Characterizing the Differential Roles of Striatal 5-HT1A Auto- and Hetero-Receptors in the Reduction of L-DOPA-Induced Dyskinesia

Experimental Neurology 292 (2017) 168–178

Contents lists available at ScienceDirect

Experimental Neurology

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

Research Paper

Characterizing the differential roles of striatal 5-HT1A auto- and hetero-receptors in the reduction of L-DOPA-induced dyskinesia

Samantha M. Meadows a, Nicole E. Chambers a, Melissa M. Conti a, Sharon C. Bossert a,CrystalTasbera, Eitan Sheena a, Mark Varney b, Adrian Newman-Tancredi b, Christopher Bishop a,⁎ a Behavioral Neuroscience Program, Department of Psychology, Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 13902-6000, USA b Neurolixis Inc., Dana Point, CA 92629, USA article info abstract

Article history: L-DOPA remains the benchmark treatment for Parkinson's disease (PD) motor symptoms, but chronic use leads to L- Received 6 September 2016 DOPA-induced dyskinesia (LID). The serotonin (5-HT) system has been established as a key modulator of LID and 5- Received in revised form 24 February 2017 HT receptors (5-HT R) stimulation has been shown to convey anti-dyskinetic effects. However, 5-HT Ragonists Accepted 22 March 2017 1A 1A 1A fi Available online 23 March 2017 often compromise clinical ef cacy or display intrinsic side effects and their site(s) of actions remain debatable. Re- cently, highly selective G-protein biased 5-HT1AR agonists, F13714 and F15599, were shown to potently target 5- Keywords: HT1A auto- or hetero-receptors, respectively. The current investigation sought to identify the signaling mechanisms LID and neuroanatomical substrates by which 5-HT1AR produce behavioral effects. In experiment 1, hemi-parkinsonian, Serotonin 1A receptor L-DOPA-primed rats received systemic injections of vehicle, F13714 (0.01 or 0.02 mg/kg), or F15599 (0.06 or Biased agonist 0.12 mg/kg) 5 min prior to L-DOPA (6 mg/kg), after which LID, motor performance and 5-HT syndrome were Serotonin syndrome rated. Both compounds significantly reduced LID, without affecting motor performance, however, acute administra- Striatum tion of F13714 significantly induced 5-HT syndrome at anti-dyskinetic doses. In experiment 2, we elucidated the Parkinson's disease Microinjection role of striatal 5-HT1AR in the effects of F13714 and F15599. Hemi-parkinsonian, L-DOPA-primed rats received bilat- Neuropharmacology eral intra-striatal microinjections of either F13714 (0, 2 or 10 μg/side)orF15599(0,10or30μg/side) 5 min prior to systemic L-DOPA (6 mg/kg). Intra-striatal effects mimicked systemic effects, suggesting that striatal 5-HT1ARsub- populations play an important role in the anti-LID and pro-5-HT syndrome profiles of F13714 and F15599. Finally,

in experiment 3, we examined the effects of F13714 and F15599 on D1 receptor (D1R) agonist-induced dyskinesia by administering either compound 5 min prior to SKF 38393 (2 mg/kg). While F13714 resulted in a mild delay in

D1R-mediated dyskinesia, F15599 had no effect. Collectively these data suggest that the F-series compounds artic-

1. Introduction gold standard for alleviating akinetic motor symptoms in Parkinson's disease (PD; Smith et al., 2012). However, in a majority of PD patients, 1 Since the 1960s, dopamine (DA ) replacement therapy, with the DA chronic L-DOPA use is complicated by the development of abnormal in- precursor L-3,4-dihydroxyphenylalanine (L-DOPA), has remained the voluntary movements (AIMs), referred to as L-DOPA-induced dyskinesia (LID; Obeso et al., 2000). Over the past several years, mounting evidence has implicated ⁎ Corresponding author. E-mail addresses: [email protected] (S.M. Meadows), nchambe4@ raphe-striatal serotonin (5-HT) neurons as surrogates to the compro- binghamton.edu (N.E. Chambers), [email protected] (M.M. Conti), sbosser1@ mised DA system, since they possess the necessary machinery to take binghamton.edu (S.C. Bossert), [email protected] (C. Tasber), esheena1@ up exogenous L-DOPA and convert it into DA for synaptic release (Arai binghamton.edu (E. Sheena), [email protected] (M. Varney), anewmantancredi@ et al., 1994; Carta et al., 2007; Kannari et al., 2003; Keber et al., 2015; neurolixis.com (A. Newman-Tancredi), [email protected] (C. Bishop). 1 DA, dopamine; L-DOPA, L-3,4-dihydroxyphenylalanine; PD, Parkinson's disease; AIMs, Nahimi et al., 2012; Nevalainen et al., 2014; Rylander et al., 2010; abnormal involuntary movement; LID, L-DOPA-induced dyskinesia; 5-HT, serotonin; DAT, Tanaka et al., 1999; Yamada et al., 2007). However, 5-HT neuron release dopamine transporter; 5-HT1AR, serotonin 1A receptor; MSNs, medium spiny neurons; ChI, of DA becomes unregulated due to a lack of feedback mechanisms, like cholinergic interneuron; 6-OHDA, 6-hydroxydopamine; FAS, forepaw adjusting steps; ALO, DA transporters (DAT) and DA D2 auto-receptors (Maeda et al., 1999), axial, limb, and orolingual; HPLC-ED, high performance liquid chromatography with electro- prolonging activation of sensitized DA receptors and exacerbating LID chemical detection; DOPAC, 3,4-dihydroxyphenlyacetic acid; M.A.D., median absolute devia- tion; S.E.M., standard mean error; DRN, dorsal raphe nucleus; PET, positron emission expression with chronic L-DOPA treatment (Maeda et al., 2003; Maeda topography; PPD, preprodynorphin; GAD67, glutamic acid decarboxylase 67 kDa isoform. et al., 2005). As such, researchers have sought to temper L-DOPA-

