FFAR1/GPR40 Contributes to the Regulation of Striatal Monoamine Releases and Facilitation of Cocaine-Induced Locomotor Activity in Mice

The free fatty acid receptor 1 (FFAR1) is suggested to function as a G protein-coupled receptor (GPR40) for medium-to-long-chain free fatty acids. Previous studies on the expression of FFAR1 revealed that the nigrostriatal region is one of the areas which express abundant FFAR1 mRNA/protein in the central nervous system (CNS). However, the role of FFAR1 in the CNS has been still largely unclarified. Here, we examined a possible functional role of FFAR1 in the control of extracellular concentrations of striatal monoamines and cocaine-induced locomotor activity. Microdialysis analysis revealed that the basal level of extracellular dopamine (DA) was significantly elevated, while the basal serotonin (5-HT) level tended to be reduced in the striatum of FFAR1 knockout (−/−) mice. Interestingly, local application of a FFAR1 agonist, GW9508, markedly augmented the striatal 5-HT release in FFAR1 wild-type (+/+) mice, whereas topical application of a FFAR1 antagonist, GW1100, significantly reduced the 5-HT release. However, the enhanced 5-HT release was completely lost in −/− mice. Although acute administration of cocaine enhanced the locomotor activity in both +/+ and −/− mice, the magnitude of the enhancement was significantly reduced in −/− mice. In addition, intraperitoneal injection of GW1100 significantly decreased the cocaine-induced locomotor enhancement. These results suggest that FFAR1 has a facilitatory role in striatal 5-HT release, and the evoked 5-HT release might contribute to enhance cocaine-induced locomotor activity.


INTRODUCTION
The free fatty acid receptor 1 (FFAR1) is a G-protein-coupled receptor (also known as GPR40), which is originally demonstrated to be abundantly expressed in pancreatic β-cells and activated by medium-to-long-chain (C12-C22) free fatty acids (FFAs) (Briscoe et al., 2003;Itoh et al., 2003;Kotarsky et al., 2003;Stoddart et al., 2008;Mancini et al., 2013). Since FFAs have long been recognized as important regulators of glucose homeostasis via their ability to stimulate insulin secretion in the presence of glucose, FFAR1 became a promising therapeutic target for type 2 diabetes treatment since its deorphanization, and a number of small-molecule FFAR1 agonists are under development as drugs for type 2 diabetes (Ghislain and Poitot, 2016;Hara, 2016;Kimura et al., 2020).
Previous studies, including our reports, have demonstrated the presence of FFAR1 in the human, primate, and rodent central nervous system (CNS) based on mRNA measurements and immunohistochemical analyses and suggested that FFAR1 plays important physiological roles in the CNS (Briscoe et al., 2003;Ma et al., 2007;Ma et al., 2008;Boneva and Yamashima, 2012;Nakamoto et al., 2012;Nakamoto et al., 2013;Zamarbide et al., 2014;Karki et al., 2015;Nakamoto et al., 2015;Nakamoto et al., 2017). For instance, we have demonstrated that hypothalamic FFAR1 exerts an antinociceptive effect through the facilitated release of endogenous opioid peptides (Nakamoto et al., 2013). In addition, we have further shown that FFAR1 is found to be expressed on descending noradrenergic and serotonergic neurons in the locus coeruleus and rostral ventromedial medulla (RVM), respectively, and the local application of a FFAR1 agonist, GW9508, into these areas evokes a descending endogenous pain control system (Nakamoto et al., 2015;Nakamoto et al., 2017). Furthermore, we have recently found that the disfunction of FFAR1 signaling also contributes to the emotional-related behaviors (Nishinaka et al., 2014;Aizawa et al., 2016;Aizawa et al., 2017;Aizawa et al., 2018) and FFAR1-deficient (FFAR1−/−) mice showed altered monoamine levels in several brain areas including hippocampus, midbrain, hypothalamus, and medulla oblongata (Aizawa et al., 2016). These results suggest that one of the functions of FFAR1 in the CNS might control the central monoaminergic system. Thus, to further pursue this hypothesis in this study, we have tested whether FFAR1 plays a role in the regulation of striatal monoamine release. Moreover, we analyzed cocaine-induced locomotor stimulation activity in both FFAR1−/− mice and mice treated with a FFAR1 antagonist to evaluate the functional significance of striatal FFAR1, since cocaine is a well-known uptake blocker of the monoamine transporters, and resultant increases in striatal monoamines are important for the cocaine-induced enhanced locomotor responses (Kelly and Iversen, 1976;Joyce et al., 1983;Reith et al., 1997;Cornish and Kalivas, 2001;Han and Gu, 2006;Lüscher and Ungless, 2006).

