su gesting that 5-HT1A receptors participate in mediating the effects of quetiapine. Quetia ine stimulates 5-HT1A receptors and inhibits 5-HT2A receptors. The 8-OH-DPAT an EMD281024 results indicate that the inhibition of hyperactivity was mainly mediated 5-HT1A receptor stimulation. Therefore, the results of the present study infer a 5-HT1A r ceptor-mediated mechanism that underlies the effects of quetiapine on PSDD. In additi to providing clues to the mechanism of action of quetiapine on PSDD, 5-HT1A recepto may be a target for effectively treating PSDD. 5-HT1A receptors are a major mediator of the actions of 5-HT. The 5-HT1A receptor a metabotropic Gprotein-coupled receptor that is highly expressed in 5-HT neurons as presynaptic inhibitory autoreceptor. 5-HT1A receptors are expressed in many brain regio that are innervated by 5-HT neurons, including the frontal cortex, septum, amygdala, hi pocampus, and hypothalamus, as postsynaptic heteroreceptors [19]. We tested two HT1A receptor agonists in the present study. 8-OH-DPAT but not tandospirone mitigat hyperactivity in DD mice. Tandospirone acts on postsynaptic 5-HT1A receptors [20,21]. contrast, 8-OH-DPAT is a full 5-HT1A receptor agonist [22,23]. The present results sugge that the presynaptic function of 5-HT1A receptors may be important for the inhibition hyperactivity in DD mice. Previous studies demonstrated the importance of the presy aptic function of 5-HT1A receptors rather than their postsynaptic functions for the trea ment of a low level of social interaction, anxiety, and cognitive dysfunction [23,24]. Thu presynaptic 5-HT1A receptors may also play an important role in improving PSDD.
Baseline 5-HT levels are elevated in the striatum in DD mice [16]. The early disru

Discussion
In the present study, we found that quetiapine, a drug that is used to treat psychotic symptoms of PD [13], effectively suppressed hyperactivity in DD mice.
Although quetiapine is a MARTA, it does not target muscarinic receptors. As expected, we confirmed that scopolamine did not block the effects of quetiapine. Therefore, hyperactivity should be inhibited through other molecular targets beyond muscarinic receptors. Among multiple receptors that are targeted by quetiapine, the present study focused on 5-HT receptors. WAY100635 partially inhibited the effects of quetiapine, suggesting that 5-HT 1A receptors participate in mediating the effects of quetiapine. Quetiapine stimulates 5-HT 1A receptors and inhibits 5-HT 2A receptors. The 8-OH-DPAT and EMD281024 results indicate that the inhibition of hyperactivity was mainly mediated by 5-HT 1A receptor stimulation. Therefore, the results of the present study infer a 5-HT 1A receptor-mediated mechanism that underlies the effects of quetiapine on PSDD. In addition to providing clues to the mechanism of action of quetiapine on PSDD, 5-HT 1A receptors may be a target for effectively treating PSDD. 5-HT 1A receptors are a major mediator of the actions of 5-HT. The 5-HT 1A receptor is a metabotropic Gprotein-coupled receptor that is highly expressed in 5-HT neurons as a presynaptic inhibitory autoreceptor. 5-HT 1A receptors are expressed in many brain regions that are innervated by 5-HT neurons, including the frontal cortex, septum, amygdala, hippocampus, and hypothalamus, as postsynaptic heteroreceptors [19]. We tested two 5-HT 1A receptor agonists in the present study. 8-OH-DPAT but not tandospirone mitigated hyperactivity in DD mice. Tandospirone acts on postsynaptic 5-HT 1A receptors [20,21]. In contrast, 8-OH-DPAT is a full 5-HT 1A receptor agonist [22,23]. The present results suggest that the presynaptic function of 5-HT 1A receptors may be important for the inhibition of hyperactivity in DD mice. Previous studies demonstrated the importance of the presynaptic function of 5-HT 1A receptors rather than their postsynaptic functions for the treatment of a low level of social interaction, anxiety, and cognitive dysfunction [23,24]. Thus, presynaptic 5-HT 1A receptors may also play an important role in improving PSDD.
Baseline 5-HT levels are elevated in the striatum in DD mice [16]. The early disruption of central dopaminergic pathways is known to increase striatal 5-HT content [25]. The stimulation of presynaptic 5-HT 1A receptors results in a decrease in 5-HT release. Therefore, high 5-HT levels may be a cause of hyperactivity, and the suppression of these high levels may be a mechanism by which hyperactivity is reduced. In humans, 5-HT neurons are degenerated in PD [26]. The degeneration of 5-HT neurons occurs more slowly than the degeneration of dopaminergic neurons, which may result in a 5-HT-dominant state that is a common pathological feature of PSDD and hyperactivity in DD mice.
Psychiatric symptoms have been studied in other DD animals. DD animals that exhibit psychiatric symptoms include 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)lesioned marmosets [27], human α-synuclein transgenic rats [18], and 6-hydroxydopaminelesioned rats [28]. However, psychotic behaviors appeared when these animals received drugs that stimulate dopamine receptors. Therefore, these models may not be suitable for studying the pathogenesis of psychosis in a dopamine-depleted state. With regard to 5-HT and psychosis, L-DOPA-stimulated psychosis-like behavior in MPTP-lesioned monkeys was abolished after 5-HT injury by 3,4-methylenedioxy-N-methamphetamine (MDMA) [29]. Although the animals in this previous study received L-DOPA, the fact that serotonergic neuron injury can improve neuropsychiatric-like behaviors in DD animals appears to support the present results, in which a decrease in serotonergic signaling could ameliorate psychotic-like behavior in DD animals.
We found that the number of Fos-expressing neurons significantly increased in the MRN in DD mice that exhibited hyperactivity. In contrast, few Fos-positive cells were detected in the RLi. The MRN is a major nucleus of 5-HT neurons. However, some 5-HT neurons and other neurons release other neurotransmitters, such as γ-aminobutyric acid (GABA) and glutamate [30]. Interestingly, the MRN mediates motor activity through both the agonism and antagonism of several neurotransmitter receptors, including GABA, glutamate, and opioid receptors [30]. Therefore, any of these neurons in the MRN may be involved in hyperactivity in DD mice. Future studies will determine which neurons are specifically activated in hyperactive DD mice.
One limitation of this study is that c-Fos is an indirect marker of neuronal activity because it is expressed after action potentials spike in neurons [31]. The appearance of c-Fos-positive cells that is attributable various nonspecific causes should also be considered. Future studies, such as calcium imaging, will be required to analyze neuronal activity more precisely. Additionally, both DD mice and wildtype mice exhibited a decrease in locomotor activity in the novel environment when they were treated with quetiapine and 8-OH-DPAT. Therefore, it is unknown whether quetiapine and 8-OH-DPAT specifically suppressed hyperlocomotion in DD mice.