http://dx.doi.org/10.1016/j.expneurol.2017.03.013 0014-4886/© 2017 Elsevier Inc. All rights reserved. S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178 169 derived DA release from striatal 5-HT terminals in order to alleviate LID The present study sought to determine whether systemic and/or expression. local striatal administration of functionally selective 5-HT1ARbiasedag- The most studied candidate for modulating raphe-striatal function onists would provide anti-LID effects, with minimal side-effect profiles. has been the 5-HT1A receptor (5-HT1AR). These inhibitory, G-protein Additionally, the DA D1R agonist SKF38393 was used as a tool to deter- coupled receptors are ubiquitously expressed throughout the brain mine the contribution of 5-HT1A heteroreceptors on D1R-bearing MSNs (Barnes and Sharp, 1999). 5-HT1AR agonists have been shown to sub- to the anti-dyskinetic actions of the F-compounds. The current findings stantially reduce LID in parkinsonian rat and primate models (Bibbiani demonstrate that both compounds exert anti-LID effects in the striatum, et al., 2001; Carta et al., 2007; Eskow et al., 2007; Lindenbach et al., but with varying 5-HT syndrome profiles and differential effects on D1R 2015). However, the clinical use of 5-HT1AR agonists, like sarizotan agonist stimulated dyskinesia highlighting the roles of 5-HT1ARpopula- and tandospirone, has been limited by modest effects and worsening tions in the dyskinetic brain. parkinsonism in some patient populations (Goetz et al., 2007; Kannari et al., 2002; Merck KGaA, 2006; Olanow et al., 2004; Svenningsson et 2. Materials and methods al., 2015). These findings are likely attributable to off-target antagonistic effects at DA D2,D3, and D4 receptors and excessive agonist stimulation 2.1. Animals of 5-HT1A auto-receptors which may suppress L-DOPA-derived ‘false ’ neurotransmitter DA release to an extent that compromises L-DOPA ef- The study utilized adult male Sprague Dawley rats (N = 35; 225– fi cacy (Bartoszyk et al., 2004; Eskow et al., 2009; Hamik et al., 1990; 250 g upon arrival, Harlan Farms, NY, USA) housed in plastic cages Kannari et al., 2001). The pre-clinical effects of more selective 5- (22 cm high, 45 cm deep, and 23 cm wide) with free access to standard HT1AR agonists, like +8-OH-DPAT, are also complicated by increased laboratory chow (Rodent Diet 5001; Lab Diet, Brentwood, MO, USA) and fl rigidity and akinesia, though this may, in part, re ect the induction of water. The colony room was maintained on a 12 h light/dark cycle (light 5-HT syndrome, a debilitating motor side effect seen in rodents and on at 0700 h) at a temperature of 22–23 °C. Animals were maintained in primates attributed to post-synaptic 5-HT1AR stimulation (Diaz and accordance with the guidelines of the Institutional Animal Care and Use Maroteaux, 2011; Iravani et al., 2006; Jacobs and Klemfuss, 1975; Committee of Binghamton University and the ‘Guide for the Care and Lindenbach et al., 2015). Although 5-HT1AR-mediated 5-HT Use of Laboratory Animals’ (Institute of Laboratory Animal Resources, syndrome is rare in humans (Habberzettl et al., 2013), such collec- National Academic Press, 2011). tive limitations have slowed the translation of 5-HT1ARagoniststo clinical employment. 2.2. Medial forebrain bundle 6-hydroxydopamine lesion and bilateral 5-HT R populations express functional G-protein heterogeneity, 1A striatal cannulation surgery likely responsible for regionally-specific 5-HT1AR agonist effects. Ago- nists could act at auto-receptors on 5-HT neurons, directly blunting All rats underwent surgery to receive unilateral 6-hydroxydopamine ‘false neurotransmitter’ DA release, or at hetero-receptors residing on (6-OHDA) lesions to the left medial forebrain bundle. Five minutes prior non-5-HT neurons, reducing LID by altering other neurotransmitter sys- to surgery and the morning after, rats were injected with buprenex tems, like glutamate (Dupreetal.,2011;Eskowetal.,2007;Lindgrenet (buprenorphine HCl; 0.03 mg/kg, i.p.; Reckitt Benckiser Pharmaceuti- al., 2010; Ostock et al., 2011). Relatedly, 5-HT auto- and hetero-recep- 1A cals Inc., Richmond, VA) to minimize pain. Rats were anesthetized tors have differential G-protein-mediated effects on downstream signal with inhalant isoflurane (2–3%; Sigma) in oxygen (2.5 L/min) and transduction, allowing the identification of functionally selective ‘bi- placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, ased’ 5-HT R agonists for more precise pharmacologic and neuroana- 1A CA, USA) with the incisor bar positioned at 5.0 mm below the interaural tomical manipulations (Newman-Tancredi, 2011; Kenakin, 2011). line. The targeted site, relative to bregma, was: AP, −1.8 mm; ML, While 5-HT auto-receptors reside on raphe-striatal projection neu- 1A +2.0mm;DV,−8.6 mm (Paxinos and Watson, 1998). A 10 μLHamilton rons, there is little to no evidence that they exist in the striatum syringe attached to a 26 gauge needle was lowered into the target (Azmitia et al., 1996). On the other hand, striatal 5-HT hetero-recep- 1A through a small burr hole in the skull and then used to deliver 6- tors are more abundant and seem to be located on a combination of OHDA (3 μg/μL; Sigma) dissolved in 0.9% NaCl + 0.1% ascorbic acid at cortico-striatal glutamatergic neurons (Cruz et al., 2004; Huot et al., arateof2μL/min, for a total volume of 4 μL. The needle was withdrawn 2012), medium spiny neurons (MSNs; Ghavami et al., 1999), and cho- 5 min post-injection to allow time for fluid diffusion. During the same linergic interneurons (ChIs; Virk et al., 2016). Given the central role of surgery, animals in experiment 2 were fitted with bilateral 15 mm the striatum in LID, precise pharmacologic tools, such as the G-protein guide cannulae (22 gauge, C313/G/SPC; Plastics One Inc., Roanoke, biased agonists F13714 and F15599, are invaluable for clarifying the VA), targeting the dorsal striatum (AP, +0.4 mm; ML, ±2.9 mm; DV, role(s) of these local 5-HT R populations. Initial characterizations of 1A −3.6 mm; relative to bregma; Paxinos and Watson, 1998). Cannulae F13714 and F15599 revealed regionally specific electrophysiological were affixed to the skull with screws and Jet Denture Repair Acrylic and neurochemical effects in raphe and cortex, respectively, leading to (Lang Dental, Wheeling, IL). The guide cannulae were then fitted with the conclusion that at low doses these compounds are selective for ei- 28-gauge inner stylets (Plastics One) to avoid cannulae obstruction ther 5-HT auto- or hetero-receptor (Assié et al., 2006; Buritova et al., 1A and infection. Upon completion, animals in experiment 1 and 3 were 2009; Lladó-Pelfort et al., 2010). Each compound exhibits a unique con- dual-housed, while rats in experiment 2 were single-housed, placed in trol of signal transduction responses whereby F13714 shows secondary clean cages and given a 21-day recovery period with ad lib food and potency for stimulating h5-HT receptor internalization, purportedly 1A water. Prior to all testing, animals were handled to minimize stress dur- inducing rapid onset of 5-HT desensitization, while F15599 has sec- 1A ing the experiment. ondary potency for G-protein activation in extra-raphe structures (Assié et al., 2006; Newman-Tancredi et al., 2009). Previous studies have assessed the ability of these compounds to modify LID (Iderberg 2.3. Pharmacological treatments and priming et al., 2015) and elicit rodent 5-HT syndrome (Assié et al., 2010), yet very little work has examined the temporal profile for 5-HT syndrome Three weeks post-lesion surgery, rats were tested on the Forepaw and its association with LID reductions. Moreover, while some striatal Adjusting Steps test (FAS; see below) on 2 separate occasions to estab- microdialysis data has been collected on these compounds, no previous lish baseline motor performance and lesion severity. Lesioned animals studies have delivered these drugs directly into the striatum to deter- in experiments 1 (Fig. 1A), 2 (Fig. 1B), and 3 (Fig. 1C) were administered mine site-specific contributions of 5-HT1ARsub-populationstoLID L-DOPA methyl ester (L-DOPA; 6 mg/kg, s.c.; Sigma) + DL-serine 2- reduction. (2,3,4-trihydroxybenzyl) hydrazide hydrochloride (benserazide; 170 S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178

Fig. 1. Experimental timeline and design for systemic administration and striatal microinjection of biased 5-HT1A receptor agonists F13714 and F15599. In Experiments 1 and 2, animals were rendered hemi-parkinsonian via medial forebrain bundle administration of 6-OHDA, whilst animals in Experiment 2 underwent extended surgery to implant bilateral striatal cannulae for microinjections. After 3 weeks of recovery, animals received daily L-DOPA (6 mg/kg, s.c.) for 14 days to establish stable dyskinesia. Forepaw Adjusting Steps (FAS) baseline was recorded on priming day 1 prior to first L-DOPA treatment and ALO AIMs were recorded on Days 1, 8, and 14. In a within subjects counter-balanced design, animals in Experiment 1 (A) received Vehicle, F13714 (0.01, or 0.02 mg/kg, s.c.) or F15599 (0.06, 0.12 mg/kg, s.c.) 5 min prior to L-DOPA (6 mg/kg, s.c.), after which AIMs, 5-HT Syndrome, and FAS were monitored. In Experiment 2 (B), animals were split into 2 equally dyskinetic cohorts. Each cohort received bilateral striatal microinjections of one of the F-series compounds in a within-subjects-counterbalanced design 5 min prior to L-DOPA (6 mg/kg, s.c.): Cohort 1 received Vehicle or F13714 (2 or 10 μg) and Cohort 2 received Vehicle or F15599 (10 or 30 μg). ALO AIMs and 5-HT syndrome were recorded on each testing day for 10 min every 180 min. In Experiment 3, L-DOPA-primed animals received a systemic injection of Vehicle, F13714 (0.01, or 0.02 mg/kg, s.c.) or F15599 (0.06, 0.12 mg/kg, s.c.) 5 min prior to SKF38393 (2 mg/kg, s.c.). Following drug administration, animals were observed for dyskinesia. Animals in Experiments 1, 2, and 3 were sacrificed off-treatment, after a significant wash-out period, and bilateral posterior striata (Post STR) were dissected for high performance liquid chromatography (HPLC) and anterior striata (Ant STR) were dissected for cannulae verification.

15 mg/kg, s.c.; Sigma) dissolved in 0.9% NaCl + 0.1% ascorbic acid once the conclusion of the study, rats were decapitated off-treatment and bi- daily for 14 days to induce stable LID (Conti et al., 2014; Putterman et al., lateral striata were collected for lesion veriﬁcation (Fig. 5). 2007). On days 1, 8, and 14 of L-DOPA priming, Axial, Limb, and

Orolingual (ALO) AIMs (see below) were recorded to establish stable 2.5. Experiment 2: effects of striatal administration of 5-HT1A biased ago- dyskinesia expression. On day 14, animals with ALO AIMs scores b 30 nists on LID expression were excluded from the studies. Both 5-HT1AR agonists, F13714 and F15599, were dissolved in distilled water (vehicle) for all experiments. Experiment 2 sought to distinguish how striatal populations of 5-

All systemic drugs were administered subcutaneously at a volume of HT1A receptors differentially modify LID. DA-lesioned rats with bilateral 1 mL/kg. Bilateral striatal microinjections were administered over striatal cannulae that also met ALO AIMs criteria (n = 17) were divided 2 min at a ﬂow rate of 0.5 μL/min to a total volume of 1.0 μL. into 2 equally dyskinetic cohorts. Prior to microinjections, animals were towel wrapped in the presence of the microinjection apparatus to habit- uate them to the restraint and sounds associated with the process. On

2.4. Experiment 1: effects of systemic administration of 5-HT1A biased ago- test days, both cohorts received bilateral striatal injections: cohort 1 nists on LID and L-DOPA motor efﬁcacy (n = 9) received F13714 (0, 2, or 10 μgin1μL) and cohort 2 (n = 8) were administered F15599 (0, 10, or 30 μgin1μL) in a within-subjects,

The goal of the first experiment was to verify the ability of 5-HT1AR counterbalanced design. Doses were based on previous work with these biased agonists F13714 (selective to 5-HT1A auto-receptors) and and like compounds (Bishop et al., 2009; Stein et al., 2013). For microin- F15599 (selective to 5-HT1A hetero-receptors) to alter LID expression. jections, animals were wrapped in a small towel, dummy cannulae were In a within-subjects, counterbalanced design, L-DOPA-primed rats removed and 16 mm injectors were inserted into each cannula, thereby meeting AIMs criteria (n = 10) were injected with vehicle, F13714 extending 1 mm past the guide tips. Injections persisted at a flow rate of (0.01 or 0.02 mg/kg, s.c.), or F15599 (0.06 or 0.12 mg/kg, s.c.) 5 min 0.5 μL/min for 2 min. This flow rate and volume has been shown to cause prior to L-DOPA (6 mg/kg, s.c.) + Benserazide (15 mg/kg, s.c.). Doses the liquid to only permeate the dorsal striatum (Lindenbach et al., were established by previous work (Iderberg et al., 2015). ALO AIMs 2011). Injectors were left in the guide cannulae for an additional and 5-HT syndrome (described below) were recorded every 10 min 3 min to allow the diffusion of the compounds before removal. Five mi- for 180 min post-L-DOPA. FAS were recorded 60 min after L-DOPA to ex- nutes later, L-DOPA (6 mg/kg, s.c.) + Benserazide (15 mg/kg, s.c.) was amine the biased agonists' effects on L-DOPA's therapeutic efficacy. Rats administered. ALO AIMs and 5-HT syndrome were recorded every were tested 2 days per week, separated by 3-day wash-out periods. At 10 min for 180 min after L-DOPA administration. Each cohort was tested S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178 171

