Brain Research Bulletin 153 (2019) 59–73

Contents lists available at ScienceDirect

Brain Research Bulletin

journal homepage: www.elsevier.com/locate/brainresbull

Lactobacillus plantarum PS128 ameliorates 2,5-Dimethoxy-4- iodoamphetamine-induced tic-like behaviors via its influences on the T microbiota–gut-brain-axis

Jian-Fu Liaoa, Yun-Fang Chenga,b,e, Shiao-Wen Lic, Wang-Tso Leed, Chih-Chieh Hsua,e, ⁎ ⁎⁎ Chien-Chen Wua,e, One-Jang Jenge, Sabrina Wangf, , Ying-Chieh Tsaia,b, a Institute of Biochemistry and Molecular Biology, National Yang-Ming University, 155, Section 2, Linong Street, Beitou Dist., Taipei City 11221, Taiwan, ROC b Microbiome Research Center, National Yang-Ming University, 155, Section 2, Linong Street, Beitou Dist., Taipei City 11221, Taiwan, ROC c Molecular Medicine Research Center, Chang Gung University, No. 259, Wenhua 1st Rd., Guishan District, Taoyuan City 33302, Taiwan, ROC d Department of Pediatrics, National Taiwan University Hospital, No. 8, Zhongshan S. Rd., Zhongzheng Dist., Taipei City 100, Taiwan, ROC e Bened Biomedical Co., Ltd., 2F-2, No. 129, Sec. 2, Zhongshan N. Rd., Zhongshan Dist., Taipei City 104, Taiwan, ROC f Institute of Anatomy and Cell Biology, National Yang-Ming University, 155, Section 2, Linong Street, Beitou Dist., Taipei City 11221, Taiwan, ROC

ARTICLE INFO ABSTRACT

Keywords: We previously reported a novel psychobiotic strain of Lactobacillus plantarum PS128 (PS128) which could Microbiome-gut-brain-axis ameliorate anxiety-like& depression-like behaviors and modulate cerebral dopamine (DA) and serotonin (5-HT) Psychobiotic in mice. Here, we examine the possibility of using PS128 administration to improve tic-like behaviors by using a Lactobacillus plantarum PS128 5-HT2A and 5-HT2C receptor agonist 2,5-Dimethoxy-4-iodoamphetamine (DOI). PS128 was orally administered Dopamine to male Wistar rat for 2 weeks before two daily DOI injections. We recorded the behaviors immediately after the Tics second DOI injection and compared the results with control and haloperidol treatment groups. PS128 sig- Tourette syndrome nificantly reduced tic-like behaviors and pre-pulse inhibition deficit in a threshold-dose of 109 CFU per day. Brain tissue analysis showed that DOI induced abnormal DA efflux in the striatum and prefrontal cortex, while PS128 ingestion improved DA metabolism and increased norepinephrine (NE) levels in these two regions. In addition, PS128 ingestion increased DA transporter and β-arrestin expressions and decreased DOI-induced phosphorylation of DA and cAMP regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) at Thr34 and extracellular regulated protein kinases (ERK). PS128 ingestion also modulated peripheral 5-HT levels and shaped the cecal microbiota composition, which helps to alleviate DOI-induced dysbiosis. These results sug- gested that PS128 ameliorated DOI-induced tic-like hyper-active behaviors via stabilizing cerebral dopaminergic pathways through its modulation of host’s microbiota–gut–brain axis. Thus, we believe there are potentials for utilizing psychobiotics to improve syndromes caused by DA dysregulation in DA-related neurological disorders and movement disorders such as Tourette syndrome.

1. Introduction through neural, immune, and endocrine pathways, and theses bidirec- tional communication pathways constitute the microbiota–gut-brain- Over the past decade, a multitude of studies had revealed that gut axis (MGBA) which is vital for both physical and mental health (Collins microbiota communicates with the central nervous system (CNS) et al., 2012; Cryan and Dinan, 2012; van de Wouw et al., 2017).

Abbreviations: CNS, central nervous system; GI tract, gastrointestinal tract; DA, dopamine; 5-HT, serotonin; NE, norepinephrine; DOI, 2,5-Dimethoxy-4-iodoam- phetamine; Hal, haloperidol; CFU, colony-forming units; BMC, back muscle contraction; WDS, wet dog shake; PPI, prepulse inhibition; PFC, prefrontal cortex; DAT, dopamine transporter; DARPP-32, dopamine and cAMP regulated phosphoprotein of molecular weight 32 kDa; ERK, extracellular regulated protein kinases; TS, Tourette syndrome ⁎ Corresponding author. ⁎⁎ Corresponding author at: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, 155, Section 2, Linong Street, Beitou District, Taipei 11221, Taiwan, ROC. E-mail addresses: [email protected] (J.-F. Liao), [email protected] (Y.-F. Cheng), [email protected] (S.-W. Li), [email protected] (W.-T. Lee), [email protected] (C.-C. Hsu), [email protected] (C.-C. Wu), [email protected] (O.-J. Jeng), [email protected] (S. Wang), [email protected] (Y.-C. Tsai). https://doi.org/10.1016/j.brainresbull.2019.07.027 Received 25 April 2019; Received in revised form 21 July 2019; Accepted 23 July 2019 Available online 25 July 2019 0361-9230/ © 2019 Published by Elsevier Inc. J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Probiotics, live microorganisms that confer health benefits on the host, relationship between different neuromodulator systems (Handley and have wide application in fermented food products and health supple- Dursun, 1992; Bronfeld et al., 2013). In this regard, DOI had been used ments. Numerous studies have exploited the beneficial effects of pro- to generate sensorimotor gating deficit (Sipes and Geyer, 1994; Kehne biotics which include improvements in gastrointestinal (GI) tract dis- et al., 1996), stereotype behaviors, perseverative behaviors, and re- orders (McKernan et al., 2010; Liu et al., 2011), metabolic disorders gional contraction or reflex of muscles in rodents (Fone et al., 1991; (Huang et al., 2013), defecation impediments (Riezzo et al., 2018), and Wright et al., 1991; Dursun and Handley, 1996; Vickers et al., 2001). modulating immune systems (Mei et al., 2013; Liu et al., 2014). Pro- Furthermore, some animal studies had demonstrated that DOI appli- biotics could also benefit the homeostasis of MGBA of the host, which cation could increase dopaminergic neuron activity and DA release in could improve mental stabilities (Kim et al., 2018). For example, Bifi- the ventral tegmental area (VTA) and medial prefrontal cortex (mPFC) dobacterium infantis 35624 had been shown to modulate cerebral (Iyer and Bradberry, 1996; Bortolozzi et al., 2005; Pehek and Hernan, monoamines metabolism, alter peripheral interleukin-6 (IL-6) levels, 2015). On the other hand, DOI might inhibit striatal DA release (Ng and alleviate depression-like behaviors in the maternal separation (MS) et al., 1999) just like other 5-HT2C agonists (Alex et al., 2005; Burke model of rat (Desbonnet et al., 2010). A recent study showed that both et al., 2014). From our previous mice studies we had demonstrated that live and heat-killed Lactobacillus paracasei PS23 could reverse corti- PS128 administrate could influence brain monoamines mainly in the costerone-induced monoamines and neurotropic proteins reduction in PFC and striatum (Liu et al., 2016a, 2016c), therefore the DOI induction hippocampus, and alleviate anxiety- and depression-like behaviors in model seemed to be an ideal model to study the possible effects of mice (Wei et al., 2019). Furthermore, Lactobacillus rhamnosus GG had PS128 on irregular neurotransmissions, especially on mesocortical and been shown to reduce obsessive-compulsive disorder (OCD)-like beha- nigrostriatal DA pathways. vior in a 5-HT1A/1B receptor agonist RU-24969 treated mice model and In the current study we focused on assessing whether PS128 could the effects were comparable to selective serotonin reuptake inhibitor ameliorate the DOI-induced tic-like behaviors in rat and further ex- (SSRI) fluoxetine (Kantak et al., 2014). In view of their effects on amined the possible mechanisms underlying the effects of PS128 by modulating behaviors, neurotransmitters and neurotrophic factors, analyzing the changes in central monoamine system and peripheral 5- these probiotics strains are now named “psychobiotics” (Dinan et al., HT concentration. In addition, we further analyzing the gut microbiota 2013). Many believed that psychobiotics have great potential to im- in each experimental group to investigate the possible effects of PS128 prove psychiatric symptoms in patients with mental disorders (Zhou on the gut-brain axis. and Foster, 2015; Foster et al., 2016; Bermudez-Humaran et al., 2019). We previously reported that chronic administration of Lactobacillus 2. Material and methods plantarum PS128 (PS128), a psychobiotic isolated from Fu-tsai (Chao et al., 2009; Liu et al., 2015), could ameliorate anxiety-like behaviors in 2.1. Experimental animals and housing naïve mice and germ-free mice (Liu et al., 2016a, 2016c). In addition, in an early-life stress mice model oral administration of PS128 could im- Male Wistar rats (6 weeks old, weight 220–330 g) were purchased prove depression-like behaviors, modulate dopamine (DA) and ser- from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan). Rats were quar- otonin (5-HT) levels in prefrontal cortex (PFC) and striatum, and antined after transportation and then housed in the specific pathogen- change serum levels of corticosterone and IL-6 (Liu et al., 2016c). free room at the Laboratory Animal Center of National Yang-Ming Furthermore, in a recent clinical study on autism spectrum disorder University. The room was kept at a constant temperature (22 ± 1℃) (ASD) patients aged 7–15 in Taiwan, PS128 significantly ameliorated and humidity (55–65%) with a 12 h light/dark cycle and the water and anxiety, hyperactivity/impulsivity and opposition/defiance behaviors chow were provided ad libitum (Lab Diet Autoclavable Rodent Diet and improved the total score of the Swanson, Nolan, and Pelham-IV- 5010, PMI Nutrition International, MO, USA). All animal studies were Taiwan version (SNAP-IV) psychometric scale in younger population conducted in accordance with National Institutes of Health Guide for (aged 7–12) compared with the placebo group (Liu et al., 2019). In the Care and Use of Laboratory Animals. All animal experimental pro- another study of triathlon athletes, PS128 supplementation alleviated cedures were reviewed and approved by the Institutional Animal Care intensive exercise-induced oxidative stress and inflammatory state. and Use Committee, National Yang-Ming University (IACUC No. Moreover, PS128 supplementation substantially increased the plasma 10404080). branched-chain amino acids levels and improved exercise performance of these athletes (Huang et al., 2019). These mice and human studies of 2.2. Preparation of L. plantarum PS128 PS128 reveal not only its novel effects on physical and mental health but also its great potential for improving neurological disorders invol- PS128 was inoculated in Man Rogosa Sharpe (MRS) broth (Difco ving DA and 5-HT neurotransmissions. Hence, it warrants the further Corp., MD, USA), cultured at 37℃ for 18 h, and then harvested by exploration of the potential of PS128 administration on relieving neu- centrifugation at 6000×g for 10 min. The pellet was re-suspended in rological disorders associated with the imbalance of dopaminergic or MRS broth supplemented with 12.5% glycerol to a final concentration serotonergic neurotransmission such as Tourette syndrome (TS), at- of 1010 colony-forming units per milliliter (CFU/mL). The re-suspended tention deficit hyperactivity disorder (ADHD), obsessive compulsive solution was then aliquoted in freezertubes and stored at −80℃. Before disorders (OCD) and schizophrenia. Therefore, here we used a 5-HT2A the oral administration, aliquot of PS128 were thawed in 37℃ water and 5-HT2C receptor agonist 2,5-Dimethoxy-4-iodoamphetamine (DOI)- bath for 1 h and then centrifuged at 6000×g for 10 min. The super- induction model to study the potential of PS128 on alleviating tic-like natant was removed and the bacterial pellet was re-suspended in saline. behaviors and the underlying DA and 5-HT changes. Many studies had utilized pharmacological manipulations in animal 2.3. Experimental procedures and tic-like behavior counting models to study the relationship between various neuromodulators and neurological disorders. The 5-HT receptor (5-HTRs) agonists had been All rats were randomly assigned into different experimental groups used in preclinical studies as in vivo models for TS (Wright et al., 1991; and acclimatized for 1 week before the experiment procedure started. Handley and Dursun, 1992; Vickers et al., 2001; McCairn and Isoda, The PS128 group received daily oral gavage of PS128 of intended CFU 2013; Roessner et al., 2013), OCD (Flaisher-Grinberg et al., 2008; for 15 continuous days while control group received saline (1 mL per Kantak et al., 2014; Alonso et al., 2015) and schizophrenia (Aghajanian day) and positive control group received haloperidol (1 mg/kg in 1 mL and Marek, 2000; Weiss and Feldon, 2001; Valsamis and Schmid, 2011; saline per day; Sigma–Aldrich, MO, USA) during the same period. For Mouri et al., 2013) studies. Some authors suggested that the 5-HT-in- tic-like behaviors induction, rats were given intravenous (i.v.) injection duced behaviors in animal model of TS could highlight the complex of DOI (200 μg/kg; Sigma–Aldrich, MO, USA) as a primary treatment on