Drugs
We L-DOPA for injection was dissolved in ascorbic acid solution to 1.4 mg/mL and administered at 50 mg/kg. Ascorbic acid solution was made by dissolving ascorbic acid in saline at a concentration of 2.5 mg/mL. Pimavanserin (5 mg/kg), tandospirone (3 mg/kg), quetiapine (10,20,40, and 80 mg/kg), and EMD281014 (10 mg/kg) were dissolved in one-tenth the required amount of dimethylsulfoxide and diluted to the final volume with saline or purified water. Clozapine (10 mg/kg) was dissolved in a minimum volume of 0.1 N HCl and diluted to the required volume in saline or purified water. Paroxetine (8 mg/kg), 8-OH-DPAT (10 mg/kg), WAY100635 (0.2 and 1 mg/kg), scopolamine (0.1 mg/kg), oxotremorine-M (0.1 mg/kg), and trihexyphenidyl (3 mg/kg) were dissolved in saline or purified water.

Dopamine-Deficient Mice
Dopamine-deficient mice were created as described previously [14]. We used DD mice (n = 10-29) and wildtype (WT) mice (n = 10-21), which were littermates that were created by crossing heterozygous/heterozygous DD mice on a C57BL/6J genetic background. The experimental procedures and housing conditions were approved by the Institutional Animal Care and Use Committee (Animal Experimentation Ethics Committee, Tokyo Metropolitan Institute of Medical Science, approval no. 12-43). All of the animals were cared for and humanely treated according to our institutional animal experimentation guidelines. All of the mice were housed in an animal facility that was maintained at 23 • C ± 1 • C and 55% ± 5% relative humidity under a 12 h/12 h light/dark cycle (lights on at 8:00 AM, lights off at 8:00 PM). Food and water were available ad libitum. Because newborn rats cannot eat sufficient food that contains L-DOPA during the neonatal period, L-DOPA was administered intraperitoneally 6 days per week until the DD mice reached 6 weeks of age. Afterward, the mice were given paste food or MediGel that was supplemented with L-DOPA in addition to their usual food pellets. We examined male and female mice at 10-58 weeks of age.
The paste food that was supplemented with L-DOPA was prepared by mixing 1000 mg of L-DOPA, 500 mg of ascorbic acid, and 250 mg of benserazide in 2 kg of powdered food. The paste was created by adding water to the mixture. The paste food was changed daily. The gel food that was supplemented with L-DOPA was created by dissolving 60 mg of L-DOPA and 15 mg of benserazide in 1 mL of ascorbic acid, prepared as described above, and adding it to the MediGel. We maintained the mice on this gel food for up to 3 days. We used both the paste food and gel food, depending on the specific experimental conditions.