MATERIALS AND METHODS
Animals FFAR1−/− mice were generated by homologous recombination (Aizawa et al., 2016;Nakamoto et al., 2017). Briefly, exon 1 of the Ffar1 gene was replaced with a PKG-neo cassette. Frozen Ffar1 −/− fertilized oocytes were inoculated into pseudopregnant foster mother (ICR) at Center for Animal Resources and Development, Kumamoto University (CARD ID 1882) and bred at the Institute of Laboratory Animal Science Research Support Center, Kagoshima University. FFAR1−/− mice were backcrossed to C57BL/6J background for >10 generations and maintained by crossbreeding of heterozygous mice. Only male mice were used in this study. To confirm ablation of Ffar1, we performed PCR on genomic DNA from tail or ear auricle using the following primers: 40-S1: 5′ TAG GAC TGG CTT CTG GTG CT 3′, 40-AS2: 5′ CCT CCT GAG TTG TGG TGG AT 3′, 3 primerneo: 5′ GGC TAT TCG GCT ATG ACT GG 3'. This primer pair yielded a 1,166-bp DNA fragment for +/+ mice and a 1,300-bp fragment for −/− mice. Primers were obtained from Integrated DNA Technologies, KK (Tokyo, Japan).
We also employed the quantitative real-time PCR method (see below) to evaluate the presence or absence of FFAR1 in the striatum with +/+ mouse pancreas tissue samples as a positive control. As depicted in Supplementary information (Supplementary Figure S1), the transcript level of FFAR1 was high in the pancreas, but significant expression was also detected in the striatum, where, as expected, the expression was undetectable in FFAR1−/− mice. These results are consistent with our previous immunoblot analysis in the mouse CNS (Nakamoto et al., 2012) and qPCR analysis in the human brain by Briscoe et al. (2003).
Mice were housed under controlled temperature (24 ± 1°C) and humidity (55 ± 10%) with a 12 h light-dark cycle with food and water freely available. All mice used aged 8-22 weeks at the start of each experiment. The animal experiments were approved by the Animal Care Committees of Kagoshima University (approval no. MD16077) and were conducted in accordance with the ethical guidelines for the study of experimental pain in conscious animals of the International Association of the Study of Pain.

Open-Field Test
The open-field test was made of polyvinyl chloride plates and was 50 cm×50 cm×40 cm in size. Each FFAR1+/+ and −/− mouse was transferred to the center of the field, and its locomotor activity was measured for 5 min using a color tracking system (CompACT VAS; Muromachi Kikai, Tokyo, Japan) as described previously (Saegusa et al., 2000).

Cocaine-Induced Locomotor Activity
On the test day, FFAR1+/+ and −/− mice were habituated to the open-field test chamber for 63 min before cocaine (20 mg/kg) injection. The spontaneous locomotion was recorded for 3 min in every 10 min. After i.p. injection of cocaine, locomotor activity was recorded for another 33 min.
In the case of examining the effects of the FFAR1 antagonist (GW1100), vehicle or GW1100 (10 mg/kg) was i.p.-injected into FFAR1+/+ mice after the 63 min habituation, and locomotor activity was similarly measured for 33 min. Then, cocaine (20 mg/kg) was given and activity was monitored for a further 33 min. GW1100 was initially dissolved in S 99.9% DMSO (5 mg/ml) and then diluted with saline (1 mg/ml, final DMSO concentration ∼20%). Thus, ∼ 20% DMSO in saline was employed as vehicle control.