60 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Fig. 1. Effects of L. plantarum PS128 on tic-like behaviors and startle response. (A) Experimental procedure flowchart. We evaluated (B) back muscle contraction (BMC) counts, (C) wet dog shake (WDS) counts, (D) total tic-like behaviors (sum of BMC, WDS, and stereotyped behaviors), and (E) percentage of prepulse inhibition (PPI) after i.p. injection of either PBS or DOI on day 15 in each treatment group (n = 10/group). (F) The treatment effects of 3 different dosages of PS128 on total tic-like behaviors and (G) percentage of PPI (n = 6/group) were evaluated. Hal, haloperidol (1 mg/kg) group; 128 L, PS128 low dose (108 CFU/day); 128 M, PS128 medium dose (109 CFU/day), 128H, PS128 high dose (1010 CFU/day). Data were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. #p < 0.05, ###p < 0.001, compared to the Saline group; *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Saline + DOI group. day 14 and intraperitoneal (i.p.) injection of DOI (1 mg/kg in phos- were also collected for further analysis. phate-buffered saline, PBS) as the secondary treatment on day15 From the 35-min recording period we counted the numbers of back (Fig. 1A). The control group received PBS 100 μL (i.v.) on day 14 and on muscle contraction (BMC), wet dog shake (WDS) and stereotyped be- day 15 each. After the i.p. injection, rats were individually transferred haviors. The BMC were counted only if a clear-cut powerful contraction to another clear cage for video recording immediately. After video re- sweeping from the back of the neck to the tail was present (Fone et al., cording, all rats were anesthetized by i.p. injection of pentobarbital 1991; Cheer et al., 1999). The WDS were counted when a paroxysmal sodium (50 mg/kg) and placed in a clean cage until rats were un- shudder of the head, neck and trunk were present (Bedard and Pycock, conscious and unresponsive. Then the blood samples were collected by 1977; Fone et al., 1991; Handley and Dursun, 1992; Cheer et al., 1999). cardiac puncture and brain, intestinal tissues, and cecal content samples Perseverative head or body grooming or self-gnawing were counted as

61 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73 stereotyped behaviors. During the primary setup stage of DOI induction same volume. Before samples were injected into the HPLC-ECD system model, the counters were trained for explicitly identify the BMC, WDS, for analysis, all samples were first filtered by 4 mm syringe filter fitted and stereotyped behaviors. As for the formal experimental stage, the with 0.22 μm polyvinylidene difluoride (PVDF) membrane (Millex-GV, recording video file names were converted into random number-code Millipore, USA). Diluted filtrates (20 μL) were injected into the chro- and were analyzed by at least two different counters. matographic system. The concentrations of monoamines and their metabolites in the samples were interpolated using standards 2.4. Startle response recording for prepulse inhibition (PPI) evaluation (Sigma–Aldrich, MO, USA) ranging from 1 to 100 ng/mL with DataApex Clarity chromatography software (version 5.0.3.180, DataApex Ltd., We used the Med Associates Startle Reflex System (SOF-825 Startle Prague, Czech). Reflex, Med Associates, Virginia, USA) to evaluate the PPI. All experi- ment configuration and system setting were based on the manu- 2.6. Western blot analysis facturer’s guidelines and related references (Jaworski et al., 2005; Valsamis and Schmid, 2011). We give the rats three training sessions The striatum and PFC of rats were lysed by sonication in RIPA lysis before the oral administration of PS128, saline, or haloperidol. Rat was buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Sodium deoxycholate, 1% fixed in an acrylic animal holder on a calibrated platform in a sound- NP-40, 0.1% sodium dodecyl sulfate, SDS; pH = 7.5) containing pro- proof chamber with 68 dB ambient background noise. After 5 min ac- tease inhibitor (Roche cOmplete™, Mini, EDTA-free Protease Inhibitor climatization, rat was given 32 startle-eliciting sound bursts (120 dB, Cocktail, Merck, Darmstadt, German) and phosphatase inhibitor (Halt™ 40 ms). In 24 of the 32 startle stimuli we gave a prepulse stimulus Phosphatase Inhibitor Cocktail, Thermo Fisher, MA, USA). Tissue lysate 100 ms before the startle stimulus. The prepuse stimulus was a 20 ms was placed on ice and homogenized by vortex in a 5-min interval for pure tone of 75, 80 or 85 dB; there were 8 trials for each intensity. The 30 min, then the lysates were centrifuged at 12,000×g for 10 min. remaining 8 trials were startle stimulus alone trials. The inter-trial in- Protein concentration in supernatant were determined by the Bradford terval was set at 1 min. Then on day 15, 5 min after the i.p. injection of Assay (Bio-Rad, CA, USA) using bovine serum album as standards PBS or DOI we give the rats three more startle sessions which was the (Sigma–Aldrich, MO, USA) ranging from 2 to 10 μg/mL. Protein sample same as the training session. The startle responses were recorded and was loaded onto a 12% SDS-PAGE gel for electrophoresis then the se- calculated by the Startle Reflex Software. The PPI was evaluated by parated proteins were transferred onto the Roche PVDF membrane calculating the difference between the startle response without the (Merck, Darmstadt, German) and incubated with blocking buffer (5% prepulse stimulation (startle alone, S1) and the startle response with skim milk in tris-buffered saline containing 0.1% Tween-20, TBST) for prepulse stimulation (prepulse and startle, S2), the difference then used 1 h at room temperature. Subsequently, the membranes were probed to calculate the percentage change over S1. PPI (%) = [(S1 - S2) / S1] * with the primary antibodies overnight at 4 °C. The primary antibodies 100. used were anti-DAT (1:500), anti-D2R (1:1000), anti-pDARPP32-34 (1:500), anti-pDARPP32-75 (1:500), anti-pERK (1:1000), anti- 2.5. Quantification of monoamines and their metabolites by high- DARPP32 (1:1000) (Santa Cruz Biotechnology, CA, USA), anti-ERK performance liquid chromatography – electrochemical detection (HPLC- (1:1000), and anti-β-arrestin (1:1000) (Cell Signaling Technology, MA, ECD) USA). After washing twice with TBST, membrane was incubated with secondary antibody conjugated with horseradish peroxidase in blocking 2.5.1. Instrumentation and conditions buffer for 1 h at room temperature. Then the membrane was washed The HPLC-ECD system consisted of a S1130 HPLC pump system twice in TBST and the signal from the antibody-protein complex was (Sykam, Eresing, German), an on-line S5300 sample injector (Sykam, obtained by enhanced chemiluminescence (ECL) of Millipore Eresing, German), a DECADE II SDC electrochemical detector (Antec, Immobilon™ Western Chemiluminescent HRP Substrate (Millipore Zoeterwoude, Netherlands), and a reversed-phase column (Kinetex C18, Corporation, MA, USA). Images were analyzed using Luminescent 2.6 μm, 100 × 2.1 mm I.D.; Phenomenex, CA, USA). The potential for Image Analyzer (LAS-4000, FUJIFILM Holdings Corporation, Tokyo, the glassy carbon working electrode was set at +700 mV with respect Japan) and the NIH ImageJ software (https://imagej.nih.gov/ij/). to an Ag/AgCl reference electrode at room temperature (25 °C). For the quantification of dopamine (DA), 3,4-dihydroxyphenylacetic acid 2.7. Analysis of cecal microbiome by 16S rRNA pyrosequencing (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5- HIAA), and 5-hydroxytryptamine or serotonin (5-HT), the mobile phase 2.7.1. DNA extraction from cecal samples of rats was pumped at a constant flow rate of 0.2 mL/min. The mobile phase Bacterial DNA was extracted and purified by previously published contained 0.1 M NaH2PO4, 2 mM KCl, 0.74 mM 1-octanesulfonic acid methods with slight modifications (Nakayama et al., 2015; Li et al., (sodium salt), 0.03 mM ethylenediaminetetraacetic acid (EDTA), and 2017). Briefly, 100 mg of cecal sample was washed three times by PBS

8% methanol and was adjusted to pH 3.74 with H3PO4 (Cheng et al., and re-suspended in extraction buffer (100 mM Tris-HCl, 40 mM EDTA, 2000; Liu et al., 2016c). For the quantification of norepinephrine (NE) 1% SDS; pH 9.0) with 0.3 g of glass beads (0.1 mm in diameter) and and 3-Methoxy-4-hydroxyphenylglycol (MHPG), the mobile phase was 500 μLofbuffer-saturated phenol. The mixture was vigorously shaken pumped at a constant flow rate of 0.1 mL/min and contained 0.1 M for 30 s in the FastPrep FP120 homogenizer (Q-Biogene, CA, USA) at a

NaH2PO4, 2 mM KCl, 1.8 mM 1-octanesulfonic acid, 0.1 mM EDTA, and speed setting of 5.0 m/s. The cecal sample was then centrifuged at 10% methanol and was adjusted to pH 3.00 with H3PO4 (Bidel et al., 12,000×g for 5 min. After centrifugation, 400 μL of the supernatant 2016). was added into an equal amount of phenol-chloroform-isoamyl alcohol (25:24:1, v/v). The mixture was shaken again in the FastPrep FP120 at 2.5.2. Sample preparation and data analysis a speed of 4.0 m/s for 45 s then centrifuged again at 12,000×g for Rats were anesthetized by pentobarbital sodium injections (50 mg/ 5 min. The DNA was precipitated with 3 M sodium acetate (pH 5.4) and kg, i.p.). The striatum and prefrontal cortex (PFC) were separated from isopropanol. The DNA was then air-dried and dissolved in TE buffer the brain. The ileum and colon segments were pulverized and collected (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). The concentration of DNA was (approximately 0.1 g, wet weight). All rat tissue samples were lysed by adjusted to 10 ng/μL and the samples were stored at −20 °C. sonication in the perchloric acid (PCA) buffer (0.1% perchloric acid,

0.1 mM EDTA, and 0.1 mM Na2S2O5) then centrifuged at 12,000×g for 2.7.2. 454 pyrosequencing of 16S rRNA genes and QIIME pipeline 10 min and the supernatant were collected. The serum samples were sequences analysis mixed with PCA buffer containing 10% trichloroacetic acid (TCA) of the The V1-V2 region of bacterial 16S rRNA gene was amplified by the

62 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

first PCR reaction with a bacterial universal primer set (27F: 5′-AGR- Differences between experimental groups were analyzed by one-way GTTTGATYMTGGCTCAG-3′; 338R: 5′-TGCTGCCTCCCGTAGG analysis of variance (ANOVA) with Tukey’s post hoc test, the AGT-3′).The reaction mixture (25 μL) contained DNA (10 ng), 10 mM Kruskal–Wallis Rank Sum Test (Alpha diversity), or the Analysis of si-

Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 μM of each deox- milarity (ANOSIM) test (Beta diversity); p < 0.05 was considered sta- ynucleoside triphosphate (dNTP), 5 pmol of each primer, and 0.625 U tistically significant. ExTaq HS (Takara Bio, Shiga, Japan). The first PCR condition was as follow: 98℃ for 2.5 min; 15 cycles of 98℃ for 15 s, 50℃ for 30 s, and 3. Results 72℃ for 20 s; and finally 72℃ for 5 min. The DNA amplicons from the first PCR reaction were barcoded by second PCR reaction with bar- 3.1. L. plantarum PS128 ameliorates tic-like behaviors and rescues the PPI coding primer set of 27Fmod with 128 different 10-bp barcode se- deficits in the DOI-induced rat model of TS quence tags (Nakayama, 2010) and 338R in the reaction mixture. The reaction mixture (50 μL) contained DNA (10–100 ng), 10 mM Tris-HCl We utilized the DOI-induced tic-like behaviors model by injecting