Open-Field Test
Because the amount of food with L-DOPA could vary between mice, they received a subcutaneous injection of 50 mg/kg L-DOPA 3 days before the study, and they were given DietGel without L-DOPA for the remaining 3 days to standardize conditions during the test. This treatment regimen caused brain dopamine levels to fall below the limit of detection [15]. The open-field test (OFT) was performed by recording locomotion for 6 h using a Supermex apparatus (Muromachi Kikai, Tokyo, Japan). Each mouse was placed in an illuminated translucent chamber (350 mm × 400 mm × 250 mm). A sensor monitor was attached on top of the apparatus, and movements were automatically recorded and summed every 10 min. After a 3 h habituation period, the drugs were administered subcutaneously, and locomotor activity was monitored continuously for another 3 h. When two drugs were administered, WAY 100635 or scopolamine was administered 30 min before quetiapine administration. After the OFT, the mice were again treated with 50 mg/kg L-DOPA. Between the separate OFT sessions, the mice were allowed to rest for at least 2 weeks and maintained on L-DOPA-containing food.

Tissue Preparation
We conducted the OFT 72 h after the last 50 mg/kg L-DOPA injection. The drugs were administered 3 h after the start of the OFT, and brains were removed 1 h later (i.e., brains were removed 4 h after the start of the OFT). The mice were divided into three groups: no injection group (n = 5 WT, n = 7 DD), quetiapine group (n = 5 WT, n = 6 DD), and 8-OH-DPAT group (n = 6 WT, n = 6 DD). These mice were deeply anesthetized with pentobarbital, first refluxed with phosphate-buffered saline (PBS), and then fixed transcardially with 4% paraformaldehyde (PFA) using a perfusion pressure pump. The brain was removed and immersed in 4% PFA overnight. The solution was replaced with PBS and stored at 4 • C. Paraffin-embedded tissue sections (5 µm-thick) were cut with a sliding microtome.

Immunohistochemistry
The paraffin-embedded sections were deparaffinized, rehydrated, and immersed in distilled water. The sections were then autoclaved in 0.01 M citrate buffer (pH 6.0) for antigen activation. The sections were immersed in 0.3% hydrogen peroxide to remove endogenous peroxidase and treated with 5% normal goat serum for blocking. The sections were then stained with polyclonal rabbit anti-c-fos antibody (Synaptic Systems, Goettingen, Germany; 1:5000 dilution) for 3 days at 4 • C. Three days later, we stained the brain slices with biotin-linked goat anti-rabbit polyclonal antibody (Vector Laboratories, Burlingame, VT, USA; 1:200 dilution). We then stained the brain slices with avidin-biotin complex (Vector Laboratories; 1:100 dilution). We then applied 0.06% 3,3 -diaminobenzidine solution with 0.00012% hydrogen peroxide. Finally, the slides were dewatered, permeabilized, sealed, and observed by microscopy.

Cell Counting
Immunostained slides were photographed using an inverted fluorescence phasecontrast microscope (BZX800, Keyence, Osaka, Japan). Fos-positive neurons in the MRN and RLi were counted using the hybrid cell counting function in BZX800 analyzer software. The number of cells was counted in the range of 100 µm × 400 µm for the MRN and 200 µm × 200 µm for the RLi.

Statistical Analysis
We calculated the sample size based on an alpha error of 0.05, power of 0.9, and effect size of 0.8 using G*Power 3.1.9.7 [32]. Based on the results of the power analysis, we used more than 10 mice for each locomotor test. The statistical analysis was performed using two-way analysis of variance. Individual post hoc comparisons were performed using the Scheffé test. Sample normality and homogeneity were assessed using the Kolmogorov-Smirnov test and Leven's test, respectively. Values of p < 0.05 were considered statistically significant. The data were analyzed using Bell-Curve for Microsoft Excel software (Social Survey Research Information 3.20, Tokyo, Japan).

Conclusions
In the present study, we found that quetiapine suppressed abnormal hyperlocomotion in DD mice. Quetiapine is often used clinically to treat PSDD, but its mechanism of action has not been clarified. The present findings suggest that 5-HT 1A receptors may mediate the therapeutic effects of quetiapine. An increase in activity in the MRN may be a pathological mechanism of PSDD.