Quantitative Real-Time PCR
Total RNA was isolated from the striatal tissue including the area in vivo microdialysis experiments performed by using Sepasol RNA I Super G reagent (Nacalai Tesque, Kyoto, Japan) and treated with DNase (Promega, Madison, WI) to avoid DNA contamination. cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to manufacturer's instructions. Quantitative PCR was performed with Thunderbird SYBR qPCR Mix (Toyobo Life Science, Osaka, Japan) using a Thermal Cycler Dice (Real Time System TP800, Takara Bio Inc., Shiga, Japan). Amplification of target cDNA was normalized to GAPDH expression. Relative levels of target mRNA expression were calculated using the standard curve method. At least, two independent qPCR experiments of two individual striatum samples were performed. The sequences of primer pairs are described below. Primers were obtained from Integrated DNA Technologies, KK:

Statistical Analysis
Experimental data are expressed as mean ± SEM. Single comparisons were made using Student's two-tailed unpaired t-test corrected for unequal variance (Welch's correction) when appropriate. All remaining data were compared using one-way analysis of variance (ANOVA) with repeated measures followed by Dunnett's post hoc test or two-way repeated measures ANOVA, with genotype or drug treatment as the between-subject factor and time as the within-subject factor, followed when necessary, by Tukey-Kramer post hoc test. p < 0.05 was considered statistically significant.

Baseline Levels of Monoamines and Their Metabolites in the Striatum
To investigate whether FFAR1 indeed regulates monoamine levels, we employed in vivo microdialysis method in this study. At first, we examined baselines levels of extracellular DA, 5-HT and their metabolites within the striatum of awake normal FFAR1+/+ and −/− mice. We found that baseline DA level in the striatum was significantly higher in −/− mice compared with +/+ mice, while a trend for decreased 5-HT baseline levels was observed in −/− mice ( Figures 1A,B). The baseline levels of all DA metabolites, DOPAC, 3-MT, and HVA, also showed significant increases in −/− mice, although the level of 5-HT metabolite, 5-HIAA, was not changed (Figures 1C-F).

FFAR1 Regulated Extracellular 5-HT Level
The in vivo microdialysis experiments suggested that FFAR1 in fact directly/indirectly regulated at least striatal DA and 5-HT levels. To evaluate how FFAR1 control striatal DA and 5-HT levels, further in vivo microdialysis experiments were performed by in situ application of a FFAR1 agonist, GW9508, and an antagonist, GW1100 (Briscoe et al., 2006;Stoddart et al., 2007) ( Figure 2). Although we observed a slight transient reduction of DA level in FFAR1−/− mice after local application of GW9508 (100 μM in the perfusing Ringer's solution) (Figure 2A), GW9508 perfusion resulted in different changes in 5-HT release between FFAR1 +/+ and −/− mice ( Figure 2B). In +/+ mice, GW9508 significantly increased the 5-HT level in the striatum. Intriguingly, the stimulated release of 5-HT was almost completely lost in −/− mice. In contrast to what was found in the GW9508 application, local treatment of GW1100 significantly decreased the 5-HT level in +/+ mice ( Figure 2D). Despite the downregulation of 5-HT release by GW1100 being remarkable, DA level was not significantly affected by the FFAR1 antagonist ( Figure 2C).