2 days per week with 3 days of wash-out between test days. Two days 2.7.3. Forepaw Adjusting Steps (FAS) after the last cohort concluded testing, animals were sacrificed via de- The FAS test is a measure of rat forelimb akinesia, modeling clinical capitation. The brains were immediately extracted and posterior striata symptoms of PD. Rats with N80% striatal DA depletion perform poorly were collected, frozen at −80 °C, and later analyzed for monoamine on the task and L-DOPA treatment partially reverses lesion-induced def- analysis using HPLC-ED to confirm DA depletion. To verify cannulae icits (Chang et al., 1999; Conti et al., 2016). The experimenter held both placement, anterior striata were coronally bisected and immediately hind legs and one forepaw such that the rat was bearing its weight on frozen in methylbutane (−30 °C) and stored at –20 °C. Coronal sections the forepaw to be tested. Rats were then moved across the table at a (18 μm) containing injection sites were sliced on a cryostat and post- speed of 90 cm/10 s, during which an additional experimenter recorded fixed with 4% paraformaldehyde (Fisher Scientific, Hanover Park, IL) be- the number of adjusting steps taken on the weight-bearing forepaw. fore cresyl violet (FD Neurotechnologies, Baltimore, MD) staining for in- Rats were dragged for 6 trials per forepaw: 3 backhand (lateral steps jection site confirmation. away from the torso) and 3 forehand (lateral steps toward the torso) trials, alternating the starting forepaw between animals. For analysis, data was represented as the sum of the 3 trials per direction for each forepaw 2.6. Experiment 3: effects of 5-HT1A biased agonists on D1Ragonist-induced (i.e. left forehand). Fewer steps indicate greater lesion-induced akinesia. dyskinesia 2.8. High-performance liquid chromatography for monoamine tissue The aim of experiment 3 was to clarify the effects of 5-HT1ARbiasedag- analysis onists on D1R-mediated dyskinesia. In a within-subjects, counterbalanced design, L-DOPA-primed, hemi-parkinsonian rats (n = 8) were adminis- After dissection, tissue from experiments 1 and 2 was immediately tered vehicle, F13714 (0.01 or 0.02 mg/kg, s.c.), or F15599 (0.06 or frozen on dry ice and stored at −80 °C until preparation for high-perfor- 0.12 mg/kg, s.c.) 5 min prior to the D1R agonist SKF38393 (2 mg/kg, s.c.; mance liquid chromatography with electrochemical detection (HPLC- SKF). Beginning 10 min after injection of the SKF, and persisting every ED). Analysis of DA and 3,4-dihydroxyphenlyacetic acid (DOPAC) was 10 min thereafter, animals were rated for AIMs until 180 min post-injec- conducted according to the protocol of Kilpatrick et al. (1986). Samples fi tion. Testing days were separated by 3 days to allow for a suf cient were homogenized in 0.1 M perchloric acid, 1% ethanol, and 0.02% wash-out period. Two days after testing concluded, animals were EDTA. Homogenates were spun at 4 °C for 30 min at 14000 rpm. Ali- fi fi sacri ced off-treatment and striatal tissue was harvested for con rmation quots of the resulting supernatant were then analyzed for levels of DA of DA depletion via HPLC. and DOPAC using reverse-phase HPLC coupled to electrochemical detection as described in Bishop et al. (2012) and Barnum et al. (2012). 2.7. Behavioral analyses Monoamines and metabolites were detected as a generated current as a function of time by EZCHROM ELITE software via a ScientificSoftware, χ 2.7.1. Abnormal Involuntary Movements (AIMs) Inc. (SS240 ) module. The resulting plots represented monoamines and Rats were monitored for rodent dyskinesia, using a protocol de- metabolites as peak areas, which were calculated using the EZCHROM scribed previously (Bishop et al., 2012) and initially modified from ELITE software, and then compared to a standard curve made from sam- − Lundblad et al. (2002). On test days (0900–1500 h), animals were ples of known monoamine and metabolite concentrations (1E 6to − placed in individual plexiglass cylinders (20 × 25 cm) immediately fol- 1E 9 M) to determine picograms (pg) of compound. Values were nor- malized to tissue weight and lesion deficits reported as percent deple- lowing L-DOPA administration. Beginning 10 min post-L-DOPA a trained observer, blind to treatment condition, assessed each rat for the pres- tion: ence of axial, limb, and orolingual (ALO) AIMs. Axial AIMs are characterized by the forced turning of the torso 90o, relative to trunk, M %Depletion ¼ 100 1− lesion contralateral to lesion. Forelimb AIMs refer to the excessive, uncontrol- Mintact lable movement of the fist and forelimb contralateral to lesion. Orolingual AIMs were quantified as repetitive tongue protrusions and jaw tremors in the absence of an external stimulus (i.e. bedding or food). Each AIM is given a severity score: 0, behavior not present; 1, be- 2.9. Statistical analysis havior present b50% of the rating period; 2, behavior present for ≥50% but b100% of the observation period; 3, behavior present for entire rat- ALO AIMs and 5-HT syndrome (expressed as medians ± median ab- ing period, but can be interrupted by a loud stimulus (pencil tap on cyl- solute difference; M.A.D.) were analyzed using non-parametric Fried- inder); or 4, behavior present for the entire observation period and man ANOVAs for treatment differences. Parametric one-way ANOVAs undisrupted by the stimulus. Observations occurred for 60 s/animal. An- were used to analyze FAS (treatment) data. Paired samples t-tests were used to evaluate HPLC (intact vs. lesion) data (expressed as imals were observed every 10 min post-L-DOPA for a total duration of 180 min. Data was analyzed as a summation of axial, limb, and means ± standard mean error; SEM). When appropriate, non-paramet- fi orolingual scores to create a single ALO AIMs score for each rating, ric Wilcoxon and parametric Fisher least signi cant difference post-hoc with a maximum of 12. ALO scores for an animal were summed across tests were employed. All statistical analyses were performed with the time to create a singular ALO sum for the test day. use of SPSS Statistics 22 (IBM, Armonk, NY, USA) with alpha set to 0.05.

3. Results 2.7.2. 5-HT syndrome Due to the high afﬁnity and speciﬁcity of the compounds tested for 3.1. Experiment 1

5-HT1AR, rats were also observed for signs of 5-HT syndrome. Using a modified protocol from Lindenbach et al. (2015), trained observers 3.1.1. Systemically administered F13714 and F15599 reduce LID, with vary- coded the presence of lower lip retraction, flat body posture, forepaw ing 5-HT syndrome profiles treading, and straub tail. The scale rated each subtype as either 0 (not In order to establish the effectiveness of the F-series compounds to present) or 1 (present). Observations of 5-HT syndrome were recorded diminish established LID, L-DOPA-primed hemi-parkinsonian rats concurrent with ALO AIMs. The maximum score for each time point was were administered vehicle, F13714, or F15599 5 min prior to L-DOPA 4 and all of the time points were summed to create a single sum for and then tested for AIMs and 5-HT syndrome. Statistical analyses re- analysis. vealed that F13714 dose-dependently reduced total ALO AIMs (χ2(2) 172 S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178

Fig. 2. Effects of systemically delivered biased 5-HT1A receptor agonists F13714 and F15599 on LID (A & C, respectively) and 5-HT syndrome (B & D, respectively). L-DOPA primed, 6- hydroxydopamine (6-OHDA) lesioned rats (n = 10) received, in a within subjects, counterbalanced design, either Vehicle, the biased 5-HT1A auto-receptor agonist F13714 (0.01 or

0.02 mg/kg, s.c.) or the biased 5-HT1A hetero-receptor agonist F15599 (0.06 or 0.12 mg/kg, s.c.) 5 min prior to L-DOPA (6 mg/kg, s.c.). Axial, limb and orolingual abnormal involuntary movements (ALO AIMs) and 5-HT syndrome were scored for 1 min every 10 min for 180 min thereafter. Values are expressed as medians [+ median absolute difference (M.A.D.)]. Non-parametric (AIMs, 5-HT Syndrome) statistics were run with post-hocs as appropriate. *p b 0.05 Veh vs High; +p b 0.05 Low vs High; ^p b 0.05 Veh vs Low.