(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 10 pmol of DOI (1 mg/kg, i.p.) to rat (Bedard and Pycock, 1977; Fone et al., 1991; each primer, and 1.25 U Ex Taq HS. The PCR condition was set as Wright et al., 1991; Cheer et al., 1999; Tizabi et al., 2001). We observed follow: 98℃ for 2.5 min; 20 cycles at 98℃ for 15 s, 54℃ for 30 s, and remarkable BMC responses 5 min after injection. Intermittent WDS and 72℃ for 20 s; and finally 72℃ for 5 min. The final PCR products were a few stereotype behaviors like grooming were also noted during the purified by QIAquick PCR Purification Kit (Qiagen, CA, USA) according 60 min observation period (Fig. S1A & B). With the intention to achieve to the manufacturer’s protocol. The DNA amplicons were sequenced on stabile behavioral induction, we administrated daily DOI (1 mg/kg, i.p.) the genome sequencer (GS) FLX 454 pyrosequencer (Roche, Basel, injection for four days and recorded the tic-like behaviors every day Switzerland) at the Veteran Yang-Ming (VYM) Genome Research Center immediately after each injection. We noticed that both BMC and WDS of National Yang-Ming University. occurrence were significantly enhanced in the 2nd day compared with The 16S rRNA gene sequences were processed by using the the 1st day while the tic-like behavior counts showed no significant Quantitative Insights Into Microbial Ecology (QIIME v1.9.0) pipeline to differences among the 2nd, 3rd and 4th day, indicating that two daily classify microbial constituents and compare memberships between DOI injections could successfully induce stable tic-like behaviors (Fig. samples (Caporaso et al., 2010). The following quality check para- S1C & D). Following this 2-day continuous injection protocol, we also meters were used: minimum quality score of 27, no primer mismatch, tested the combinations of different injection methods, i.e. i.v. + i.v., read length of 300 to 400 bp, maximum 3 ambiguous bases, and 6 bases i.v. + i.p., i.p. + i.p. We found that the i.v. + i.p. group showed more in homopolymer runs. A total of 193,711 sequences were used to significant tic-like behavioral expressions than other groups (Fig. S1E). construct operational taxonomic units (OTUs) consisting of sequences Therefore, we employed the two-time DOI-induction method (1st-day with 97% sequence identity. Chimeric sequences were removed by i.v. 200 μg/kg + 2nd-day i.p. 1 mg/kg) and recorded the tic-like be- ChimeraSlayer (Haas et al., 2011) and OTUs comprising fewer than haviors or startle responses immediately after the 2nd-day injection to three reads were excluded from further analyses (2854 OTUs). As a evaluate the effects of PS128 administration (Fig. 1A). Haloperidol, a result, 1391 OTUs with quality-filtered sequences were obtained. Tax- DA antagonist commonly used in tics treatment, was administrated in onomy classification was assigned according to the Greengenes re- another group of rat by oral gavage (1 mg/kg, daily) as the positive ference sequence database (gg_13_5) (DeSantis et al., 2006) with a control. In our preliminary experiments we already demonstrated that confidence threshold value of 80% with the uclust consensus taxonomy the haloperidol treatment could mitigate the tic-like behaviors and PPI assigner (Edgar, 2010). The bacterial composition of each sample was deficit (Fig. S1F & G, Fig. 1E), which is consistent with previous studies determined for each taxonomic level by applying the summarize_tax- (Handley and Dursun, 1992; Gaynor and Handley, 2001). a_through_plots.py function of QIIME to the OTU table with the as- The two-time administration of DOI successfully induced prominent signed taxonomy dataset. BMC (F(4, 44) = 42.5, p < 0.001, Fig. 1B) and WDS (F(4, 45) = 6.63, p < 0.05, Fig. 1C). We quantify the total tic-like behaviors as the sum of 2.7.3. Principal component analysis BMC, WDS, and the stereotype behavior counts (Fig. 1D). DOI induction

The bacterial family diversity of each sample was analyzed based on also reduced the percentage of PPI (F(4, 32) = 13.3, p < 0.001, Fig. 1E). relative abundance of each family by principal component analysis Oral gavage of PS128 (1010 CFU, daily) alleviated the DOI-induced

(PCA). The R package (ggbiplot) was used to generate PCA plots by BMC (F(4, 44) = 42.5, p < 0.01), total tic-like behaviors (F(4, 51) = 22.4, using the first two principal components according to group. p < 0.01), and the reduction on percentage of PPI (F(4, 32) = 13.3, p < 0.05) but not on WDS (Fig. 1B–E). Oral gavage of PS128 of same 2.7.4. Alpha- and beta-diversities of bacterial communities dosage to normal rats did not produce any abnormal behaviors com- Alpha diversity (as measured by the number of species observed, the pared with the saline control group (Fig. 1B–E). We further evaluated phylogenetic diversity whole-tree, and Shannon indices) and Beta di- the effective dosage by daily oral gavage of low (108 CFU), medium versity were estimated with QIIME. The average values of the number (109 CFU), and high (1010 CFU) doses of PS128 and found that both of observed OTUs, the phylogenetic diversity whole tree, and the medium and high dose groups showed improvement on total tic-like

Shannon indices over 10 iterations were calculated for each rarefied behaviors (F(5, 63) = 13.2, p < 0.01, p < 0.01, respectively) and PPI OTU composition by using 1000 reads per sample. Beta diversity ana- deficits (F(5, 31) = 7.98, p < 0.05, p < 0.05, respectively) whereas the lysis was performed using phylogenetic distance matrices simulated by low dose group did not show any obvious effect on DOI-induced be- UniFrac analysis (Lozupone and Knight, 2005). To visualize bacterial haviors (Fig. 1F–G). community populations between groups, a PcoA plot was generated by using the make_2d_plots script bundled with QIIME. The QIIME script 3.2. L. plantarum PS128 modulates the metabolism of monoamines in the compare_categories using an Analysis of similarity (ANOSIM) (Clarke, striatum and prefrontal cortex 1993) test was used to consider whether there were significant differ- ences in sample groupings using distance matrices. To investigate the effects of PS128 on the aminergic neuro- transmitters in brains of normal and DOI-treated rats, we used HPLC- 2.8. Statistical analysis ECD to measure levels of monoamines and their metabolites in the striatum and prefrontal cortex (PFC). The results showed that PS128

All data were expressed as mean ± SEM. All statistical analyses ingestion alone could increase NE (F(4, 45) = 6.51, p < 0.001) and were performed using Prism (version 7, Prism, CA, USA) or R software. caused a lower NE turnover rate (F(4, 44) = 3.57, p < 0.05) in the

63 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Table 1 Monoamines and their metabolites in the striatum and prefrontal cortex.

Group Saline PS128 DOI (+)

Saline Hal PS128

Monoamine and metabolites (ng/g wet tissue) in striatum DA 10209 ± 560 10327 ± 921 7056 ± 486## 5651 ± 675## 11080 ± 850** DOPAC 1172 ± 86 1027 ± 99 883 ± 102 1274 ± 136 1492 ± 223** HVA 534 ± 52 568 ± 50 481 ± 40 849 ± 115* 847 ± 110* 5-HT 307 ± 20 342 ± 31 315 ± 27 311 ± 24 334 ± 20 5-HIAA 354 ± 24 393 ± 35 441 ± 30 382 ± 31 433 ± 49 NE 241 ± 32 585 ± 102### 365 ± 51 354 ± 31 539 ± 22## MHPG 49 ± 14 43 ± 9 41 ± 7 33 ± 5 41 ± 8

Monoamine turnover ratio (%) in the striatum (DOPAC + HVA) : DA 18.3 ± 0.7 16.9 ± 0.8 21.6 ± 0.8 39.0 ± 2.91* 25.4 ± 4.1 5-HIAA : 5-HT 122.4 ± 12.6 120.1 ± 12.1 131.1 ± 18.8 122.8 ± 10.7 125.4 ± 16.2 MHPG : NE 19.2 ± 3.1 8.3 ± 2.1# 13.8 ± 4.1 10.2 ± 1.8 8.1 ± 1.8#

Monoamine and metabolites (ng/g wet tissue)in the prefrontal cortex DA 147 ± 18 193 ± 32 324 ± 37## 321 ± 40## 176 ± 20* DOPAC 29 ± 3 37 ± 8 33 ± 6 77 ± 6###,*** 63 ± 10#,* HVA 27 ± 2 35 ± 4 36 ± 2 88 ± 6###,*** 54 ± 10# 5-HT 215 ± 34 232 ± 23 203 ± 27 223 ± 17 252 ± 26 5-HIAA 222 ± 38 226 ± 31 242 ± 49 187 ± 21 250 ± 37 NE 169 ± 7 195 ± 8# 249 ± 12## 179 ± 7* 207 ± 6# MHPG 22 ± 2 18 ± 2 18 ± 1 19 ± 2 21 ± 3

Monoamine turnover ratio (%) in the prefrontal cortex (DOPAC + HVA) : DA 44.2 ± 2.4 37.8 ± 4.8 27.8 ± 2.1# 62.2 ± 4.6#,*** 49.0 ± 3.7** 5-HIAA : 5-HT 103.3 ± 9.2 96.8 ± 9.2 120.7 ± 18.8 84.9 ± 8.8 94.9 ± 7.8 MHPG : NE 13.6 ± 1.1 9.8 ± 0.8## 7.2 ± 0.7### 11.5 ± 0.6** 9.6 ± 0.7##

Concentrations of monoamines (ng/g wet tissue), metabolites (ng/g wet tissue), and turnover ratio (%) are expressed as mean ± SEM. DA, dopamine; DOPAC, 3,4- dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HT, serotonin or 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; NE, norepinephrine; MHPG, 3- Methoxy-4-hydroxyphenylglycol; Hal, haloperidol (1 mg/kg) group. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. #p < 0.05, ##p < 0.01, ###p < 0.001, compared to the Saline group; *p < 0.05, **p < 0.01, ***p < 0.001, compared to the Saline + DOI group (n = 10/group). striatum compared with the Saline group (Table 1). DOI induction re- pathways. Compared with the Saline group, the DOI injection increased duced the striatal DA level (F(4, 44) = 11.3, p < 0.01) which was re- the levels of phosphorylated ERK (p-ERK, F(4, 29) = 8.16, p < 0.05) and versed by PS128 ingestion (F(4, 44) = 11.3, p < 0.01) but not by halo- DARPP-32 at Thr34 (p-Thr34-DARPP-32, F(4, 31) = 5.67, p < 0.05), but peridol treatment (Table 1). PS128 ingestion also increased the striatal did not affect the DA transporter (DAT), D2R, and β-arrestin expression

DOPAC (F(4, 45) = 2.82, p < 0.01), HVA (F(4, 45) = 4.92, p < 0.05), and levels in the striatum (Figs. 2A–D&4 A & C). Both PS128 and halo- NE levels (F(4, 45) = 6.51, p < 0.01) of DOI-treated rats, whereas ha- peridol treatments reversed the effects of DOI on ERK (F(4, 29) = 8.16, loperidol increased the striatal DOPAC (approaching statistical sig- p < 0.05, p < 0.05, respectively, Fig. 4A) and DARPP-32 Thr34 phos- nificance), HVA (F(4, 45) = 4.92, p < 0.05)and DA turnover ratio (F(4, phorylation (F(4, 31) = 5.67, p < 0.05, p < 0.05, respectively, Fig. 4C). 45) = 14.4, p < 0.05) compared with the Saline + DOI group (Table 1). PS128 treatment also increased the DAT (F(4, 37) = 4.76, p < 0.05, In the PFC, PS128 ingestion alone slightly increased DA and NE and p < 0.05, respectively, Fig. 2A & B) and β-arrestin levels (F(4, decreased NE turnover ratio (F(4, 45) = 9.72, p < 0.05) compared with 37) = 5.93, p < 0.05, p < 0.05, respectively, Fig. 2D) in the striatum of the Saline group (Table 1). DOI induction significantly increased DA both normal and DOI-treated rats. In the PFC, similar treatments effects