Locomotor Activity in a Novel Environment (5 min Open-Field Test)
Thus, to further explore the functional significance of striatal FFAR1, we employed a cocaine-induced locomotor enhancement model in this study and examined the behavioral phenotype of FFAR1−/− mice. Moreover, we also tested the effects of GW1100 on cocaine-evoked locomotor enhancement. First, we checked baseline locomotor activity under a novel condition (Figure 3). Naïve FFAR1+/+ and −/− mice were placed in a testing chamber (open-field box) and locomotor activity was recorded for 5 min.
In accord with our previous report (Aizawa et al., 2016), FFAR1−/ − mice spent more time in the center region of the field when compared with +/+ mice ( Figure 3D and Supplementary Figure  S2), although there was no significant difference between +/+ and −/− mice in the distance traveled ( Figure 3B). In this study, we also analyzed additional parameters of the locomotor activity, the locomotion time, and speed and found that locomotion speed was slightly but significantly slower in −/− mice ( Figures 3A,C).
Cocaine-Induced Acute Locomotor Enhancement Was Attenuated in FFAR1−/− and FFAR1 Antagonist-Treated +/+ Mice Then, we next studied the functional role of FFAR1 by examining locomotor activity in response to acute cocaine injections in both FFAR1+/+ and −/− mice. Locomotor stimulation is one of the most characteristic effects of psychostimulants including cocaine (Kelly and Iversen, 1976;Joyce et al., 1983;Reith et al., 1997;Cornish and Kalivas, 2001;Han and Gu, 2006;Lüscher and Ungless, 2006). Both FFAR1+/+ and −/− mice underwent a 63 min habituation in a testing open-field box prior to cocaine (20 mg/kg) injection. Except for locomotion speed, there were no significant differences in the other three parameters (locomotion time, distance traveled, and time spent in the center region) during the habituation period (Figure 4 and Supplementary Figure S3). An acute administration of cocaine significantly increased locomotor activities in both +/+ and −/− mice. However, the overall locomotor responses to cocaine in terms of three parameters (locomotion time, distance traveled, and locomotion speed) were significantly reduced in −/− mice relative to +/+ mice ( Figures 4A-C). Interestingly, we observed a change in the pattern of horizontal locomotion. Although we could not detect a significantly increased percentage of the time spent in the center region in −/− mice before cocaine injection with this experimental condition (3 min measurement in every 10 min), we could detect significantly increased ambulation of −/− mice in the center area of the open-field chamber after cocaine injection ( Figure 4D).
As shown in Figure 5, pretreatment of FFAR1+/+ mice with GW1100 (10 mg/kg, i.p.) for 30 min also significantly reduced cocaine-induced enhancement of mobility time, distance travelled, and locomotion speed ( Figures 5A-C). Although we could not detect a significant increase in the time spent in the center region, there was a tendency toward an increment of this parameter ( Figure 5D).

Cocaine-Induced Increases in Extracellular DA and 5-HT Levels Were Apparently Normal in FFAR1−/− Mice
Since impaired cocaine action on striatal monoamine transmission could be responsible for the blunted locomotor responses seen in FFAR1−/− mice, we examined the ability of acute systemic cocaine injection to elevate extracellular DA and 5-FIGURE 2 | Reciprocal control of striatal 5-HT release by FFAR1 ligands. A FFAR1 agonist, GW9508, increased, but a FFAR1 antagonist, GW1100, decreased striatal 5-HT release without significant changes striatal DA release in FFAR1+/+ mice. However, the facilitatory effect of 5-HT release by GW9508 was completely lost in −/− mice. GW9508 (100 μM) and GW1100 (100 μM) were locally applied through a microdialysis probe as indicated by black lines. We also depicted control data from both +/+ and HT using in vivo microdialysis approach. As shown in Figures  6A,B, cocaine induced similar magnitude of potentiation in striatal DA and 5-HT levels in both +/+ and −/− mice, suggesting that the function of DA and 5-HT transporters appeared to be normal in −/− mice.

Expression Levels of Monoamine Transporters and DA Receptors in the Striatum
Finally, we examined the expression levels of DA and 5-HT transporters as well as D1 and D2 DA receptors using qPCR and western blot methods. Comparisons of the levels of these mRNAs and proteins between normal +/+ and −/− striata showed no significant differences (Figure 7).