N 19.538, p b 0.05; Fig. 2A), whereas F15599 only significantly reduced microinjection of the compounds, prior to L-DOPA. Changes in the se- LID at the highest dose (χ2(2) N 13.400, p b 0.05; Fig. 2C). verity of rodent dyskinesia and 5-HT syndrome were quantified and Time course analyses were performed to assess the temporal rela- then summed for the biased agonists F13714 (Fig. 4A) and F15599 tionship between 5-HT syndrome and LID. When animals received (Fig. 4C). Rats receiving intra-striatal F13714 exhibited an overall F13714 at 0.02 mg/kg, dyskinesia suppression lasted 120 min (Fig. 2A; dose-dependent attenuation of LID (χ 2(2) = 15.9, p b 0.05; Fig. 4A). all p b 0.05), but was accompanied by severe 5-HT syndrome for the Statistical analysis revealed however, that F13714 also dose-dependent- first 100 min (Fig. 2B; all p b 0.05). When administered the lower ly induced 5-HT syndrome (χ2(2) = 17.5, p b 0.05; Fig. 4B). Administra- dose of F13714, 0.01 mg/kg, the anti-dyskinetic effects were maintained tion of the high dose (30 μg/μL) of F15599 yielded significant reductions for 80 min (Fig. 2A: all p b 0.05) with mild 5-HT syndrome persisting for in LID (χ2(2) = 7.8, p b 0.05; Fig. 4C), and trended toward being dose- 30 min (Fig. 2B; all p b 0.05). The higher dose of F15599 moderately re- dependent (p = 0.09). Interestingly, rats treated with F15599 manifest duced LID for 90 min (Fig. 2C; all p b 0.05), while producing mild 5-HT less LID and mild 5-HT syndrome with the high dose. No behavioral ef- syndrome for 60 min (Fig. 2D; all p b 0.05). Importantly the low dose of fects were observed with the low dose (10 μg/μL; Fig. 4D). F15599 (0.06 mg/kg, s.c.) reduced LID onset for the first 50 min (Fig. 2C; An evaluation of the time courses of 5-HT syndrome and LID severi- all p b 0.05), and was not accompanied by any significant level of 5-HT ty, as shown in Fig. 4, clarify the degree to which the two behaviors syndrome (Fig. 2D). temporally coincided. Rats administered the high and low dose of F13714 exhibited LID suppression to 150 min and 50 min, respectively 3.1.2. F13714 and F15599 do not affect L-DOPA's motor efficacy on the FAS (Fig. 4A; all p b 0.05), whilst moderate to severe 5-HT syndrome test persisted for approximately the same duration (Fig. 4B; all p b 0.05).

Given reports of lost L-DOPA efficacy with pan-5-HT1AR agonists, the In comparison, after administration of F15599, rats that received the FAS test was performed to determine if the co-administration of biased high dose exhibited a delay in the onset of LID until the 90-minute b 5-HT1AR agonists F13714 and F15599 affected L-DOPA-related motor time period (Fig. 4C; all p 0.05), with only mild displays of 5-HT improvements. As displayed in Fig. 3, L-DOPA treatment significantly in- syndrome for 40 min (Fig. 4D). creased total lesioned forehand steps and this was not significantly al- b tered by F13714 or F15599 (F(5,54) = 3.958, p 0.05). 3.3. Experiment 3

3.2. Experiment 2 3.3.1. F13714, but not F15599 reduces D1R agonist-induced dyskinesia To clarify the impact of biased 5-HT1ARs on post-synaptic D1R-stim- 3.2.1. Intra-striatal F13714 and F15599 dose-dependently convey anti-dys- ulated dyskinesia, L-DOPA primed, hemi-parkinsonian rats were admin- kinetic and side effect proﬁles istered vehicle, F13714 (0.01 or 0.02 mg/kg, s.c.), or F15599 (0.06 or

The role of striatal populations of 5-HT1ARs in the anti-dyskinetic ef- 0.12 mg/kg, s.c.) 5 min prior to SKF (2 mg/kg, s.c.), after which dyskine- fects of F13714 and F15599 was determined via intra-striatal sia was measured. Statistical analyses revealed that while the high dose S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178 173

Fig. 3. Effects of the systemically administered biased 5-HT1A receptor agonists F13714 (A & B) and F15599 (C & D) on forepaw adjusting steps (FAS). Prior to L-DOPA priming, the baseline performance of L-DOPA-naive 6-hydroxydopamine (6-OHDA) lesioned rats (n = 10) on the FAS task was recorded. After 14 days of L-DOPA priming, rats received, in a within subjects, counterbalanced design, either the biased 5-HT1A auto-receptor agonist F13714 (Vehicle, 0.01 or 0.02 mg/kg, s.c.) or the biased 5-HT1A hetero-receptor agonist F15599 (Vehicle, 0.06 or 0.12 mg/kg, s.c.) 5 min prior to L-DOPA (6 mg/kg, s.c.). Forepaw adjusting steps were recorded 60 min after L-DOPA administration to measure L-DOPA's anti-parkinsonian efﬁcacy. Values are expressed as the mean overall steps [+ standard error of the mean (S.E.M.)]. Parametric ANOVAs were run with post-hocs as appropriate. *p b 0.05 vs all. of F13714 reduces DA-agonist induced dyskinesia (χ 2(2) = 6.75, p b experiment 2, cohort 1 animals (F13714-treated; n = 9) showed

0.05; Fig. 6A), F15599 did not significantly alter dyskinesia expression 99.2% DA (Mintact = 8136.58, Mlesion = 64.08, t8 = −9.745, p b 0.05) (Fig. 6B). Time course analysis shows that F13714 significantly and 86.3% DOPAC (Mintact = 3074.77, Mlesion = 421.45, t8 = −3.694, p reduced ALO AIMs for the first 30 min (all p b 0.05), but effects subside b 0.05) depletion as a result of 6-OHDA lesion. Similar effects were after that. seen in cohort 2 (F15599-treated; n = 8), with lesion-specificDA

(Mintact = 9527.52, Mlesion =86.17,t7 = −8.298, p b 0.05) and 3.4. Post-mortem lesion efficacy and cannulae verification DOPAC (Mintact = 2499.84, Mlesion = 84.97, t7 = −9.41, p b 0.05) deficits of 99.1% and 96.6%, respectively. Anterior striatum was sliced and ana- The severity of 6-OHDA lesions was measured by post-mortem anal- lyzed for cannulae placements using cresyl violet staining. All injection ysis of DA and DOPAC in lesion (left) versus intact (right) striata. Rats in- sites were verified to be within dorsal striata and are represented sche- cluded in experiment 1 showed significant reductions in striatal DA matically in Fig. 5. Animals in experiment 3 exhibited DA and DOPAC

(Mintact = 8706.60, Mlesion =51.50,t9 = −27.547, p b 0.05) and depletions of 99.4% (Mintact = 12,757.4, Mlesion = 70.73, t7 = −9.291, DOPAC (Mintact = 1856.58, Mlesion = 105.06, t9 = −21.594, p b 0.05), p b 0.05) and 96.4% (Mintact = 3100.86, Mlesion = 131.96, t7 = − 99.4% and 94.3%, respectively, between their lesion and intact STR. In 15.328, p b 0.05), respectively. 174 S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178

Fig. 4. Effects of intrastriatal microinjections of the biased 5-HT1A receptor agonists F13714 and F15599 on LID (A & C, respectively) and 5-HT syndrome (B & D, respectively). Two groups of L-DOPA primed, 6-hydroxydopamine (6-OHDA) lesioned rats (n = 8–9/group) received, in a within subjects, counterbalanced design, intrastriatal microinjections of either the biased 5-

HT1A auto-receptor agonist F13714 (Vehicle, 2 or 10 μgin1μL) or the biased 5-HT1A hetero-receptor agonist F15599 (Vehicle, 10 or 30 μgin1μL) after which rats were treated with L-DOPA (6 mg/kg, s.c.). Axial, limb and orolingual abnormal involuntary movements (ALO AIMs), rotations and 5-HT syndrome were scored every 10 min for 3 h thereafter. Values are expressed as medians [+ median absolute difference (M.A.D.)].Non-parametric Friedman's ANOVAs were run with post-hocs as appropriate. *p b 0.05 Vehicle vs High; +p b 0.05 Low vs High; ^p b 0.05 Vehicle vs Low.

4. Discussion widespread clinical application has been limited due to worsening parkinsonism and potential side effects (Bibbiani et al., 2001; Dupre et al., The role of the 5-HTergic system in LID has been well-characterized 2007; Goetz et al., 2007; Grégoire et al., 2009; Kannari et al., 2002; leading to the study of 5-HT1AR agonists as possible therapeutic agents. Lindenbach et al., 2015; Olanow et al., 2004; Yoshida et al., 1998). A While the anti-dyskinetic actions of 5-HT1AR agonists have been exam- need therefore remains to test and investigate the mechanisms of selec- ined and conﬁrmed in several pre-clinical models and PD patients, tive and highly efﬁcacious 5-HT1AR agonists that may exhibit superior

Fig. 5. A schematic of coronal section of rat brain depicting bilateral striatal microinjection sites. The schematic was modiﬁed from a coronal slice at +0.48 mm relative to bregma from

Paxinos and Watson (1998). Dark and light ovals indicate cannulae placements for animals receiving the 5-HT1A receptor biased agonists F13714 and F15599, respectively. Relevant anatomical structures are: CC, corpus callosum; LV, lateral ventrical; CPu, caudate putamen. S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178 175

implicating the dorsal raphe nucleus (DRN) as a viable target for LID management (Berger et al., 1985; Eskow et al., 2009; Navailles et al., 2010), we could not exclude the likelihood that F13714 may also exert effects in other areas of the basal ganglia. Recent functional magnetic resonance imaging data has shown that in an intact rodent model, administration of F13714 results in greater blood oxygenation level dependent signaling in the thalamus, motor cortex, and ventrolateral striatum, exemplifying multiple alternative regions for the anti-LID effects of the compound (Becker et al., 2016). Additionally, in primates, positron emission tomography (PET) with [18F]F13714 showed pronounced binding potential in the DRN, caudate, putamen, and thalamus, recapitulating the fMRI effects seen in rodent (Yokoyama et al., 2016). Due to the established role of the striatum in LID manifestation, the

present work posited that stimulation of striatal 5-HT1ARs via F13714 was a viable locus for the anti-LID effects seen from systemic administration (Dupre et al., 2008; Dupre et al., 2011; Rylander et al., 2010).