(F(4, 43) = 7.39, p < 0.01) and NE levels (F(4, 45) = 13.7, p < 0.01) and of haloperidol and PS128 on the levels of DAT, β-arrestin, p-ERK, and p- decreased the turnover rates of DA (F(4, 45) = 12.1, p < 0.05) and NE Thr34-DARPP-32 were observed in the DOI-treated rats (Figs. 3 & 4 B& (F(4, 45) = 9.72, p < 0.001) in the PFC (Table 1). Haloperidol treatment D). However, only PS128 administration restored DOI-induced reduc- did not reverse the DOI-induced DA elevation, but showed a reduction tion of phosphorylated DARPP32 at Thr75 (p-Thr75-DARPP-32, F(4, in NE (F(4, 45) = 13.7, p < 0.05) compared with the Saline + DOI 31) = 16.2, p < 0.05) in the PFC, but not haloperidol (Fig. 4D). group. The haloperidol treated DOI group (Hal + DOI) also showed increased DOPAC (F(4, 43) = 8.70, p < 0.001), HVA (F(4, 44) = 16.2, 3.4. L. plantarum PS128 modulates the level of peripheral 5-HT p < 0.001) and turnover rates of DA (F(4, 45) = 12.1, p < 0.001) and NE (F(4, 45) = 9.72, p < 0.01, Table 1). PS128 ingestion on the other hand, Recent studies have revealed that gut microbiota regulates en- suppressed DA elevation in DOI-treated rats (F(4, 43) = 7.39, p < 0.05). dogenous 5-HT biosynthesis in the gut, which might be an important However, PS128 + DOI group also showed increased DOPAC (F(4, player in the MBGA pathway (O’Mahony et al., 2015; Reigstad et al., 43) = 8.70, p < 0.05), HVA (approaching statistical significance), and 2015; Yano et al., 2015). In order to investigate effects of PS128 on the DA turnover ratio (F(4, 43) = 7.39, p < 0.01) compared with the peripheral serotonergic system, we measured the levels of 5-HT in the Saline + DOI group (Table 1). There was no significant difference in the ileum, colon, and serum following PS128 administration in normal and 5-HT, 5-HIAA, and 5-HT turnover ratios among all groups in both DOI-treated rats. We found that the oral gavage of PS128 clearly in- striatum and PFC. creased the levels of 5-HT in the ileum (F(4, 31) = 13.5, p < 0.001), colon (F(4, 38) = 7.62, p < 0.001), and serum (F(4, 29) = 3.63, p < 0.05) 3.3. L. plantarum PS128 increases the level of DAT and β-arrestin and in normal rats (Fig. 5A–C). The peripheral 5-HT levels were not affected regulates DA-related signaling transduction in the striatum and PFC of rats by DOI treatment in normal rat. However, PS128 administration did not increase the peripheral 5-HT level in DOI-treated rats, whereas the

Since the oral gavage of PS128 mainly regulated the metabolism of haloperidol apparently increased the 5-HT level in the ileum (F(4, DA (Table 1), we next examined changes in the DA transmission 31) = 13.5, p < 0.001) but not in the colon and serum of DOI-treated

64 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Fig. 2. DA transmission- or signaling-associated protein expression levels in the striatum. (A) The representative western blots of striatal DAT, D2R and β-arrestin expression. The quantification of (B) DAT, (C) D2R and (D) β-arrestin expression level in the striatum were analyzed by western blot experiments. Hal, haloperidol (1 mg/kg) group; CON, saline-fed control group. The fold-changes over the Saline group were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. #p < 0.05 compared to the Saline group (n = 10/group). rats (Fig. 5A–C). the relative abundance of each bacterial family, we further demon- strated the principal component analysis (PCA) could decompose the data on bacterial composition into two factors that explained 81.2% of 3.5. L. plantarum PS128 modulates the cecal microbiota of rats and the variance. Principal component 1 (PC1) was heavily loaded with alleviates the DOI-induced dysbiosis Prevotellaceae, whereas principal component 2 (PC2) was heavily loaded with Bacteroidaceae and negatively loaded with Lactobacillaceae For cecal microbiota analysis, the OTUs were classified into known (Fig. 6B). Samples from the Saline group and the PS128 + DOI group taxa (13 phyla, 22 classes, 35 orders, 59 families, 70 genera, and 40 were distributed equally in the four quadrants of the biplot, while species) and unclassified groups. At the phylum level, the PS128 treated samples from the Saline + DOI group tended to form a cluster in the group showed a significant elevation in the Proteobacteria population PC1-negative and PC2-negative regions. Samples from the PS128 group fi (F(4, 30) = 9.59, p < 0.05) and a signi cant reduction both in the and the Hal + DOI group were distributed in the PC2-positive and PC2- Firmicutes/Bacteroidetes ratio (F/B ratio, F(4, 27) = 3.59, p < 0.05) and negative regions, respectively (Fig. 6B). Further species diversity ana- the Bacteroidetes/Proteobacteria ratio (B/P ratio, F(4, 29) = 6.88, lysis showed that PS128 administration and DOI treatment led to lower p < 0.05) among all rat groups (Table 2). The PS128 + DOI group also and higher Alpha diversity indices respectively than saline group, al- had a lower F/B ratio compared with both the Saline and Saline + DOI though these differences did not reach a statistical significance. Both groups (approaching statistical significance). The B/P ratio was also the PS128 and PS128 + DOI groups showed lower indices compared increased in the Hal + DOI group compared with the Saline + DOI with the Saline + DOI group (Kruskal–Wallis Rank Sum Test, p < 0.05, group (F(4, 29) = 6.88, p < 0.05, Table 2). p < 0.05, respectively), while the Hal + DOI group did not lead to a At the family level, we observed seven predominant bacterial po- significant change (Fig. 6C). The weighted UniFrac analysis indicated pulations among all rat groups (Table 2 & Fig. 6A). The PS128 treated that the samples from the five experimental groups showed significant group exhibited significantly more Bacteroidaceae compared with the differences (Fig. 6D, ANOSIM; p < 0.05). Differences in the microbiota Saline group (F(4, 30) = 3.96, p < 0.05, Table 2). In the DOI treated communities between the rat groups could also be observed in the PCoA groups, both the Hal + DOI and PS128 + DOI groups showed a sig- plots, which also showed that samples from the PS128 and nificant increase in Prevotellaceae compared with Saline + DOI group PS128 + DOI groups tended to form a cluster (Fig. 6D). (F(4, 28) = 6.03, p < 0.001, p < 0.05, respectively, Table 2). Further- more, both the PS128 and PS128 + DOI groups showed a significant decrease in Lachnospiraceae compared with the Saline + DOI group (F(4, 4. Discussion 29) = 9.56, p < 0.001, p < 0.05, respectively, Table 2). The proportions of these predominant families also tended to display an equal dis- In the current study we used DOI induction model to assess the tribution only in the Saline + DOI group (Table 2 & Fig. 6A). Based on potential of using the prominent psychobiotic strain Lactobacillus

65 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Fig. 3. DA transmission- or signaling-associated protein expression levels in the prefrontal cortex. (A) The representative western blots of striatal DAT, D2R and β-arrestin expression. The quantification of (B) DAT, (C) D2R and (D) β-arrestin expression level in the prefrontal cortex were analyzed by western blot experiments. Hal, haloperidol (1 mg/kg) group; CON, saline-fed control group. The fold-changes over the Saline group were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. #p < 0.05 compared to the Saline group; *p < 0.05 compared to the Saline + DOI group (n = 10/group). plantarum PS128 to alleviate tic-like hyper-active behaviors and neu- essential to ingest certain amount of live PS128. rotransmissions in rat. We had reliably observed BMCs, WDSs, stereo- The 5-HT-associated models are often used in psychopharmacolo- type behaviors and PPI deficit after two-daily DOI injection and these gical studies to explore the complex relationships between different behaviors could be ameliorated by PS128 administration and haloper- monoamine systems (Iyer and Bradberry, 1996; Gobert and Millan, idol treatment (Fig. 1A–E). Our DOI-induction procedure could induce 1999; Bowers et al., 2000; Burke et al., 2014). Brain monoamine ana- perseverative behaviors, regional contraction or reflex, stereotype be- lysis in our DOI-induction model revealed that after DOI induction the haviors, and sensorimotor gating deficits in rat. Based on the pervious DA efflux in PFC was increased whereas in the striatum the DA efflux animal studies and current results we believe that pharmacological was decrease (Table 1), suggesting both the mesocortical and nigros- manipulation by DOI injection could be used to mimic neurological triatal DA pathways were involved and regulated differently (Iyer and syndromes caused by irregular neurotransmissions (Handley and Bradberry, 1996; Bortolozzi et al., 2005; Pehek and Hernan, 2015). Dursun, 1992; Cheer et al., 1999; Gaynor and Handley, 2001; Tizabi These changes might underlie the tic-like hyper-active behaviors and et al., 2001; Hayslett and Tizabi, 2003; Flaisher-Grinberg et al., 2008; sensorimotor gating deficits (Sipes and Geyer, 1994; Bowers et al., Malkova et al., 2014). Therefore, the DOI-induction model could serve 2000; Wong et al., 2008; Pehek and Hernan, 2015). On the other hand, as a useful rodent model to study the potentials of using psychobiotic haloperidol treatment had no significant effects on DOI-induced DA strains to ameliorate the neurological syndromes caused by aberrant elevation or reduction but showed elevated DA metabolites in both the neurotransmission. The major concern of using live bacteria adminis- striatum and PFC, suggesting haloperidol exerted its effects by in- tration to alleviate symptoms is safety and the effective dosage on creasing the DA turnover rate (Table 1). In contrast, PS128-fed rats not normal physiology. In our dose-response trial, we found that once the only showed elevated DA metabolites, but also suppressed the DOI-in- PS128 ingestion dosage is 109 CFU/day or over, the amelioration effects duced DA changes, which implied that PS128-treated rats had a more on DOI-induced behaviors and deficit became apparent. In addition, the stable DA system than saline-treated rats in response to DOI stimulation effects of 109 and 1010 CFU/day treatments were similar, suggesting (Table 1); and these effects might underlie the behavioral improvement. that the efficacy of PS128 is not dose-dependent, but more like We also found that PS128 ingestion alone could significantly increase threshold dependent (Fig. 1F–G). We had previously demonstrated that the NE levels in both the striatum and PFC, however there was no sy- it was safe to use PS128 at a 109 CFU/day dosage, and the equivalent nergistic effect on DOI-induced NE elevation in the PFC (Table 1). The heat-killed PS128 did not produce any improvement effects (Liu et al., DOI-induced changes in the NE system might participate in modulating 2016a). We speculated that the novel psychotropic effects of PS128 the locomotor activity and sensorimotor gating deficits (Fishman et al., might be originated from the effects of its symbiosis in GI tract of the 1983; Oades et al., 1986; Mitchell et al., 2006) however, since PS128 host, but not mediated by its structural components such as surface could relieve excessive hyperkinetic behaviors without changing DOI- exopolysaccharide. Therefore, in order to show its beneficial effects it is induced NE elevation, the improvement effect is probably not mediated

66 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Fig. 4. DA signaling-associated kinase protein phosphorylation levels in the striatum and prefrontal cortex. The representative western blot and quantification of the ERK phosphorylation level in the striatum (A) and prefrontal cortex (B). Furthermore, the representative western blot and quantification of the DARPP-32 phosphorylation level in the striatum (C) and prefrontal cortex (D). The kinase proteins phosphorylation levels were analyzed by western blot experiments. Hal, haloperidol (1 mg/kg) group; CON, saline-fed control group. The fold-changes over the Saline group were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. #p < 0.05 compared to the Saline group; *p < 0.05 compared to the Saline + DOI group (n = 10/group). by the NE system. Considering that DA is the direct precursor of NE, the occurrence of these two pathways contributes to the signaling diversity increase in NE might be a consequence of DA elevation caused by DOI of DRs, mediating several signaling regulations under different condi- and PS128 ingestion through independent mechanisms (Iyer and tions and produce different period of activation (Del’guidice et al., Bradberry, 1996; Gobert and Millan, 1999; Bortolozzi et al., 2005; Liu 2011; Urs et al., 2016). Since, the DRs downstream kinases such as et al., 2016a, 2016c). Although we need more studies to clarify the DARPP-32 and mitogen-activated protein kinase (MAPK) can act as exact mechanisms underlying the interactions between these mono- integrators and coincidence detectors during the DRs signaling or the amine systems, our results nevertheless suggested that psychobiotic activation of other neurotransmitter systems (Svenningsson et al., 2004; PS128 could assist the CNS function of the host to cope with abnormal Valjent et al., 2005), therefore we also examined D2R and the DRs neurotransmission and ameliorate behavioral ailments. downstream kinases in the striatum and PFC in each experimental The expression of DAT could directly affect DA synaptic levels, group. PS128-fed rats with or without DOI induction all showed greater therefore DAT plays a key role in regulating DA transmission. On the DAT expression in the striatum and PFC compared to saline-fed con- receiving side, the D2R is widely found in both medium spiny neurons trols; D2R expression, on the other hand, did not show significance (MSN) and DA neurons in the basal ganglia, and is an important difference between each groups (Figs. 2A–C&3 A–C). The DAT ele- treatment target in disorders exhibiting aberrant DA functions such as vation in PFC and striatum of PS128-fed rats could explain the in- TS and schizophrenia (Minzer et al., 2004; Masri et al., 2008; Urs et al., creased DA metabolites after DOI induction (Table 1) and no tic-like 2016, 2017). The signaling pathway of DRs is mainly through distinct G hyper-active behaviors in PS128-alone group (Figs. 1B–D, 2 B&3 B). protein-dependent and β-arrestin-mediated pathways. The co- The higher DAT expression could also explain why in our previous