DISCUSSION
The main findings of the current study include the following: 1) FFAR1 tonically regulated striatal 5-HT release: activation of FFAR1 facilitated 5-HT release, but inhibition of this receptor downregulated the release, and 2) FFAR1−/− mice and mice treated with the FFAR1 antagonist (GW1100) demonstrated reduced locomotor activity in response to cocaine administration. To our knowledge, this study provides the first direct in vivo evidence for FFAR1 as an important regulator of striatal 5-HT level. Although further extensive studies are necessary to delineate the mechanism underlying the reduced locomotor responses to cocaine, we could speculate that the facilitatory role of FFAR1 in the striatal 5-HT release would be likely to contribute to the enhancement of acute cocaineinduced locomotor activity. We found that FFAR1−/− mice displayed a significant increase in the basal level of extracellular DA, as well as its metabolites, with a trend for the decreased basal level of 5-HT. Thus, the FFAs-FFAR1 system was suggested to play an important role in striatal DA and 5-HT effluxes. In fact, previous studies using animals deficient in n-3 polyunsaturated FFAs reported that the deficiency enhanced anxiety-like behaviors in mice (Harauma and Moriguchi, 2011) and modified striatal DA availability or basal DA release in rats (Zimmer et al., 2000;Bondi et al., 2014), which may be at least partly consistent with both our previous (Aizawa et al., 2019) and present data. Conversely, n-3 FFAs, such as eicosapentaenoic acid and docosahexaenoic acid, were suggested to reduce stress and anxiety-like behaviors in animal studies (Song et al., 2007;Aizawa et al., 2019), and supplementation of n-3 FFAs reduced the risk of progression to psychotic disorder and psychiatric morbidity (Amminger et al., 2015). Moreover, our group recently demonstrated that repeated intracerebroventricular administration of GW9508 reduced the duration of immobility behavior in the forced swim test, suggesting that activation of brain FFAR1 may downregulate depression-related behavior (Nishinaka et al., 2014). However, it may be worth noting here that FFAR1−/− mice spent more time in the center region compared with +/+ mice in this open-field test (performed at Kagoshima University; Figure 3D), which concurs with our previous report performed at Kobe Gakuin University (Aizawa et al., 2016). Center versus surround occupancy and locomotion in open-field chambers is a measure of anxiety-like behavior and is sensitive to anxiolytic treatments (Prut and Belzung, 2003), although the interpretation of center/surround measure as an anxiety measure has also been a matter of debate due to the failure of effectiveness in several mouse strains (particularly C57BL/6J strain) to prototypical anxiolytic drugs (Thompson et al., 2015). The increase in basal DA and its metabolite levels observed in the naïve FFAR1−/− mice might reflect activation of DA neurons such as the increase in DA release, synthesis, and metabolism, and decrease in DA reuptake. However, we could speculate that the striatal DA reuptake activity would be apparently normal, since expression analysis of DA transporter suggested no alterations in FFAR1−/− mice, and cocaine increased DA levels to a similar extent as in +/+ mice (see below). However, further investigations are necessary to determine DA reuptake activity more precisely in −/− mice striatum.
The most important finding in this study is that activation of FFAR1 facilitated 5-HT release and inhibition of this receptor reduced the release in the striatum. Our previous study has demonstrated that FFAR1 colocalized with NeuN (a neuron marker) positive cells and also with tryptophan hydroxylase (a serotonergic neuron marker) positive cells in the RVM (Nakamoto et al., 2015). Thus, it would be possible that axon terminals derived from FFAR1-positive RVM serotonergic neurons contributed to the 5-HT release in the striatum. However, since subcellular localization of FFAR1 in the striatum has not been fully elucidated yet, anatomical data such as whether midbrain DA neurons are under the control of FFAR1-positive serotonergic neurons would be required in future experiments.