The present investigation also employed the biased 5-HT1AR agonist, F15599, which purportedly targets 5-HT1A hetero-receptors of the forebrain at doses under 0.04 mg/kg, with increasing activation of auto-receptors as dose increases (Lladó-Pelfort et al., 2010; Newman-Tancredi et al., 2009). In the current study, systemic administration of F15599 delayed LID onset at 0.06 and 0.12 mg/kg, but with pronounced effects on overall LID observed at the higher dose. Consis- tent with previous findings in rat and macaques (Huot et al., 2015; Iderberg et al., 2015), the current results suggest that anti-dyskinetic efficacy of F15599 is dependent on full hetero- and mild auto-receptor re- cruitment. Systemic administration of F15599 suppressed LID with minimal 5-HT syndrome and no detrimental effects on motor performance, demonstrating an optimal preclinical therapeutic profile that, in future work, may bypass the detrimental effects of indiscriminate

5-HT1A agonists on L-DOPA's promotor beneﬁts. Fig. 6. Effects of systemically delivered biased 5-HT1A receptor agonists F13714 and Interestingly, local striatal administration of F13714 and F15599 F15599 on DA-agonist-induced dyskinesia (A & B, respectively). L-DOPA primed, 6- conﬁrmed that the anti-dyskinetic actions and the onset of 5-HT syn- OHDA lesioned rats (n = 8) received, in a within subjects counterbalanced design, drome from these compounds are both mediated at least in part within either the biased 5-HT1A auto-receptor agonist F13714 (Vehicle, 0.01 or 0.02 mg/kg, s.c.) the striatum, providing additional support to prior work with non-bi- or the biased 5-HT1A hetero-receptor agonist F15599 (Vehicle, 0.06 or 0.12 mg/kg, s.c.) 5 min prior to L-DOPA (6 mg/kg, s.c.). Axial, limb and orolingual abnormal involuntary ased 5-HT1AR agonists (Bishop et al., 2009; Dupre et al., 2011). While movements (ALO AIMs) and 5-HT syndrome were scored for 1 min every 10 min for the effects of F13714 may be interpreted as support for terminal 180 min thereafter. Values are expressed as medians [+ median absolute difference raphe-striatal 5-HT1ARs, it is unlikely due to their terminal absence in (M.A.D.)]. Non-parametric Friedman ANOVAs were run with post-hocs as appropriate. *p b 0.05 Veh vs High; +p b 0.05 Low vs High; ^p b 0.05 Veh vs Low. numerous neuroanatomical investigations (Mengod et al., 1996; Pompeiano et al., 1992; Vergé et al., 1986). Thus, it is likely that

F13714 and F15599 are both acting at 5-HT1A hetero-receptors in striatum. Whilst this may seem antithetical, given the reported specificity of fi profiles for therapeutic application. Our present findings reinforce the F13714 for 5-HT1A auto-receptors, the variable 5-HT syndrome pro les between the compounds suggest that they differentially stimulate sub- role of the striatal 5-HT1AR in LID. Furthermore, our study builds upon populations of hetero-receptors, maintaining unique pharmacologic recent work demonstrating that systemic stimulation of 5-HT1A auto- or hetero-receptors via F13714 and F15599, respectively, can differen- profiles. Newman-Tancredi et al. (2009) have reported that, in vitro fi tially modulate LID expression without compromising L-DOPA efficacy and ex vivo, F13714 and F15599 have regional speci city for either (Iderberg et al., 2015). Finally, for the first time, we have shown that dis- the raphe or the cortex, respectively, suggesting ‘biased’ activation of auto- versus hetero-receptors. It appears, however, that in striatum, tinct striatal subpopulations of 5-HT1ARs contribute to the behavioral ef- the ‘biased’ nature of these compounds is less clear, implicating that in- fects of these compounds, providing further validation of 5-HT1ARas promising clinical targets. stead of auto- versus hetero-receptor ‘biased’ agonism, the ‘biased’ nature of these drugs may refer more to their potential to potently and

preferentially target different subpopulations of 5-HT1A hetero-recep- 4.1. Localization of anti-LID actions of F13714 and F15599 tors. 5-HT1A hetero-receptors are located on a number of different neuronal populations across the patch-matrix of the striatum. Interestingly,

F13714 is a highly efficacious biased 5-HT1AR agonist with preferen- 5-HT1ARs are reportedly up-regulated in striosomes of parkinsonian and tial stimulation of pre-synaptic somatodendritic auto-receptors in the dyskinetic primates (Frechilla et al., 2001; Huot et al., 2012), which may dorsal raphe nucleus (DRN; Assié et al., 2006; Newman-Tancredi et al., contribute to the anti-LID efficacy of 5-HT1AR agonists. This primarily 2009). In the present study, we showed that systemic administration implicates two neuronal populations for the effects of the compounds: of F13714 dose-dependently reduced LID without compromising the GABAergic MSNs and glutamatergic pyramidal neurons. therapeutic effects of L-DOPA. Based on the signal transduction profile A very likely candidate for the anti-LID effects seen with striatal ad- of F13714 (Newman-Tancredi et al., 2009), systemic administration of ministration was somatodendritic post-synaptic hetero-receptors on this compound may act to reduce LID by activating 5-HT1A auto-recep- GABAergic MSNs (Ghavami et al., 1999). In the dyskinetic striatum, tors thereby attenuating ectopic ‘false neurotransmitter’ DA release preprodynorphin (PPD) mRNA, a marker of direct pathway activation, from raphe-striatal serotonergic neurons (Carta et al., 2007; Lindgren and glutamic acid decarboxylase 67 kDa isoform (GAD67) mRNA, a et al., 2010). While these effects support a large body of research marker of GABA release, are both increased, suggesting that direct 176 S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178

pathway overactivity drives excessive movement (Cenci et al., 1998). have contributed to the failure of previous 5-HT1AR-related anti-dyski- Several experiments have used non-specific5-HT1AR agonists to reduce netic strategies (Kannari et al., 2002). In this study, we had the unique LID and normalize striatal GAD67 and PPD (Bishop et al., 2009; opportunity to investigate 5-HT1AR-mediated 5-HT syndrome in rat Tomiyama et al., 2005). However, Dupre et al. (2013) showed that using 5-HT1AR biased agonists as investigational probes. We found these compounds do not have to normalize aberrant direct pathway both drug- and dose-dependent 5-HT syndrome was more pronounced gene expression to effectively reduce DA agonist-induced dyskinesia. with F13714. Prior work from our group has closely examined 5-HT Therefore, in the present work we tested the efficacy of the F-com- syndrome in rat with an emphasis on its impact in the clinical popula- pounds to reduce D1-agonist (SKF38393)-induced dyskinesia, at a tion (Lindenbach et al., 2015). It seems that while the clinical literature dose previously shown to produce dyskinesia (Iderberg et al., 2013). In- does not explicitly mention 5-HT syndrome, it may also account for terestingly, systemically administered F13714 significantly reduced non-motor adverse effects, like nausea, vomiting, and excessive sweat- total ALO AIMs, suggesting mild actions at 5-HT1AR located on D1R-ex- ing, reported by dyskinetic patients receiving 5-HT1A agonists (Politis et pressing MSNs. This confirms our hypothesis that F13714 loses its spec- al., 2014). In agreement with the current data, F13714 has been previ- ificity for 5-HT1A auto-receptors in striatum, however the data doesn't ously reported to elicit significantly greater 5-HT syndrome than explain the long duration of the anti-LID effects seen in Experiment 1. F15599 (Assié et al., 2010). While this is surprising, due to the

Even more intriguing, F15599 failed to alter D1R-induced dyskinesia, established role of 5-HT1A hetero-receptors in 5-HT syndrome, the in- implying that F15599 doesn't modulate LID through reduction of post- duction of these behaviors observed following both acute systemic synaptic D1-bearing MSN activity. While testing the F-series compounds and intra-striatal F13714 over F15599 provides additional evidence against DA-agonist induced dyskinesia was useful in identifying the that these compounds maintain functional selectivity for unique popu- contribution of 5-HT1ARs located on D1-bearing MSNs, DA-agonist-in- lations of striatal 5-HT1A hetero-receptors (Lucki, 1992; Manos, 2000; duced dyskinesia does not recapitulate other changes to striatal signal- Nakayama et al., 2014; Volpi-Abadie et al., 2013). However this could ing that drive LID, like excessive extracellular striatal glutamate also have led to recent reports of motor performance deficits on the (Robelet et al., 2004; Dupre et al., 2011). Thus, while we can conclude rotarod task at doses of F13714 equal to or higher than those eliciting the relative contributions of D1 MSNs to the effects of F13714, the effect 5-HT syndrome in our study (Iderberg et al., 2015). For example, flat size was modest, at best, and the conclusion that F13714 acts exclusive- body posture, one of the cardinal signs of rat 5-HT syndrome, may ly post-synaptically is unlikely. have promoted deficits in rotarod performance that we did not observe

A substantial body of research has shown pre-synaptic 5-HT1A het- with the stepping test (Boyer and Shannon, 2005; Ener et al., 2003; ero-receptors are located on corticostriatal glutamatergic pyramidal Jacobs and Klemfuss, 1975). neuron axon hillocks (Azmitia et al., 1996; Huot et al., 2012; Mengod This said, severe 5-HT syndrome is quite rare in humans and often et al., 1996). Due to its known afﬁnity for cortical 5-HT1ARs, systemically requires either increased 5-HT tone and/or selective actions at 5-HT2 re- administered F15599 may attenuate motor cortex M1-mediated over- ceptors for full expression (Boyer and Shannon, 2005; Gillman, 2010). activation of the striatum by decreasing cortico-striatal pyramidal neu- How the prominent rat 5-HT syndrome observed in the present study ron excitability (Newman-Tancredi et al., 2009; Lladó-Pelfort et al., predicts clinical translation of 5-HT1AR agonists as anti-dyskinetic ther- 2010; Ostock et al., 2011). However, AIMs data from the intra-striatal apies is unclear. Patients are routinely prescribed anti-dyskinetic medi- microinjections mirrored the effects of the systemic administration, cation chronically and most features of rodent 5-HT syndrome abate suggesting a substantial role for the striatum in these effects. Thus, over time (Habberzettl et al., 2013; Iderberg et al., 2015). Nonetheless,

F15599 may stimulate pre-synaptic 5-HT1A hetero-receptors located it does necessitate close observation of motor symptoms recapitulating on cortico-striatal terminals, resulting in a normalization of overactive 5-HT syndrome upon clinical application of high-efﬁcacy 5-HT1ARago- striatal glutamatergic activity associated with LID (Ahmed et al., 2011; nists for LID management.