67 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

expression and reversed the DOI-induced p-Thr75-DARPP changes in both the striatum and PFC (Figs. 2D, 3 D&4 D). Besides its desensiti- zation role, β-arrestin also serves as polyvalent GPCR-associated scaf- folding proteins, having complex and multiple functions in regulating the final outcome of monoamine receptor stimulation especially for DRs (Del’guidice et al., 2011; Urs et al., 2016). It is possible that both the DAT and β-arrestins elevation might be the consequence of dopami- nergic activation during the long-term PS128 ingestion. These results might imply that PS128 ingestion could help the host to establish a more flexible and adaptive DA system in response to DOI challenges. In the MGBA, endocrine, immune, and nervous systems all have been considered as possible mediators for psychobiotics to influence the host’s brain (Cryan and Dinan, 2012; Zhou and Foster, 2015; Foster et al., 2016). Our results showed that PS128 ingestion significantly increased the enteric and peripheral 5-HT level in naïve rats but not in DOI induction rats, suggesting the modulation ability of PS128 on host’s enteric serotonergic system during the different situation (Fig. 5). Gut microbiota or their metabolic products, such as short chain fatty acids (SCFAs), are essential for peripheral 5-HT biosynthesis (O’Mahony et al., 2015; Reigstad et al., 2015; Yano et al., 2015). Although per- ipheral serotonergic systems are separated from the CNS, they could modulate immunity, regulate energy balance, and cellular processes, and may affect CNS physiology indirectly (Flood et al., 2012; Baganz and Blakely, 2013; Namkung et al., 2015; Pawlak et al., 2017). Per- ipheral 5-HT may also act on the 5-HTRs on vagal afferent fibers in the GI tract and transmit information from the enteric nervous system to the CNS, forming the gut–brain neuronal circuit (Li et al., 2000; Bonaz et al., 2018). Many studies had demonstrated a close relationship be- tween the vagus nerve and the dopaminergic system in the CNS (Ziomber et al., 2012; Surowka et al., 2015). Some psychobiotic strains like Lactobacillus rhamnosus JB-1 had lose the neurochemical and be- havioral effects in vagotomized animal, demonstrating the important role of vagus nerve in the bidirectional communication of the MGBA (Bravo et al., 2011). Furthermore, a recent study provided direct evi- dences establishing vagal gut-brain axis as an integral component of the neuronal reward pathway, and suggesting vagal stimulation might be a possible treatments strategy in affective disorders (Han et al., 2018). In our previous study using PS128-fed GF mice, the striatal DA elevation had suggested that PS128 in GI tract of the host tract could stimulate vagus nerve and influence the mesolimbic pathway through vagal gut- brain neuronal circuit (Liu et al., 2016a). Since we observed PS128 modulated peripheral 5-HT and the central dopaminergic pathways (Table 1 & Fig. 5), the vagus nerve might be an essential pathway for PS128 to influence the DA mesolimbic system (Fig. 7). However, this hypothesis will require further study to substantiate. Past animal studies indicated that gut microbiota could affect brain development and modulate behaviors; changes in microbiota compo- sition could also affect cerebral protein expression and behaviors even in mature animals (Bercik et al., 2011; Diaz Heijtz et al., 2011). DOI Fig. 5. Peripheral 5-HT levels in the ileum, colon, and serum of experimental induction caused a dispersive cecal bacterial community as indicated by rats. the clustering in PCA plots and the high Alpha diversity indices. The 5-HT level of the (A) ileum, (B) colon, and (C) serum were measured using However, these changes were not seen in PS128-fed DOI rats (Table 2, the HPLC-ECD method (n = 10/group). Data were expressed as mean ± SEM Fig. 6A–C). Since the rats received DOI by i.p. injection only 1 h before ’ # and analyzed by one-way ANOVA with Tukey s post hoc test. p < 0.05, sacrifice, DOI might not interact with intestinal bacteria directly but ##p < 0.01, ###p < 0.001, compared to the Saline group; &p < 0.05, &&& might exert its effects by interfering with 5-HTRs in the GI tract to p < 0.001, compared to the PS128 group. modulate intestinal secretion and motility (Gershon and Tack, 2007). Interestingly, both the PS128 and PS128 + DOI groups had lower Alpha studies we had observed PS128-fed rodents have present higher DA diversity indices compared to other groups and showed a clustering level but did not show hyperactive motor behaviors (Liu et al., 2016a, tendency in their PCoA plots distribution (Fig. 6C–D). These results ff 2016c). Some natural chemical also showed their tics-reducing e ects indicate a similar cecal microbiota community among all PS128-fed rats via regulating DAT expression which increases DA metabolism, and had and the microbiota community was not signifiacantly ffected by the potential for tics treatment (Lv et al., 2009, 2012; Wang et al., 2013; DOI treatment. The ratios between major bacterial phyla had been re- Zhang and Li, 2015). We also found enhanced p-Thr34-DARPP-32 and garded as the significant relevance in composition, ecological alteration p-ERK after DOI induction suggesting hyper-excitation in these dopa- and the indication of dysbiosis of gut microbiota, whereas PS128 ga- ff minergic projections (Fig. 4). In addition, unlike the e ects of DA an- vage also resulted in a decline in F/B and B/P ratios (Table 2). The F/B β tagonists haloperidol, PS128-fed rats exhibited a higher -arrestin ratio elevation has been shown to be closely related to pathological

68 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Table 2 Relative abundance of bacterial composition at the phylum level, family level, and ratios of bacteria communities.

Group Saline PS128 DOI (+)

Saline Hal PS128

Phylum level (relative abundance (%)) Firmicutes 61.5 ± 4.4 48.3 ± 4.1 63.5 ± 1.5 51.9 ± 5.9 52.6 ± 5.6 Bacteroidetes 35.1 ± 4.3 44.6 ± 4.0 32.2 ± 1.6 45.8 ± 6.0 43.9 ± 5.9 Proteobacteria 1.9 ± 0.5 5.5 ± 1.0# 2.2 ± 0.5 1.2 ± 0.1 2.2 ± 0.4 Tenericutes 0.43 ± 0.1 0.03 ± 0.1 0.77 ± 0.2 0.33 ± 0.1 0.40 ± 0.2 Cyanobacteria 0.38 ± 0.2 0.42 ± 0.2 0.49 ± 0.4 0.15 ± 0.1 0 Actinobacteria 0.28 ± 0.2 0.41 ± 0.2 0.24 ± 0.1 0.07 ± 0.1 0.56 ± 0.2 Others 0.26 ± 0.1 0.68 ± 0.3 0.60 ± 0.2 0.47 ± 0.3 0.21 ± 0.1

Family level (relative abundance (%)) Prevotellaceae 21.7 ± 5.7 21.8 ± 5.6 12.5 ± 3.1 33.5 ± 8.8*** 33.9 ± 6.5* Lachnospiraceae 14.5 ± 1.3 8.0 ± 1.5*** 17.8 ± 1.3 18.4 ± 3.0 11.3 ± 1.4* Lactobacillaceae 13.7 ± 3.9 7.3 ± 3.3 11.9 ± 0.8 7.1 ± 3.3 12.1 ± 2.3 Ruminococcaceae 13.4 ± 2.2 10.5 ± 1.8 13.0 ± 1.5 13.4 ± 1.6 9.9 ± 1.7 Peptostreptococcaceae 10.0 ± 1.9 12.7 ± 2.4 10.4 ± 1.5 6.5 ± 1.0 11.0 ± 1.4 Bacteroidales fS24-7 7.5 ± 3.0 8.0 ± 1.9 12.8 ± 2.1 6.6 ± 3.3 2.7 ± 1.3 Bacteroidaceae 4.1 ± 1.4 9.8 ± 2.4#,* 3.8 ± 0.8 3.3 ± 1.1 5.4 ± 0.8 Others 15.2 ± 5.4 21.9 ± 7.2 17.9 ± 6.5 11.2 ± 4.6 13.7 ± 3.0

Bacterial communities ratios F/B ratio 2.1 ± 0.4 0.95 ± 0.1# 2.02 ± 0.1 1.34 ± 0.3 1.21 ± 0.3 B/P ratio 24.2 ± 4.6 9.5 ± 1.9# 22.6 ± 5.2 38.5 ± 2.2* 23.6 ± 5.2

Cecal microbiota proportions average > 0.2% of total population at the phylum level and average > 5% of the total population at the family level were presented. F, Firmicutes;B,Bacteroidetes;P,Proteobacteria; Hal, haloperidol (1 mg/kg) group. Data were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test; #p < 0.05 to Saline group; *p < 0.05, ***p < 0.001 to Saline + DOI group. n = 7 in Saline group, n = 6 in PS128 group & PS128 + DOI group and n = 8 in Saline + DOI group & Hal + DOI group. conditions such as obesity, hypertension, and aging (Mariat et al., 2009; and Leckman, 2009; Scharf et al., 2012). The patients are presented Sanz and Moya-Perez, 2014; Yang et al., 2015). As for psychiatric with characteristic multiple motor tics and one or more phonic tics; conditions, some had reported an increase in Firmicutes or decline in while some even confounding with ADHD or OCD (Bloch and Leckman, Bacteroidetes that resulted in F/B ratio elevation in the clinical studies of 2009; Battle, 2013). TS mainly affects school-aged children, so therapy major depression disorder (Naseribafrouei et al., 2014; Chen et al., and medication require more stringent safety consideration (Bloch and 2018a, 2018b), ASD (Williams et al., 2011; Tomova et al., 2015; Strati Leckman, 2009; Roessner et al., 2011; Kurlan, 2014). Medications like et al., 2017), and schizophrenia (Castro-Nallar et al., 2015). However, alpha2-adrenergic agonists, muscle relaxants, and DA antagonists may others had reported entire opposite situations (Finegold et al., 2010; reduce tic frequency, but they may not be suitable for long-term use in Jiang et al., 2015; Liu et al., 2016b). Although the results are still developing children (Awaad, 1999; Roessner et al., 2011; Roessner controversial, clinical studies in neuropsychiatric disorders and neuro- et al., 2013). Both acupuncture and deep brain stimulation have shown degenerative condition increasingly reported the occurrence of gut good short-term effects, but acupuncture requires professional appli- dysbiosis (Hasegawa et al., 2015; Scheperjans et al., 2015; Vogt et al., cation and understandably there are concerns about children undergo 2017; Lubomski et al., 2019; Roy Sarkar and Banerjee, 2019). Out re- invasive brain surgery (Viswanathan et al., 2012; Yu et al., 2016). In sults suggest that PS128 administration could produce a more stable gut light of the positive effects of PS128 on ASD children aged 7–12 in a microbiota ecosystem to resist external stimuli, probably by influencing recent clinical study (Liu et al., 2019), we believe PS128 is safe for the enteric serotonergic system which is believed to be involved in the children application and may have beneficial effects on the disorders regulation of gastrointestinal motility and mucosal immune responses involving DA system. To our knowledge, PS128 is the first psychobiotic (Gross et al., 2012; Heredia et al., 2013; Reigstad et al., 2015; Yano strain showing supportive influences on the DA system of the host and et al., 2015). Considering the abovementioned studies and our experi- also has the potential for maintaining MGBA homeostasis of host mental results, we believe that stabilizing the gut microbiota could be (Fig. 7). According to our past studies and current results, PS128, as an beneficial for syndrome amendment and improve the quality of life in immune-regulatory probiotic strain that exhibits abilities to ameliorate neuropsychiatric patients. tics-like, anxiety-like, and depression-like behaviors (Liu et al., 2015, DOI has been used in rodent models to study TS (Cheer et al., 1999; 2016a; 2016c; Huang et al., 2019; Liu et al., 2019), has great potential Tizabi et al., 2001; Body et al., 2003; Hongyan et al., 2017), OCD for relieving TS and may be beneficial for other DA-related CNS dis- (Flaisher-Grinberg et al., 2008), schizophrenia (Kohnomi et al., 2008; orders. Malkova et al., 2014) and hallucinogen (Canal and Morgan, 2012; Schindler et al., 2012; Smith et al., 2014). Using DOI to generate a pharmacological TS rodent model was unanimously recommended in 5. Conclusion many research and review articles (Fone et al., 1991; Dursun and Handley, 1996; Swerdlow and Sutherland, 2006 ; Bronfeld et al., 2013; We demonstrated that PS128 ameliorated DOI-induced tic-like McCairn and Isoda, 2013; Godar et al., 2014). According to these stu- hyper-active behaviors and sensorimotor gating deficits caused by ir- dies, DOI-induced behaviors qualify for major criteria of a validate regular DA efflux in mesocortical and nigrostriatal DA pathways. animal model of TS, such as distinct regional contraction only after Further results suggested that PS128 stabilized cerebral dopaminergic induction, etiological validity, and responsiveness to DA antagonist pathways through its modulation of host’s microbiota–gut–brain axis. treatment (Swerdlow and Sutherland, 2006; Bronfeld et al., 2013; Our study may provide a potential new insight for psychobiotics ap- McCairn and Isoda, 2013; Godar et al., 2014). TS is an inherited neu- plication of improving syndromes caused by DA dysregulation in DA- rological disorder with childhood onset (Freeman et al., 2000; Bloch related neurological disorders and movement disorders such as TS.