It has been known that the central 5-HT system is associated with the modulation (both inhibitory and excitatory) of DA neuronal activity, and both basal and psychostimulant-induced locomotor activity (see below). The extensive body of literature demonstrates the involvement of at least two members of 5-HT 2 receptor family, 5-HT 2A receptor and 5-HT 2C receptor, in the regulation of DA release (Higgins et al., 2013;Cunningham and    Anastasio, 2014; Howell and Cunningham, 2015). There is a general consensus that 5-HT 2A receptor activation stimulates but 5-HT 2C receptor activation inhibits DA release. For instance, although 5-HT 2A receptor stimulation clearly facilitates DA release in the mesocorticolimbic system in areas key for addiction, 5-HT 2A receptor antagonists have no effect on basal DA release in these areas, suggesting that the 5-HT 2A receptor is involved primarily in the control of phasic, not tonic, DA release. Meanwhile, administration of 5-HT 2C receptor agonists decreased, whereas 5-HT 2C receptor antagonist and inverse agonists increased basal firing rates of the ventral tegmental area (VTA) DA neurons and subsequent DA release within the nucleus accumbens (NAc). A later study found that 5-HT 2C receptor knockout mice had increased basal DA levels in the NAc and dorsal striatum, which correlated with the increased tonic activity in the nigral neurons (Abdallah et al., 2009). Thus, these considerations suggest that endogenous striatal FFAR1-evoked 5-HT release might control basal (tonic) DA release through 5-HT 2C receptors.
Another important finding of this study is that FFAR1−/− mice and mice i.p.-administered with the FFAR1 antagonist (GW1100) displayed a significantly reduced locomotor response to acute cocaine. It is well documented that increases in extracellular DA levels in the striatum are related to the psychomotor-stimulating effects of cocaine and other psychostimulants (see Introduction). This suggests that altered striatal DA responsivity may change the locomotion in these mice. However, contrary to our expectations, we found in the present study that FFAR1−/− mice also showed a similar magnitude of potentiation by cocaine in DA and 5-HT levels in the striatum. These data might rule out impaired activities of DA and 5-HT transporters in FFAR1−/− mice. Furthermore, qPCR and western blot analysis suggested that the expression levels of DA and 5-HT transporters, as well as those of D1 and D2 receptors, were not significantly changed in the striatum of FFAR1−/− mice. Although a further rigorous study is necessary for possible involvement of FFAR1 signaling system in cocaine-evoked responses, we could present one hypothesis that further elevation of extracellular DA in −/− mice whose baseline DA level was significantly increased compared to +/+ mice might produce impairment of downstream DA receptor signaling mechanisms and lead to attenuated cocaine-induced locomotor enhancement. Thus, we could postulate that one essential role of the striatal FFAR1-5-HT signaling system might be to adjust DA level within an appropriate range.
Recent preclinical studies have clearly indicated that 5-HT plays an important role in the abuse-related effects of cocaine (Cunningham and Anastasio, 2014). Although we could not exclude the involvement of other 5-HT receptor subtypes (see Filip et al., 2010, for review), several lines of evidence suggested that 5-HT 2A and 5-HT 2C receptors again may be potential targets to treat some aspects of cocaine abuse (Higgins et al., 2013;Howell and Cunningham, 2015). For instance, systemic administration of 5-HT 2A receptor antagonists block (McMahon and Cunningham, 2001;Fletcher et al., 2002;Filip et al., 2004) whereas a 5-HT 2A receptor preferential agonist DOI (2,5-dimethoxy-4iodoamphetamine) and virally mediated overexpression of 5-HT 2A receptors in the VTA enhance the locomotor-stimulant effects of cocaine (Filip et al., 2004;Herin et al., 2013). Conversely, studies in rodents have reliably found that 5-HT 2C receptor agonists attenuate (Grottick et al., 2000;Filip et al., 2004), whereas 5-HT 2C receptor antagonists enhance the behavioral effects of cocaine (Fletcher et al., 2002, Fletcher et al., 2006. Thus, although further research is still needed to evaluate the possible involvement of the 5-HT receptor system in the FFAR1-5-HT signaling, we currently hypothesize that 5-HT receptor activation evoked by FFAR1-induced 5-HT release would control the magnitude of cocaine-induced hyperlocomotion.
In conclusion, the present study suggests that FFAR1 plays an important role in 5-HT release and appears to exert substantial modulatory influence on DA level in the mouse striatum. Although future studies are necessary to elucidate the precise roles of FFAR1 in cocaine action including the locomotor responses, this study would promote a better understanding of the neurophysiological function of FFAR1 as a potential therapeutic target of cocaine abuse.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care Committees of Kagoshima University (approval no. MD16077).

AUTHOR CONTRIBUTIONS
YS carried out all experiments, performed the statistical analysis, and drafted the manuscript. TK conceived and participated in the design of the study, performed in vivo microdialysis studies, and wrote the manuscript. ST, RM, and YK carried out behavioral and immunoblot analysis. AH, KN, ST, KY, KA, AM, and TO participated in the design of the study and reviewed the manuscript.

FUNDING
This work was supported by JSPS KAKENHI Grant Numbers 16K10792 (TO, KA, AM, and TK) and 19K09512 (TO and TK).