Bishop et al., 2009). In fact, general 5-HT1A agonists, like ±8-OH- DPAT, have been shown to decrease striatal glutamate when administered systemically or intrastriatally prior to L-DOPA (Dupre et al., 5. Conclusions 2011). However, previous work did not show any signiﬁcant effects on striatal glutamate in response to systemic L-DOPA or NLX-112 In conclusion, by employing the novel 5-HT1AR biased agonists (a.k.a. beﬁradol), a chemical congener of F13714 and F15599 F13714 and F15599, the present work demonstrates that stimulation

(McCreary et al., 2016). of heterogeneous striatal 5-HT1AR populations provides substantial Another possible locus for the effects of striatal F15599 and F13714 anti-LID efﬁcacy. While systemic F13714 and F15599 preferentially acti- are ChIs that have been more recently implicated in LID (Bordia et al., vate DRN and cortical 5-HT1ARs, respectively, it seems likely that their 2016; Ding et al., 2011). However, whereas a substantial percentage of actions in striatum primarily drive their anti-LID actions. Though both

ChIs in ventral striatum contain 5-HT1ARs, only a small percentage of compounds likely work through stimulation of 5-HT1A hetero-recep- ChIs (~1.5%) in the dorsal striatum contain these receptors, suggesting tors, their distinct behavioral profiles suggest variable efficacy at sub- that ChIs in the dorsal striatum are not responsible for the motor actions populations of 5-HT1ARs. Whether biased agonism is optimal for of 5-HT1AR agonists (Gonzales and Smith, 2015; Virk et al., 2016). Final- clinical application remains to be determined, but, NLX-112 (befiradol), ly, the conclusion that only one of the aforementioned mechanisms is a compound structurally related to those investigated here, is a highly working in isolation to reduce LID would be an oversimplification of selective full 5-HT1A agonist currently being examined as an anti-LID the complicated striatal circuitry, more likely that there are multiple agent. The present data suggest that such a compound may differential- points of neuroarticulation that convey the striatal effects of biased 5- ly recruit multiple populations for optimal efficacy. Thus, continued

HT1ARagonists. study of the underlying mechanisms of 5-HT1AR signaling in the dyskinetic brain will advance therapeutic prospects for treatment of LID and

4.2. Implications of rat 5-HT syndrome for clinical translation of 5-HT1AR other disorders where 5-HT dysfunction is implicated. agonists

In humans, the incidence and mechanisms of drug-induced 5-HT Disclosure statement syndrome, sometimes called 5-HT toxicity, continue to be debated (Boyer and Shannon, 2005; Gillman, 2010). The motor symptoms of SMM, NC, MC, SCB, CT, ES, and CB have no disclosures or conﬂicts of 5-HT syndrome, especially tremor and rigidity, are also symptoms of interest to report. MV and ANT are employees and stockholders at PD. Therefore, it is possible that 5-HT syndrome related effects may Neurolixis Inc. S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178 177