69 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

Fig. 6. Analysis of the cecal microbiome of experimental rats. (A) Bar graph representing the relative community composition. (B) Principal component analysis (PCA) of bacterial abundance. The percentages of variation explained by PC1 and PC2 are indicated on the axes, and three bacterial families are indicated by arrows and family names in the PCA graph. The dash circle represents the cluster of Saline + DOI group. (C) Alpha diversities of bacterial community in individual samples in each rat group. Data were expressedas mean ± S.E.M. and analyzed by the Kruskal–Wallis Rank Sum Test. *p < 0.05 to Saline + DOI group. (D) 3D Principal coordinate analysis (PCoA) profiles, based on weighted UniFrac metric of bacterial diversity across all samples (ANOSIM; p < 0.05). The percentages of variation explained by PC1, PC2, and PC3 are indicated on the axes. The dash circle represents the cluster of PS128 group and Saline + PS128 group. n = 7 in Saline group, n = 6 in PS128 group & PS128 + DOI group, and n = 8 in Saline + DOI group & Hal + DOI group.

Relevant conflicts of interests/financial disclosures Author’s contribution statement

The authors declare that the research was conducted in the absence J.F.L., Y.F.C., S.W. and Y.C.T. conceived and organized the study. of any commercial or financial relationships that could be construed as Y.F.C., W.T.L, C.C.H., S.W. and Y.C.T. contributed to experimental de- a potential conflict of interest. sign. J.F.L. and Y.F.C. performed the experiments. S.W.L. performed the 454 pyrosequencing data analysis. J.F.L., Y.F.C. and S.W.L. performed data statistical analysis. J.F.L. wrote the first draft of the manuscript Funding agencies and Y.F.C., S.W.L., C.C.W., O.J.J., S.W. and Y.C.T. contributed to writing the manuscript. All authors read, edited and approved the final This work is supported by the Ministry of Science and Technology, manuscript. Taiwan, Republic of China (MOST 104-2320-B-010-005-MY2). The funding source had no contribution to the study design, collection, Acknowledgments analysis, interpretation of the data, or writing of the report for pub- lication. We would like to thank Koichi Watanabe for his invaluable assis- tance and advice. We thank VYM Genome Research Center, National Yang-Ming University, Taiwan, for providing 454 pyrosequencing

70 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

663–670. Bercik, P., Denou, E., Collins, J., Jackson, W., Lu, J., Jury, J., Deng, Y., Blennerhassett, P., et al., 2011. The intestinal microbiota affect central levels of brain-derived neuro- tropic factor and behavior in mice. Gastroenterology 141, 599–609 609 e591–593. Bermudez-Humaran, L.G., Salinas, E., Ortiz, G.G., Ramirez-Jirano, L.J., Morales, J.A., Bitzer-Quintero, O.K., 2019. From probiotics to psychobiotics: live beneficial bacteria which act on the brain-gut axis. Nutrients 11. Bidel, F., Corvaisier, S., Jozet-Alves, C., Pottier, I., Dauphin, F., Naud, N., Bellanger, C., 2016. An HPLC-ECD method for monoamines and metabolites quantification in cuttlefish (cephalopod) brain tissue. Biomed. Chromatogr. 30, 1175–1183. Bloch, M.H., Leckman, J.F., 2009. Clinical course of Tourette syndrome. J. Psychosom. Res. 67, 497–501. Body, S., Kheramin, S., Ho, M.Y., Miranda, F., Bradshaw, C.M., Szabadi, E., 2003. Effects of a 5-HT2 receptor agonist, DOI (2,5-dimethoxy-4-iodoamphetamine), and antago- nist, ketanserin, on the performance of rats on a free-operant timing schedule. Behav. Pharmacol. 14, 599–607. Bonaz, B., Bazin, T., Pellissier, S., 2018. The vagus nerve at the interface of the micro- biota-gut-brain axis. Front. Neurosci. 12, 49. Bortolozzi, A., Diaz-Mataix, L., Scorza, M.C., Celada, P., Artigas, F., 2005. The activation of 5-HT receptors in prefrontal cortex enhances dopaminergic activity. J. Neurochem. 95, 1597–1607. Bowers, B.J., Henry, M.B., Thielen, R.J., McBride, W.J., 2000. Serotonin 5-HT(2) receptor stimulation of dopamine release in the posterior but not anterior nucleus accumbens of the rat. J. Neurochem. 75, 1625–1633. Bravo, J.A., Forsythe, P., Chew, M.V., Escaravage, E., Savignac, H.M., Dinan, T.G., Bienenstock, J., Cryan, J.F., 2011. Ingestion of Lactobacillus strain regulates emo- tional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc. Natl. Acad. Sci. U. S. A. 108, 16050–16055. Bronfeld, M., Israelashvili, M., Bar-Gad, I., 2013. Pharmacological animal models of Tourette syndrome. Neurosci. Biobehav. Rev. 37, 1101–1119. Burke, M.V., Nocjar, C., Sonneborn, A.J., McCreary, A.C., Pehek, E.A., 2014. Striatal serotonin 2C receptors decrease nigrostriatal dopamine release by increasing GABA-A receptor tone in the substantia nigra. J. Neurochem. 131, 432–443. Canal, C.E., Morgan, D., 2012. Head-twitch response in rodents induced by the halluci- nogen 2,5-dimethoxy-4-iodoamphetamine: a comprehensive history, a re-evaluation of mechanisms, and its utility as a model. Drug Test. Anal. 4, 556–576. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., et al., 2010. QIIME allows analysis of high-throughput com- munity sequencing data. Nat. Methods 7, 335–336. Fig. 7. Schematic mechanisms of PS128 on strengthening the micro- Castro-Nallar, E., Bendall, M.L., Perez-Losada, M., Sabuncyan, S., Severance, E.G., biota–gut–brain axis function of the host. Dickerson, F.B., Schroeder, J.R., Yolken, R.H., et al., 2015. Composition, taxonomy ffi fl and functional diversity of the oropharynx microbiome in individuals with schizo- The symbiosis of su cient live PS128 in GI tract of the host could in uence phrenia and controls. PeerJ 3, e1140. both the enteric serotonergic system and gut microbiota. The modulation of Chao, S.H., Wu, R.J., Watanabe, K., Tsai, Y.C., 2009. Diversity of lactic acid bacteria in enteric 5-HT could have an impact on the ENS, GI tract motility, mucosa suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan. Int. J. Food function, and stimulate vagus nerve. These effects help to shape and stabilize Microbiol. 135, 203–210. fi the enteric ecosystem. In addition, PS128 in GI tract might also influence the Cheer, J.F., Cadogan, A.K., Marsden, C.A., Fone, K.C., Kendall, D.A., 1999. Modi cation ffi of 5-HT2 receptor mediated behaviour in the rat by oleamide and the role of can- mesocortical and nigrostriatal DA pathways to form a more e cient DA system nabinoid receptors. Neuropharmacology 38, 533–541. in CNS via the vagal gut-brain neuronal circuit. The long-term ingestion of Chen, J.J., Zheng, P., Liu, Y.Y., Zhong, X.G., Wang, H.Y., Guo, Y.J., Xie, P., 2018a. Sex adequate amount PS128 could strengthen the MGBA function of the host to differences in gut microbiota in patients with major depressive disorder. cope with gut dysbiosis and improve on abnormal neurotransmission and be- Neuropsychiatr. Dis. Treat. 14, 647–655. havioral ailments. Chen, Z., Li, J., Gui, S., Zhou, C., Chen, J., Yang, C., Hu, Z., Wang, H., et al., 2018b. Comparative metaproteomics analysis shows altered fecal microbiota signatures in patients with major depressive disorder. Neuroreport 29, 417–425. service. We also thank the experimental services and animal cares from Cheng, F.C., Kuo, J.S., Huang, H.M., Yang, D.Y., Wu, T.F., Tsai, T.H., 2000. Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by micro- the Animal Behavioral Facility and Laboratory Animal Center at dialysis-microbore liquid chromatography. J. Chromatogr. A 870, 405–411. National Yang-Ming University. Clarke, K., 1993. Nonparametric Multivariate Analyses of Changes in Community Structure. Collins, S.M., Surette, M., Bercik, P., 2012. The interplay between the intestinal micro- Appendix A. Supplementary data biota and the brain. Nat. Rev. Microbiol. 10, 735–742. Cryan, J.F., Dinan, T.G., 2012. Mind-altering microorganisms: the impact of the gut mi- Supplementary material related to this article can be found, in the crobiota on brain and behaviour. Nat. Rev. Neurosci. 13, 701–712. Del’guidice, T., Lemasson, M., Beaulieu, J.M., 2011. Role of Beta-arrestin 2 downstream online version, at doi:https://doi.org/10.1016/j.brainresbull.2019.07. of dopamine receptors in the basal ganglia. Front. Neuroanat. 5, 58. 027. DeSantis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., Huber, T., Dalevi, D., et al., 2006. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72, 5069–5072. References Desbonnet, L., Garrett, L., Clarke, G., Kiely, B., Cryan, J.F., Dinan, T.G., 2010. Effects of the probiotic Bifidobacterium infantis in the maternal separation model of depression. Aghajanian, G.K., Marek, G.J., 2000. Serotonin model of schizophrenia: emerging role of Neuroscience 170, 1179–1188. glutamate mechanisms. Brain Res. Brain Res. Rev. 31, 302–312. Diaz Heijtz, R., Wang, S., Anuar, F., Qian, Y., Bjorkholm, B., Samuelsson, A., Hibberd, Alex, K.D., Yavanian, G.J., McFarlane, H.G., Pluto, C.P., Pehek, E.A., 2005. Modulation of M.L., Forssberg, H., et al., 2011. Normal gut microbiota modulates brain develop- dopamine release by striatal 5-HT2C receptors. Synapse 55, 242–251. ment and behavior. Proc. Natl. Acad. Sci. U. S. A. 108, 3047–3052. Alonso, P., Lopez-Sola, C., Real, E., Segalas, C., Menchon, J.M., 2015. Animal models of Dinan, T.G., Stanton, C., Cryan, J.F., 2013. Psychobiotics: a novel class of psychotropic. obsessive-compulsive disorder: utility and limitations. Neuropsychiatr. Dis. Treat. 11, Biol. Psychiatry 74, 720–726. 1939–1955. Dursun, S.M., Handley, S.L., 1996. Similarities in the pharmacology of spontaneous and Awaad, Y., 1999. Tics in Tourette syndrome: new treatment options. J. Child Neurol. 14, DOI-induced head-shakes suggest 5HT2A receptors are active under physiological 316–319. conditions. Psychopharmacology (Berl.) 128, 198–205. Baganz, N.L., Blakely, R.D., 2013. A dialogue between the immune system and brain, Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. spoken in the language of serotonin. ACS Chem. Neurosci. 4, 48–63. Bioinformatics 26, 2460–2461. Battle, D.E., 2013. Diagnostic and statistical manual of mental disorders (DSM). Codas 25, Finegold, S.M., Dowd, S.E., Gontcharova, V., Liu, C., Henley, K.E., Wolcott, R.D., Youn, E., 191–192. Summanen, P.H., et al., 2010. Pyrosequencing study of fecal microflora of autistic Bedard, P., Pycock, C.J., 1977. "Wet-dog" shake behaviour in the rat: a possible quanti- and control children. Anaerobe 16, 444–453. tative model of central 5-hydroxytryptamine activity. Neuropharmacology 16, Fishman, R.H., Feigenbaum, J.J., Yanai, J., Klawans, H.L., 1983. The relative importance