Funding source Cruz, D.A., Eggan, S.M., Azmitia, E.C., Lewis, D.A., 2004. Serotonin1A receptors at the axon initial segment of prefrontal pyramidal neurons in schizophrenia. Am. J. Psychiatry 161 (4), 739–742. This research did not receive any specific grant from funding agen- Diaz, S.L., Maroteaux, L., 2011. Implication of 5-HT(2B) receptors in serotonin syndrome. cies in the public, commercial, or not-for-profitsectors. Neuropharmacology 61 (3), 495–502. Ding, Y., Won, L., Britt, J.P., Lim, S.A., McGehee, D.S., Kang, U.J., 2011. Enhanced striatal cholinergic neuronal activity mediates L-DOPA-induced dyskinesia in parkinsonian mice. Proc. Natl. Acad. Sci. U. S. A. 108 (2), 840–845. Acknowledgements Dupre, K.B., Eskow, K.L., Negron, G., Bishop, C., 2007. The differential effects of 5-HT(1A) receptor stimulation on dopamine receptor-mediated abnormal involuntary move- – The authors would like to thank David Lindenbach for expertise with ments and rotations in the primed hemiparkinsonian rat. Brain Res. 1158, 135 143. Dupre, K.B., Eskow, K.L., Barnum, C.J., Bishop, C., 2008. Striatal 5-HT1A receptor stimula- the striatal microinjection surgeries and technique. The authors would tion reduces D1 receptor-induced dyskinesia and improves movement in the also like to recognize Benjamin Austin Hettinger and Libby Gross for as- hemiparkinsonian rat. Neuropharmacology 55 (8), 1321–1328. sistance with collecting behavioral data. Dupre, K.B., Ostock, C.Y., Eskow Jaunarajs, K.L., Button, T., Savage, L.M., Wolf, W., Bishop, C., 2011. Local modulation of striatal glutamate efflux by serotonin 1A receptor stimulation in dyskinetic, hemiparkinsonian rats. Exp. Neurol. 229 (2), 288–299. References Dupre, K.B., Ostock, C.Y., George, J.A., Eskow Jaunarajs, K.L., Hueston, C.M., Bishop, C., 2013. Effects of 5-HT1A receptor stimulation on D1 receptor agonist-induced striatonigral Ahmed, I., Bose, S.K., Pavese, N., Ramlackhansingh, A., Turkheimer, F., Hotton, G., ... Brooks, activity and dyskinesia in hemiparkinsonian rats. ACS Chem. Neurosci. 4 (5), D.J., 2011. Glutamate NMDA receptor dysregulation in Parkinson's disease with dys- 747–760. kinesia. Brain 134, 979–986. Ener, R.A., Meglathery, S.B., Van Decker, W.A., Gallagher, R.M., 2003. Serotonin syndrome Arai, R., Karasawa, N., Geffard, M., Nagatsu, T., Nagastu, I., 1994. Immunohistochemical ev- and other serotonergic disorders. Pain Med. 4 (1), 63–74. idence that central serotonin neurons produce dopamine from exogenous L-DOPA in Eskow, K.L., Gupta, V., Alam, S., Park, J.Y., Bishop, C., 2007. The partial 5-HT(1A) agonist the rat, with reference to the involvement of aromatic L-amino acid decarboxylase. buspirone reduces the expression and development of L-DOPA-induced dyskinesia Brain Res. 667, 295–299. in rats and improves L-DOPA efficacy. Pharmacol. Biochem. Behav. 87 (3), 306–314. Assié, M.B., Lomenech, H., Ravailhe, V., Faucillon, V., Newman-Tancredi, A., 2006. Rapid Eskow, K.L., Dupre, K.B., Barnum, C.J., Dickinson, S.O., Park, J.Y., Bishop, C., 2009. The role of desensitization of somatodendritic 5-HT1A receptors by chronic administration of the dorsal raphe nucleus in the development, expression, and treatment of L-DOPA- the high efficacy 5-HT1A agonist, F13714: a microdialysis study in the rat. Br. induced dyskinesia in hemiparkinsonian rats. Synapse 63 (7), 610–620. J. Pharmacol. 149, 170–178. Frechilla, D., Cobreros, A., Saldise, L., Moratalla, R., Insausti, R., Liquin, M.R., Del Rio, J., 2001. Assié, M.B., Bardin, L., Auclair, A.L., Carilla-Durand, E., Depoortère, R., Koek, W., ... Serotonin 5-HT1A receptor expression is selectively enhanced in striosomal compart- Newman-Tancredi, A., 2010. F15599, a highly selective post-synaptic 5-HT1A recep- ment of chronic parkinsonian monkeys. Synapse 39 (4), 288–296. tor agonist: in vivo profile in behavioural models of antidepressant and serotonergic Ghavami, A., Stark, K.L., Jareb, M., Ramboz, S., Ségu, L., Hen, R., 1999. Differential address- activity. Int. J. Neuropsychopharmacol. 13 (10), 1285–1298. ing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons. J. Cell Sci. 112, Azmitia, E.C., Gannon, P.J., Kheck, N.M., Whitaker-Azmitia, P.M., 1996. Cellular localization 967–976. of the 5-HT1A receptor in primate brain neurons and glial cells. Gillman, P.K., 2010. Triptans, serotonin agonists, and serotonin syndrome (serotonin tox- Neuropsychopharmacology 14 (1), 35–46. icity): a review. Headache 50 (2), 264–272. Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function. Neu- Goetz, C.G., Damier, P., Hicking, C., Laska, E., Müller, T., Olanow, C.W., ... Russ, H., 2007. ropharmacology 38 (8), 1083–1152. Sarizotan as a treatment for dyskinesias in parkinson's disease: a double-blind, place- Barnum, C.J., Bhide, N., Lindenbach, D., Surrena, M.A., Goldenberg, A.A., Tignor, S., Bishop, bo-controlled trial. Mov. Disord. 22 (2), 179–186. C., 2012. Effects of noradrenergic denervation L-DOPA-induced dyskinesia and its Gonzales, K.K., Smith, Y., 2015. Cholinergic interneurons in the dorsal and ventral stria- treatment by α- and β-adrenergic receptor antagonists in hemiparkinsonian rats. tum: anatomical and functional considerations in normal and diseased conditions. Pharmacol. Biochem. Behav. 100 (3), 607–615. Ann. N. Y. Acad. Sci. 1349, 1–45. Bartoszyk, G.D., Van Amsterdam, C., Greiner, H.E., Rautenberg, W., Russ, H., Seyfried, C.A., Grégoire, L., Samadi, P., Graham, J., Bédard, P.J., Bartoszyk, G.D., Di Paolo, T., 2009. Low 2004. Sarizotan, a serotonin 5-HT1A receptor agonist and dopamine receptor ligand. doses of sarizotan reduce dyskinesias and maintain antiparkinsonian efficacy of L- 1. Neurochemical profile. J Neural Transm (Vienna) 111 (2), 113–126. DOPA in parkinsonian monkeys. Parkinsonism Relat. Disord. 15 (6), 445–452. Becker, G., Bolbos, R., Costes, N., Redouté, J., Newman-Tancredi, A., & Zimmer, L. (2016). Habberzettl, R., Bert, B., Fink, H., Fox, M.A., 2013. Animal models of the serotonin syn- Selective serotonin 5-HT1A receptor biased agonists elicit distinct brain activation drome: a systematic review. Behav. Brain Res. 256, 328–345. patterns: a pharmacoMRI study. ScientificReports, 6. http://dx.doi.org/10.1038/ Hamik, A., Oksenberg, D., Fischette, C., Peroutka, S.J., 1990. Analysis of tandospirone (SM- srep26633 3997) interactions with neurotransmitter receptor binding sites. Biol. Psychiatry 28 Berger, T.W., Kaul, S., Stricker, E.M., Zigmond, M.J., 1985. Hyperinnervation of the striatum (2), 99–109. by dorsal raphe afferents after dopamine-depleting brain lesions in neonatal rats. Huot, P., Johnston, T.H., Koprich, J.B., Winkelmolen, L., Fox, S.H., Brotchie, J.M., 2012. Reg- Brain Res. 336 (2), 354–358. ulation of cortical and striatal 5-HT1A receptors in the MPTP-lesioned macaque. Bibbiani, F., Oh, J.D., Chase, T.N., 2001. Serotonin 5-HT1A agonist improves motor compli- Neurobiol. Aging 33 (1), 207.e9–207.e19. cations in rodent and primate parkinsonian models. Neurology 57, 1829–1834. Huot, P., Johnston, T.H., Fox, S.H., Newman-Tancredi, A., Brotchie, J.M., 2015. The highly- Bishop, C., Krolewski, D.M., Eskow, K.L., Barnum, C.J., Dupre, K.B., Deak, T., Walker, P.D., selective 5-HT(1A) agonist F15599 reduces L-DOPA-induced dyskinesia without 2009. Contribution of the striatum to the effects of 5-HT1A receptor stimulation. compromising anti-parkinsonian benefits in the MPTP-lesioned macaque. Neuro- J. Neurosci. Res. 87 (7), 1645–1658. pharmacology 97, 306–311. Bishop, C., George, J.A., Buchta, W., Goldenberg, A.A., Mohamed, M., Dickinson, S.O., ... Iderberg, H., Rylander, D., Bimpisidis, Z., Cenci, M.A., 2013. Modulating mGluR5 and Eskow Jaunarajs, K.L., 2012. Serotonin transporter inhibition attenuates L-DOPA-in- 5HT1A/1B receptors to treat L-DOPA-induced dyskinesia: Effects of combined treat- duced dyskinesia without compromising L-DOPA efficacy in hemi-parkinsonian ment and possible mechanisms of action. Exp. Neurol. 250, 116–124. rats. Eur. J. Neurosci. 36, 2839–2848. Iderberg, H., McCreary, A.C., Varney, M.A., Cenci, M.A., Newman-Tancredi, A., 2015. Activ- Bordia, T., Perez, X.A., Heiss, J.E., Zhang, D., Quik, M., 2016. Optogenetic activation of ity of serotonin 5-HT1A receptor ‘biased agonists’ in rat models of Parkinson's disease striatal cholinergic interneurons regulates L-DOPA-induced dyskinesias. Neurobiol. and L-DOPA-induced dyskinesia. Neuropharmacology 93, 52–67. Dis. 91, 47–58. Iravani, M.M., Tayarani-Binazir, K., Chu, W.B., Jackson, M.J., Jenner, P., 2006. In 1-methyl-4- Boyer, E.W., Shannon, M., 2005. The serotonin syndrome. N. Engl. J. Med. 352 (11), phenyl-1,2,3,6-tetrahydropyridine-treated primates, the selective 5-hydroxytrypta- 1112–1120. mine 1a agonist (R)-(+)-8-OHDPAT inhibits levodopa-induced dyskinesia but only Buritova, J., Berrichon, G., Cathala, C., Colpaert, F., Cussac, D., 2009. Region-specificchanges with increased motor disability. J. Pharmacol. Exp. Ther. 319, 1225–1234. in 5-HT1A agonist-induced extracellular signal-regulated kinases ½ phosphorylation Jacobs, B.L., Klemfuss, H., 1975. Brain stem and spinal cord mediation of serotonergic be- in rat brain: a quantitative ELISA study. Neuropharmacology 56 (2), 350–361. havioural syndrome. Brain Res. 100, 450–457. Carta, M., Carlsson, T., Kirik, D., Björklund, A., 2007. Dopamine released from 5-HT termi- Kannari, K., Yamato, H., Shen, H., Tomiyama, M., Suda, T., Matsunaga, M., 2001. Activation nals is the cause of L-DOPA-induced dyskinesia in parkinsonian rats. Brain 130, of 5-HT1A but not 5-HT1B receptors attenuates an increase in extracellular dopamine 1819–1833. derived from exogenously administered L-DOPA in the striatum with nigrostriatal de- Cenci, M.A., Lee, C.S., Björklund, A., 1998. L-DOPA-induced dyskinesia in the rat is associ- nervation. J. Neurochem. 76 (5), 1346–1353. ated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase Kannari,K.,Kurahashi,K.,Tomiyama,M.,Maeda,T.,Arai,A.,Baba,M.,...Matsunaga, mRNA. Eur. J. Neurosci. 10 (8), 2694–2706. M., 2002. Tandospirone citrate, a selective 5-HT1A agonist, alleviates L-DOPA- Chang, J.-W., Wachtel, S.R., Young, D., Kang, U.-J., 1999. Biochemical and anatomical char- induced dyskinesia in patients with Parkinson's disease. No To Shinkei 54 (2), acterization of forepaw adjusting steps in rat models of parkinson's disease: studies 133–137. on medial forebrain bundle and striatal lesions. Neuroscience 88 (2), 617–628. Kannari, K., Maeda, T., Tanaka, H., Arai, A., Shen, H., Matsunaga, M., 2003. L-DOPA-derived Conti, M.M., Ostock, C.Y., Lindenbach, D., Goldenberg, A.A., Kampton, E., Dell'isola, R., ... extracellular dopamine in the striatum with dopaminergic denervation: Role of sero- Bishop, C., 2014. Effects of prolonged selective serotonin reuptake inhibition on the tonergic neurons in L-DOPA metabolism. Int. Congr. Ser. 1251, 181–189. development and expression of L-DOPA-induced dyskinesia in hemi-parkinsonian Keber, U., Klietz, M., Carlsson, T., Oertel, W.H., Weihe, E., Schäfer, M.K., ... Depboylu, C., rats. Neuropharmacology 77, 1–8. 2015. Striatal tyrosine hydroxylase-positive neurons are associated with L-DOPA-in- Conti, M.M., Goldenberg, A.A., Kuberka, A., Mohamed, M., Eissa, S., Lindenbach, D., Bishop, duced dyskinesia in hemiparkinsonian mice. Neuroscience 298, 302–317. C., 2016. Effect of tricyclic antidepressants on L-DOPA-induced dyskinesia and motor Kenakin, T., 2011. Functional selectivity and biased receptor signaling. J Pharmacol and improvement in hemi-parkinsonian rats. Pharmacol. Biochem. Behav. 142, 64–71. Exp Ther 336 (2), 296–302. 178 S.M. Meadows et al. / Experimental Neurology 292 (2017) 168–178