71 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

of dopamine and norepinephrine in mediating locomotor activity. Prog. Neurobiol. PS128 in germ-free mice. Behav. Brain Res. 298, 202–209. 20, 55–88. Liu, W.H., Yang, C.H., Lin, C.T., Li, S.W., Cheng, W.S., Jiang, Y.P., Wu, C.C., Chang, C.H.,

Flaisher-Grinberg, S., Klavir, O., Joel, D., 2008. The role of 5-HT2A and 5-HT2C receptors et al., 2015. Genome architecture of Lactobacillus plantarum PS128, a probiotic strain in the signal attenuation rat model of obsessive-compulsive disorder. Int. J. with potential immunomodulatory activity. Gut Pathog. 7, 22. Neuropsychopharmacol. 11, 811–825. Liu, Y., Zhang, L., Wang, X., Wang, Z., Zhang, J., Jiang, R., Wang, X., Wang, K., et al., Flood, Z.C., Engel, D.L., Simon, C.C., Negherbon, K.R., Murphy, L.J., Tamavimok, W., 2016b. Similar fecal microbiota signatures in patients with diarrhea-predominant Anderson, G.M., Janusonis, S., 2012. Brain growth trajectories in mouse strains with irritable bowel syndrome and patients with depression. Clin. Gastroenterol. Hepatol. central and peripheral serotonin differences: relevance to autism models. 14, 1602–1611 e1605. Neuroscience 210, 286–295. Liu, Y.W., Liao, T.W., Chen, Y.H., Chiang, Y.C., Tsai, Y.C., 2014. Oral administration of Fone, K.C., Robinson, A.J., Marsden, C.A., 1991. Characterization of the 5-HT receptor heat-inactivated Lactobacillus plantarum K37 modulated airway hyperresponsiveness subtypes involved in the motor behaviours produced by intrathecal administration of in ovalbumin-sensitized BALB/c mice. PLoS One 9, e100105. 5-HT agonists in rats. Br. J. Pharmacol. 103, 1547–1555. Liu, Y.W., Liong, M.T., Chung, Y.E., Huang, H.Y., Peng, W.S., Cheng, Y.F., Lin, Y.S., Wu, Foster, J.A., Lyte, M., Meyer, E., Cryan, J.F., 2016. Gut microbiota and brain function: an Y.Y., et al., 2019. Effects of Lactobacillus plantarum PS128 on children with autism evolving field in neuroscience. Int. J. Neuropsychopharmacol. 19. spectrum disorder in Taiwan: a randomized, double-blind, placebo-controlled trial. Freeman, R.D., Fast, D.K., Burd, L., Kerbeshian, J., Robertson, M.M., Sandor, P., 2000. An Nutrients 11. international perspective on Tourette syndrome: selected findings from 3,500 in- Liu, Y.W., Liu, W.H., Wu, C.C., Juan, Y.C., Wu, Y.C., Tsai, H.P., Wang, S., Tsai, Y.C., dividuals in 22 countries. Dev. Med. Child Neurol. 42, 436–447. 2016c. Psychotropic effects of Lactobacillus plantarum PS128 in early life-stressed and Gaynor, C.M., Handley, S.L., 2001. Effects of nicotine on head-shakes and tryptophan naive adult mice. Brain Res. 1631, 1–12. metabolites. Psychopharmacology (Berl.) 153, 327–333. Liu, Y.W., Su, Y.W., Ong, W.K., Cheng, T.H., Tsai, Y.C., 2011. Oral administration of Gershon, M.D., Tack, J., 2007. The serotonin signaling system: from basic understanding Lactobacillus plantarum K68 ameliorates DSS-induced ulcerative colitis in BALB/c to drug development for functional GI disorders. Gastroenterology 132, 397–414. mice via the anti-inflammatory and immunomodulatory activities. Int. Gobert, A., Millan, M.J., 1999. Serotonin (5-HT)2A receptor activation enhances dialysate Immunopharmacol. 11, 2159–2166. levels of dopamine and noradrenaline, but not 5-HT, in the frontal cortex of freely- Lozupone, C., Knight, R., 2005. UniFrac: a new phylogenetic method for comparing mi- moving rats. Neuropharmacology 38, 315–317. crobial communities. Appl. Environ. Microbiol. 71, 8228–8235. Godar, S.C., Mosher, L.J., Di Giovanni, G., Bortolato, M., 2014. Animal models of tic Lubomski, M., Tan, A.H., Lim, S.Y., Holmes, A.J., Davis, R.L., Sue, C.M., 2019. Parkinson’s disorders: a translational perspective. J. Neurosci. Methods 238, 54–69. disease and the gastrointestinal microbiome. J. Neurol. Gross, E.R., Gershon, M.D., Margolis, K.G., Gertsberg, Z.V., Li, Z., Cowles, R.A., 2012. Lv, H., Li, A., Liu, F., Ma, H., Yao, B., 2009. Effects of gastrodin on the dopamine system of Neuronal serotonin regulates growth of the intestinal mucosa in mice. Tourette’s syndrome rat models. Biosci. Trends 3, 58–62. Gastroenterology 143, 408–417 e402. Lv, H., Lin, H.Y., Zhao, H., Li, A.Y., Lin, Y., Yao, B., 2012. Effects of ningdong granule on Haas, B.J., Gevers, D., Earl, A.M., Feldgarden, M., Ward, D.V., Giannoukos, G., Ciulla, D., DA, DRD2, and HVA in a rat model of Tourette’s syndrome. J. Tradit. Chin. Med. 32, Tabbaa, D., et al., 2011. Chimeric 16S rRNA sequence formation and detection in 283–288. Sanger and 454-pyrosequenced PCR amplicons. Genome Res. 21, 494–504. Malkova, N.V., Gallagher, J.J., Yu CZ, Jacobs R.E., Patterson, P.H., 2014. Manganese- Han, W., Tellez, L.A., Perkins, M.H., Perez, I.O., Qu, T., Ferreira, J., Ferreira, T.L., Quinn, enhanced magnetic resonance imaging reveals increased DOI-induced brain activity D., et al., 2018. A neural circuit for gut-induced reward. Cell 175, 665–678 e623. in a mouse model of schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 111, E2492–2500. Handley, S.L., Dursun, S.M., 1992. Serotonin and Tourette’s syndrome: movements such Mariat, D., Firmesse, O., Levenez, F., Guimaraes, V., Sokol, H., Dore, J., Corthier, G., as head-shakes and wet-dog shakes may model human tics. Adv. Biosci. 85, 235–253. Furet, J.P., 2009. The Firmicutes/Bacteroidetes ratio of the human microbiota changes Hasegawa, S., Goto, S., Tsuji, H., Okuno, T., Asahara, T., Nomoto, K., Shibata, A., with age. BMC Microbiol. 9, 123. Fujisawa, Y., et al., 2015. Intestinal dysbiosis and lowered serum lipopolysaccharide- Masri, B., Salahpour, A., Didriksen, M., Ghisi, V., Beaulieu, J.M., Gainetdinov, R.R., binding protein in Parkinson’s Disease. PLoS One 10, e0142164. Caron, M.G., 2008. Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction Hayslett, R.L., Tizabi, Y., 2003. Effects of donepezil on DOI-induced head twitch response is a common property of clinically effective antipsychotics. Proc. Natl. Acad. Sci. U. S. in mice: implications for Tourette syndrome. Pharmacol. Biochem. Behav. 76, A. 105, 13656–13661. 409–415. McCairn, K.W., Isoda, M., 2013. Pharmacological animal models of tic disorders. Int. Rev. Heredia, D.J., Gershon, M.D., Koh, S.D., Corrigan, R.D., Okamoto, T., Smith, T.K., 2013. Neurobiol. 112, 179–209. Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in McKernan, D.P., Fitzgerald, P., Dinan, T.G., Cryan, J.F., 2010. The probiotic vitro analyses in mice lacking tryptophan hydroxylase 1. J. Physiol. (Paris) 591, Bifidobacterium infantis 35624 displays visceral antinociceptive e ffects in the rat. 5939–5957. Neurogastroenterol. Motil. 22, 1029–1035 e1268. Hongyan, L., Zhenyang, S., Chunyan, W., Qingqing, P., 2017. Lipopolysaccharide ag- Mei, H.C., Liu, Y.W., Chiang, Y.C., Chao, S.H., Mei, N.W., Liu, Y.W., Tsai, Y.C., 2013. gravated DOI-induced Tourette syndrome: elaboration for recurrence of Tourette Immunomodulatory activity of Lactococcus lactis A17 from Taiwan fermented cab- syndrome. Metab. Brain Dis. 32, 1929–1934. bage in OVA-sensitized BALB/c mice. Evid. Complement. Alternat. Med., 287803 Huang, H.Y., Korivi, M., Tsai, C.H., Yang, J.H., Tsai, Y.C., 2013. Supplementation of 2013. Lactobacillus plantarum K68 and fruit-vegetable ferment along with high fat-fructose Minzer, K., Lee, O., Hong, J.J., Singer, H.S., 2004. Increased prefrontal D2 protein in diet attenuates metabolic syndrome in rats with insulin resistance. Evid. Tourette syndrome: a postmortem analysis of frontal cortex and striatum. J. Neurol. Complement. Alternat. Med., 943020 2013. Sci. 219, 55–61. Huang, W.C., Wei, C.C., Huang, C.C., Chen, W.L., Huang, H.Y., 2019. The beneficial ef- Mitchell, H.A., Ahern, T.H., Liles, L.C., Javors, M.A., Weinshenker, D., 2006. The effects of fects of Lactobacillus plantarum PS128 on high-intensity, exercise-induced oxidative norepinephrine transporter inactivation on locomotor activity in mice. Biol. stress, inflammation, and performance in triathletes. Nutrients 11. Psychiatry 60, 1046–1052. Iyer, R.N., Bradberry, C.W., 1996. Serotonin-mediated increase in prefrontal cortex do- Mouri, A., Nagai, T., Ibi, D., Yamada, K., 2013. Animal models of schizophrenia for pamine release: pharmacological characterization. J. Pharmacol. Exp. Ther. 277, molecular and pharmacological intervention and potential candidate molecules. 40–47. Neurobiol. Dis. 53, 61–74. Jaworski, D.M., Boone, J., Caterina, J., Soloway, P., Falls, W.A., 2005. Prepulse inhibition Nakayama, J., 2010. Pyrosequence-based 16S rRNA profiling of gastro-intestinal micro- and fear-potentiated startle are altered in tissue inhibitor of metalloproteinase-2 biota. Biosci. Microflora 29, 83–96. (TIMP-2) knockout mice. Brain Res. 1051, 81–89. Nakayama, J., Watanabe, K., Jiang, J., Matsuda, K., Chao, S.H., Haryono, P., La- Jiang, H., Ling, Z., Zhang, Y., Mao, H., Ma, Z., Yin, Y., Wang, W., Tang, W., et al., 2015. Ongkham, O., Sarwoko, M.A., et al., 2015. Diversity in gut bacterial community of Altered fecal microbiota composition in patients with major depressive disorder. school-age children in Asia. Sci. Rep. 5, 8397. Brain Behav. Immun. 48, 186–194. Namkung, J., Kim, H., Park, S., 2015. Peripheral serotonin: a new player in systemic Kantak, P.A., Bobrow, D.N., Nyby, J.G., 2014. Obsessive-compulsive-like behaviors in energy homeostasis. Mol. Cells 38, 1023–1028. house mice are attenuated by a probiotic (Lactobacillus rhamnosus GG). Behav. Naseribafrouei, A., Hestad, K., Avershina, E., Sekelja, M., Linlokken, A., Wilson, R., Rudi, Pharmacol. 25, 71–79. K., 2014. Correlation between the human fecal microbiota and depression. Kehne, J.H., Padich, R.A., McCloskey, T.C., Taylor, V.L., Schmidt, C.J., 1996. 5-HT Neurogastroenterol. Motil. 26, 1155–1162. modulation of auditory and visual sensorimotor gating: I. Effects of 5-HT releasers on Ng, N.K., Lee, H.S., Wong, P.T., 1999. Regulation of striatal dopamine release through 5- sound and light prepulse inhibition in Wistar rats. Psychopharmacology (Berl.) 124, HT1 and 5-HT2 receptors. J. Neurosci. Res. 55, 600–607. 95–106. O’Mahony, S.M., Clarke, G., Borre, Y.E., Dinan, T.G., Cryan, J.F., 2015. Serotonin, tryp- Kim, N., Yun, M., Oh, Y.J., Choi, H.J., 2018. Mind-altering with the gut: modulation of the tophan metabolism and the brain-gut-microbiome axis. Behav. Brain Res. 277, 32–48. gut-brain axis with probiotics. J. Microbiol. 56, 172–182. Oades, R.D., Taghzouti, K., Rivet, J.M., Simon, H., Le Moal, M., 1986. Locomotor activity Kohnomi, S., Suemaru, K., Kawasaki, H., Araki, H., 2008. Effect of aripiprazole on 5-HT2 in relation to dopamine and noradrenaline in the nucleus accumbens, septal and receptor-mediated wet-dog shake responses and disruption of prepulse inhibition in frontal areas: a 6-hydroxydopamine study. Neuropsychobiology 16, 37–42. rats. J. Pharmacol. Sci. 106, 645–650. Pawlak, D., Domaniewski, T., Znorko, B., Oksztulska-Kolanek, E., Lipowicz, P., Doroszko, Kurlan, R.M., 2014. Treatment of tourette syndrome. Neurotherapeutics 11, 161–165. M., Karbowska, M., Pawlak, K., 2017. The impact of peripheral serotonin on leptin- Li, S.W., Watanabe, K., Hsu, C.C., Chao, S.H., Yang, Z.H., Lin, Y.J., Chen, C.C., Cao, Y.M., brain serotonin axis, bone metabolism and strength in growing rats with experi- et al., 2017. Bacterial composition and diversity in breast milk samples from mothers mental chronic kidney disease. Bone 105, 1–10. living in Taiwan and Mainland China. Front. Microbiol. 8, 965. Pehek, E.A., Hernan, A.E., 2015. Stimulation of glutamate receptors in the ventral teg- Li, Y., Hao, Y., Zhu, J., Owyang, C., 2000. Serotonin released from intestinal en- mental area is necessary for serotonin-2 receptor-induced increases in mesocortical terochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic dopamine release. Neuroscience 290, 159–164. secretion in rats. Gastroenterology 118, 1197–1207. Reigstad, C.S., Salmonson, C.E., Rainey 3rd, J.F., Szurszewski, J.H., Linden, D.R., Liu, W.H., Chuang, H.L., Huang, Y.T., Wu, C.C., Chou, G.T., Wang, S., Tsai, Y.C., 2016a. Sonnenburg, J.L., Farrugia, G., Kashyap, P.C., 2015. Gut microbes promote colonic Alteration of behavior and monoamine levels attributable to Lactobacillus plantarum serotonin production through an effect of short-chain fatty acids on enterochromaffin

72 J.-F. Liao, et al. Brain Research Bulletin 153 (2019) 59–73

cells. FASEB J. 29, 1395–1403. 78–85. Riezzo, G., Orlando, A., D’Attoma, B., Linsalata, M., Martulli, M., Russo, F., 2018. Valjent, E., Pascoli, V., Svenningsson, P., Paul, S., Enslen, H., Corvol, J.C., Stipanovich, A., Randomised double blind placebo controlled trial on Lactobacillus reuteri DSM 17938: Caboche, J., et al., 2005. Regulation of a protein phosphatase cascade allows con- improvement in symptoms and bowel habit in functional constipation. Benef. vergent dopamine and glutamate signals to activate ERK in the striatum. Proc. Natl. Microbes 9, 51–60. Acad. Sci. U. S. A. 102, 491–496. Roessner, V., Plessen, K.J., Rothenberger, A., Ludolph, A.G., Rizzo, R., Skov, L., Strand, Valsamis, B., Schmid, S., 2011. Habituation and prepulse inhibition of acoustic startle in G., Stern, J.S., et al., 2011. European clinical guidelines for Tourette syndrome and rodents. J. Vis. Exp., e3446. other tic disorders. Part II: pharmacological treatment. Eur. Child Adolesc. Psychiatry van de Wouw, M., Schellekens, H., Dinan, T.G., Cryan, J.F., 2017. Microbiota-Gut-Brain 20, 173–196. Axis: modulator of host metabolism and appetite. J. Nutr. 147, 727–745. Roessner, V., Schoenefeld, K., Buse, J., Bender, S., Ehrlich, S., Munchau, A., 2013. Vickers, S.P., Easton, N., Malcolm, C.S., Allen, N.H., Porter, R.H., Bickerdike, M.J., Pharmacological treatment of tic disorders and Tourette Syndrome. Kennett, G.A., 2001. Modulation of 5-HT(2A) receptor-mediated head-twitch beha- Neuropharmacology 68, 143–149. viour in the rat by 5-HT(2C) receptor agonists. Pharmacol. Biochem. Behav. 69, Roy Sarkar, S., Banerjee, S., 2019. Gut microbiota in neurodegenerative disorders. J. 643–652. Neuroimmunol. 328, 98–104. Viswanathan, A., Jimenez-Shahed, J., Baizabal Carvallo, J.F., Jankovic, J., 2012. Deep Sanz, Y., Moya-Perez, A., 2014. Microbiota, inflammation and obesity. Adv. Exp. Med. brain stimulation for Tourette syndrome: target selection. Stereotact. Funct. Biol. 817, 291–317. Neurosurg. 90, 213–224. Scharf, J.M., Miller, L.L., Mathews, C.A., Ben-Shlomo, Y., 2012. Prevalence of Tourette Vogt, N.M., Kerby, R.L., Dill-McFarland, K.A., Harding, S.J., Merluzzi, A.P., Johnson, S.C., syndrome and chronic tics in the population-based Avon longitudinal study of parents Carlsson, C.M., Asthana, S., et al., 2017. Gut microbiome alterations in Alzheimer’s and children cohort. J. Am. Acad. Child Adolesc. Psychiatry 51, 192–201 e195. disease. Sci. Rep. 7, 13537. Scheperjans, F., Aho, V., Pereira, P.A., Koskinen, K., Paulin, L., Pekkonen, E., Haapaniemi, Wang, D.H., Li, W., Liu, X.F., Zhang, J.M., Wang, S.M., 2013. Chinese medicine formula E., Kaakkola, S., et al., 2015. Gut microbiota are related to Parkinson’s disease and "Jian-Pi-Zhi-Dong Decoction" attenuates Tourette Syndrome via downregulating the clinical phenotype. Mov. Disord. 30, 350–358. expression of dopamine transporter in mice. Evid. Complement. Alternat. Med., Schindler, E.A., Dave, K.D., Smolock, E.M., Aloyo, V.J., Harvey, J.A., 2012. Serotonergic 385685 2013. and dopaminergic distinctions in the behavioral pharmacology of (+/-)-1-(2,5-di- Wei, C.L., Wang, S., Yen, J.T., Cheng, Y.F., Liao, C.L., Hsu, C.C., Wu, C.C., Tsai, Y.C., methoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD). 2019. Antidepressant-like activities of live and heat-killed Lactobacillus paracasei Pharmacol. Biochem. Behav. 101, 69–76. PS23 in chronic corticosterone-treated mice and possible mechanisms. Brain Res. Sipes, T.A., Geyer, M.A., 1994. Multiple serotonin receptor subtypes modulate prepulse Weiss, I.C., Feldon, J., 2001. Environmental animal models for sensorimotor gating de- inhibition of the startle response in rats. Neuropharmacology 33, 441–448. ficiencies in schizophrenia: a review. Psychopharmacology (Berl.) 156, 305–326. Smith, D.A., Bailey, J.M., Williams, D., Fantegrossi, W.E., 2014. Tolerance and cross- Williams, B.L., Hornig, M., Buie, T., Bauman, M.L., Cho Paik, M., Wick, I., Bennett, A., tolerance to head twitch behavior elicited by phenethylamine- and tryptamine-de- Jabado, O., et al., 2011. Impaired carbohydrate digestion and transport and mucosal rived hallucinogens in mice. J. Pharmacol. Exp. Ther. 351, 485–491. dysbiosis in the intestines of children with autism and gastrointestinal disturbances. Strati, F., Cavalieri, D., Albanese, D., De Felice, C., Donati, C., Hayek, J., Jousson, O., PLoS One 6, e24585. Leoncini, S., et al., 2017. New evidences on the altered gut microbiota in autism Wong, D.F., Brasic, J.R., Singer, H.S., Schretlen, D.J., Kuwabara, H., Zhou, Y., Nandi, A., spectrum disorders. Microbiome 5, 24. Maris, M.A., et al., 2008. Mechanisms of dopaminergic and serotonergic neuro- Surowka, A.D., Krygowska-Wajs, A., Ziomber, A., Thor, P., Chrobak, A.A., Szczerbowska- transmission in Tourette syndrome: clues from an in vivo neurochemistry study with Boruchowska, M., 2015. Peripheral vagus nerve stimulation significantly affects lipid PET. Neuropsychopharmacology 33, 1239–1251. composition and protein secondary structure within dopamine-related brain regions Wright, I.K., Ismail, H., Upton, N., Marsden, C.A., 1991. Effect of isolation rearing on 5- in rats. Neuromolecular Med. 17, 178–191. HT agonist-induced responses in the rat. Psychopharmacology (Berl.) 105, 259–263. Svenningsson, P., Nishi, A., Fisone, G., Girault, J.A., Nairn, A.C., Greengard, P., 2004. Yang, T., Santisteban, M.M., Rodriguez, V., Li, E., Ahmari, N., Carvajal, J.M., Zadeh, M., DARPP-32: an integrator of neurotransmission. Annu. Rev. Pharmacol. Toxicol. 44, Gong, M., et al., 2015. Gut dysbiosis is linked to hypertension. Hypertension 65, 269–296. 1331–1340. Swerdlow, N.R., Sutherland, A.N., 2006. Preclinical models relevant to Tourette syn- Yano, J.M., Yu, K., Donaldson, G.P., Shastri, G.G., Ann, P., Ma, L., Nagler, C.R., Ismagilov, drome. Adv. Neurol. 99, 69–88. R.F., et al., 2015. Indigenous bacteria from the gut microbiota regulate host serotonin Tizabi, Y., Russell, L.T., Johnson, M., Darmani, N.A., 2001. Nicotine attenuates DOI-in- biosynthesis. Cell 161, 264–276. duced head-twitch response in mice: implications for Tourette syndrome. Prog. Yu, J., Ye, Y., Liu, J., Wang, Y., Peng, W., Liu, Z., 2016. Acupuncture for Tourette syn- Neuropsychopharmacol. Biol. Psychiatry 25, 1445–1457. drome: a systematic review. Evid. Complement. Alternat. Med., 1834646 2016. Tomova, A., Husarova, V., Lakatosova, S., Bakos, J., Vlkova, B., Babinska, K., Ostatnikova, Zhang, F., Li, A., 2015. Dual restoring effects of gastrodin on dopamine in rat models of D., 2015. Gastrointestinal microbiota in children with autism in Slovakia. Physiol. Tourette’s syndrome. Neurosci. Lett. 588, 62–66. Behav. 138, 179–187. Zhou, L., Foster, J.A., 2015. Psychobiotics and the gut-brain axis: in the pursuit of hap- Urs, N.M., Gee, S.M., Pack, T.F., McCorvy, J.D., Evron, T., Snyder, J.C., Yang, X., piness. Neuropsychiatr. Dis. Treat. 11, 715–723. Rodriguiz, R.M., et al., 2016. Distinct cortical and striatal actions of a beta-arrestin- Ziomber, A., Thor, P., Krygowska-Wajs, A., Zalecki, T., Moskala, M., Romanska, I., biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties. Michaluk, J., Antkiewicz-Michaluk, L., 2012. Chronic impairment of the vagus nerve Proc. Natl. Acad. Sci. U. S. A. 113, E8178–E8186. function leads to inhibition of dopamine but not serotonin neurons in rat brain Urs, N.M., Peterson, S.M., Caron, M.G., 2017. New concepts in dopamine D2 receptor structures. Pharmacol. Rep. 64, 1359–1367. biased signaling and implications for schizophrenia therapy. Biol. Psychiatry 81,

73