Kilpatrick, I.C., Jones, M.W., Phillipson, O.T., 1986. A semiautomated analysis method for Olanow, C.W., Damier, P., Goetz, C.G., Mueller, T., Nutt, J., Rascol, O., Serbanescu, A., ... Russ, catecholamines, indolelamines, and some prominent metabolites in microdissected H., 2004. Multicenter, open-label, trial of sarizotan in parkinson disease patients with regions of the nervous system: An isocratic HPLC technique employing coulometric levodopa-induced dyskinesias (the SPLENDID study). Clin. Neuropharmacol. 27 (2), detection and minimal sample preparation. J. Neurochem. 46 (6), 1865–1876. 58–62. Lindenbach, D., Ostock, C.Y., Eskow Jaunarajs, K.L., Dupre, K.B., Barnum, C.J., Bhide, N., Ostock, C.Y., Dupre, K.B., Jaunarajs, K.L., Walters, H., George, J., Krolewski, D., ... Bishop, C., Bishop, C., 2011. Behavioral and cellular modulation of L-DOPA-induced dyskinesia 2011. Role of the primary motor cortex in L-DOPA-induced dyskinesia and its modu- by β-adrenoreceptor blockade in the 6-hydroxydopamine-lesioned rat. lation by 5-HT1A receptor stimulation. Neuropharmacology 61 (4), 753–760. J. Pharmacol. Exp. Ther. 337 (3), 755–765. Paxinos, G., Watson, W., 1998. The Rat Brain in Stereotaxic Coordinates. fourth ed. Aca- Lindenbach, D., Palumbo, N., Ostock, C.Y., Vilceus, N., Conti, M.M., Bishop, C., 2015. Side ef- demic Press, San Diego, CA. fect profile of 5-HT treatments for parkinson's disease and L-DOPA-induced dyskine- Politis, M., Wu, K., Loane, C., Brooks, D.J., Kiferle, L., Turkheimer, F.E., ... Piccini, P., 2014. Se- sia in rats. Br. J. Pharmacol. 172 (1), 119–130. rotonergic mechanisms responsible for levodopa-induced dyskinesias in Parkinson's Lindgren, H.S., Andersson, D.R., Lagerkvist, S., Nissbrandt, H., Cenci, M.A., 2010. L-DOPA- disease patients. J. Clin. Invest. 124 (3), 1340–1349. induced dopamine efflux in the striatum and the substantia nigra in a rat model of Pompeiano, M., Palacios, J.M., Mengod, G., 1992. Distribution and cellular localization of Parkinson's disease: temporal and quantitative relationship to the expression of dys- mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding. kinesia. J. Neurochem. 112 (6), 1465–1476. J. Neurosci. 12 (2), 440–453. Lladó-Pelfort, L., Assié, M.B., Newman-Tancredi, A., Artigas, F., Celada, P., 2010. Preferential Putterman, D.B., Munhall, A.C., Kozell, L.B., Belknap, J.K., Johnson, S.W., 2007. Evaluation of in vivo action of F15599, a novel 5-HT1A receptor agonist, at postsynaptic 5-HT1A re- levodopa dose and magnitude of dopamine depletion as risk factors for levodopa-in- ceptors. Br. J. Pharmacol. 160 (8), 1929–1940. duced dyskinesia in a rat model of Parkinson's disease. J. Pharmacol. Exp. Ther. 323 Lucki, I., 1992. 5-HT1 receptors and behavior. Neurosci. Biobehav. Rev. 16 (1), 83–93. (1), 277–284. Lundblad, M., Andersson, M., Winkler, C., Kirik, D., Wierup, N., Cenci, M.A., 2002. Pharma- Robelet, S., Melon, C., Guillet, B., Salin, P., Kerkerian-Le Goff, L., 2004. Chronic L-DOPA cological validation of behavioural measures of akinesia and dyskinesia in a rat model treatment increases extracellular glutamate levels and GLT1 expression in the basal of Parkinson's disease. Eur. J. Neurosci. 15 (1), 120–132. ganglia in a rat model of Parkinson's disease. Eur. J. Neurosci. 20 (5), 1255–1266. Maeda, T., Kannari, K., Suda, T., Matsunaga, M., 1999. Loss of regulation by presynaptic do- Rylander, D., Parent, M., O'Sullivan, S.S., Dovero, S., Lees, A.J., Bezard, E., ... Cenci, M.A., pamine D2 receptors of exogenous L-DOPA-derived dopamine release in the dopami- 2010. Maladaptive plasticity of serotonin axon terminals in levodopa-induced dyski- nergic denervated striatum. Brain Res. 817, 185–191. nesia. Ann. Neurol. 68 (5), 619–628. Maeda, T., Kannari, K., Shen, H., Arai, A., Tomiyama, M., Matsunaga, M., Suda, T., 2003. Smith, Y., Wichmann, T., Factor, S.A., DeLong, M.R., 2012. Parkinson's disease therapeutics: Rapid induction of serotonergic hyperinnervation in the adult rat striatum with ex- new developments and challenges since the introduction of levodopa. tensive dopaminergic denervation. Neurosci. Lett. 343 (1), 17–20. Neuropsychopharmacology 37 (1), 213–246. Maeda, T., Nagata, K., Yoshida, Y., Kannari, K., 2005. Serotonergic hyperinnervation into Stein, D.J., Miczek, K.A., Lucion, A.B., Martins de Almeida, R.M., 2013. Aggression-reducing the dopaminergic denervated striatum compensates for dopamine conversion from effects of F15599, a novel selective 5-HT1A receptor agonist, after microinjection into exogenously administered L-DOPA. Brain Res. 1046 (1–2), 230–233. the ventral orbital prefrontal cortex, but not in infralimbic cortex in male mice. Psy- Manos, G.H., 2000. Possible serotonin syndrome associated with buspirone added to flu- chopharmacology 230 (3), 375–387. oxetine. Ann. Pharmacother. 34 (7–8), 871–874. Svenningsson, P., Rosenblad, C., Edholm Arviddsson, K., Wictorin, K., Keywood, C., McCreary, A.C., Varney, M.A., Newman-Tancredi, A., 2016. The novel 5-HT1A receptor ag- Shankar, B., ... Widner, H., 2015. Eltoprazine counteracts L-dopa-induced dyskinesias onist, NLX-11, reduces L-DOPA-induced abnormal involuntary movements in rat: a in parkinson's disease: a dose-finding study. Brain 138 (Pt 4), 963–973. chronic administration study with microdialysis measurements. Neuropharmacology Tanaka, H., Kannari, K., Maeda, T., Tomiyama, M., Suda, T., Matsunaga, M., 1999. Role of se- 105, 651–660. rotonergic neurons in L-DOPA-derived extracellular dopamine in the striatum of 6- Mengod, G., Vilaró, M.T., Raurich, A., López-Giménez, J.F., Cortés, R., Palacios, J.M., 1996. 5- OHDA-lesioned rats. Neuroreport 10 (3), 631–634. HT receptors in mammalian brain: receptor autoradiography and in situ hybridiza- Tomiyama, M., Kimura, T., Maeda, T., Kannari, K., Matsunaga, M., Baba, M., 2005. A seroto- tion studies of new ligands and newly identified receptors. Histochem. J. 28 (11), nin 5-HT1A receptor agonist prevents behavioral sensitization to L-DOPA in a rodent 747–758. model of Parkinson's disease. Neurosci. Res. 52 (2), 185–194. Merck KGaA, 2006. Sarizotan phase III studies did not meet primary efficacy Vergé, D., Daval, G., Macinkiewicz, M., Patey, A., el Mestikawy, S., Gozlan, H., Hamon, M., endpoint [Press release]. Retrieved from. http://me.merck.de/n/ 1986. Quantitative autoradiography of multiple 5-HT1 receptor subtypes in the A720D2336B86597DC1257196001CCA99/$FILE/Sarizo-e.pdf. brain of control or 5,7-dihydroxytryptamine-treated rats. J. Neurosci. 6 (12), Nahimi, A., Høltzermann, M., Landau, A.M., Simonsen, M., Jakobsen, S., Olsen Alstrup, A.K., 3474–3482. ... Doudet, D.J., 2012. Serotonergic modulation of receptor occupancy in rats treated Virk, M.S., Sagi, Y., Medrihan, L., Leung, J., Kaplitt, M.G., Greengard, P., 2016. Opposing roles with L-DOPA after unilateral 6-OHDA lesioning. J. Neurochem. 120 (5), 806–817. for serotonin in cholinergic neurons of the ventral and dorsal striatum. Proc. Natl. Nakayama, H., Umeda, S., Nibuya, M., Terao, T., Nisijima, K., Nomura, S., 2014. Two cases of Acad. Sci. U. S. A. 113 (3), 734–739. mild serotonin toxicity via 5-hydroxytryptamine 1A receptor stimulation. Volpi-Abadie, J., Kaye, A.M., Kaye, A.D., 2013. Serotonin syndrome. The Ochsner J 13 (4), Neuropsychiatr. Dis. Treat. 10, 283–287. 533–540. Navailles, S., Bioulac, B., Gross, C., De Deurwaerdère, P., 2010. Serotonergic neurons medi- Yamada, H., Aimi, Y., Nagatsu, I., Taki, K., Kudo, M., Arai, R., 2007. Immunohistochemical ate ectopic release of dopamine induced by L-DOPA in a rat model of Parkinson's dis- detection of L-DOPA-derived dopamine within serotonergic fibers in the striatum ease. Neurobiol. Dis. 38 (1), 136–143. and the substantia nigra pars reticulate in parkinsonian model rats. Neurosci. Res. Nevalainen, N., Bjerkén, S., Gerhardt, G.A., Strömberg, I., 2014. Serotonergic nerve fibers in 59 (1), 1–7. L-DOPA-derived dopamine release and dyskinesia. Neuroscience 260, 73–86. Yokoyama, C., Mawatari, A., Kawasaki, A., Takeda, C., Onoe, K., Doi, H., ... Onoe, H., 2016. Newman-Tancredi, A., 2011. Biased agonism at serotonin 5-HT1A receptors: preferential Marmoset serotonin 5-HT1A receptor mapping with a biased agonist PET probe post-synaptic activity for improved therapy of CNS disorders. Neuropsychiatry 1 (1), 18F-F13714: comparison with an antagonist tracer 18F-MPPF in awake and anesthe- 149–164. tized states. (in press). Int. J. Neuropsychopharmacol.. Newman-Tancredi, A., Martel, J.C., Assié, M.B., Buritova, J., Lauressergues, E., Cosi, C., ... Yoshida, K., Sugita, T., Higuchi, H., Hishikawa, T., 1998. Effect of tandospirone on tardive Cussac, D., 2009. Signal transduction and functional selectivity of F15599, a preferen- dyskinesia and parkinsonian symptoms. Eur Psychiatry 13 (8), 421–422. tial post-synaptic 5-HT1A receptor agonist. Br. J. Pharmacol. 156 (2), 338–353. Obeso, J.A., Olanow, W., Nutt, J.G., 2000. Levodopa motor complications in parkinson's disease. Trends Neurosci. 23 (10 Suppl), S2–S7.