Molecular Psychiatry https://doi.org/10.1038/s41380-018-0227-x ARTICLE 5-HT2C receptor blockade reverses SSRI-associated basal ganglia dysfunction and potentiates therapeutic efficacy 1,2 1 1 1 1,3 Elena Y. Demireva ● Deepika Suri ● Emanuela Morelli ● Darshini Mahadevia ● Nao Chuhma ● 1,2 1,2 1 1 1 1 Catia M. Teixeira ● Annette Ziolkowski ● Marc Hersh ● James Fifer ● Sneha Bagchi ● Alexei Chemiakine ● 1,4 1,2 1,2 1,3 1,2 Holly Moore ● Jay A. Gingrich ● Peter Balsam ● Stephen Rayport ● Mark S. Ansorge Received: 30 November 2016 / Revised: 13 July 2018 / Accepted: 24 July 2018 © Springer Nature Limited 2018 Abstract Serotonin (5-HT) selective reuptake inhibitors (SSRIs) are widely used in the treatment of depression and anxiety disorders, but responsiveness is uncertain and side effects often lead to discontinuation. Side effect profiles suggest that SSRIs reduce dopaminergic (DAergic) activity, but specific mechanistic insight is missing. Here we show in mice that SSRIs impair motor function by acting on 5-HT2C receptors in the substantia nigra pars reticulata (SNr), which in turn inhibits nigra pars compacta (SNc) DAergic neurons. SSRI-induced motor deficits can be reversed by systemic or SNr-localized 5-HT2C 1234567890();,: 1234567890();,: receptor antagonism. SSRIs induce SNr hyperactivity and SNc hypoactivity that can also be reversed by systemic 5-HT2C receptor antagonism. Optogenetic inhibition of SNc DAergic neurons mimics the motor deficits due to chronic SSRI treatment, whereas local SNr 5-HT2C receptor antagonism or optogenetic activation of SNc DAergic neurons reverse SSRI- induced motor deficits. Lastly, we find that 5-HT2C receptor antagonism potentiates the antidepressant and anxiolytic effects of SSRIs. Together our findings demonstrate opposing roles for 5-HT2C receptors in the effects of SSRIs on motor function and affective behavior, highlighting the potential benefits of 5-HT2C receptor antagonists for both reduction of motor side effects of SSRIs and augmentation of therapeutic antidepressant and anxiolytic effects. Introduction dysregulation influences psychiatric states, and most neu- ropsychiatric medications act in these two systems. How the 5-HT and DA neurotransmission are highly interconnected transmitter systems influence each other is of key importance in both normal brain function and disease. Their for understanding normal brain function, psychopathology, and pharmacotherapy. SSRIs that block the 5-HT transporter (5-HTT) are currently the most widely prescribed psychiatric drugs. They are prescribed not only as a first-line treatment These authors contributed equally: Elena Y. Demireva and Deepika Suri. for depressive disorders, but also for many other psychiatric disorders, including bipolar, obsessive-compulsive, panic Electronic supplementary material The online version of this article and generalized anxiety disorder. Despite their widespread (https://doi.org/10.1038/s41380-018-0227-x) contains supplementary fi material, which is available to authorized users. use, SSRIs face both ef cacy and side-effect challenges [1]. One hypothesis is that their therapeutic shortcomings arise * Mark S. Ansorge from 5-HTergic modulation of the DA system [2]. Indeed, [email protected] several side effects of SSRIs such as anhedonia, apathy, 1 Department of Psychiatry, Columbia University, New York, NY emotional blunting, reduced libido, akathisia, and extra- 10032, USA pyramidal motor symptoms (EPS) point to DAergic hypo- 2 Department of Developmental Neuroscience, New York State function in the behavioral and emotional effects of SSRIs Psychiatric Institute, New York, NY 10032, USA [3–8]. Animal studies support this idea, with several studies 3 Division of Molecular Therapeutics, New York State Psychiatric demonstrating inhibitory effects of pharmacologically Institute, New York, NY 10032, USA increased extracellular 5-HT on midbrain DAergic function 4 Division of Systems Neuroscience, New York State Psychiatric [9, 10]. In mice, and occasionally in predisposed patients, Institute, New York, NY 10032, USA E. Y. Demireva et al. such blunted DAergic function can induce parkinsonism weighed 25–35 g. All subjects were genotyped by PCR of [2, 11]. genomic DNA isolated from tissue samples harvested at To improve 5-HTT-based pharmacotherapy, one 18–21 days of age. approach has been to compensate for DAergic dysregula- tion by directly targeting the DA system with compounds Animal husbandry such as bupropion or amphetamines [12]. But such aug- mentation strategies, which broadly impact DA signaling, Animals were housed in groups of five per cage and pro- have had little success and can themselves have adverse vided with food and water ad libitum (except as noted). effects. A more targeted approach requires therapies that Animals were maintained on a 12 h light/dark cycle (8:00 A. directly address the mechanism by which SSRI-induced M./8:00 P.M.) Animal testing was conducted during the increase in 5-HTergic tone leads to DA hypofunction. To light phase of the light cycle. Animal testing was conducted that end, it is important to identify the 5-HT receptors in accordance with National Institutes of Health (NIH) whose activation is responsible for mediating the inhibitory Laboratory Animal Care and Welfare guidelines and effects on DA signaling. Among the fourteen 5-HT recep- approved by the Institutional Animal Care and Use Com- tors, one promising candidate is the 5-HT2C receptor, mittees of Columbia University and NYS Psychiatric which is expressed by SNr and VTA GABAergic neurons, Institute. and its antagonism reverses acute effects of SSRIs on VTA DAergic activity [13–15]. Furthermore, 5-HT2C receptor Animal use agonism reduces incentive motivation [16, 17], while antagonism increases motivation [18–20], with a hypothe- The numbers of mice used were based on a power analysis sized mechanism centered on 5-HT acting at 5-HT2C assuming a 30% mean effect size of manipulations and an receptors to reduce motivation by suppressing mesolimbic acceptable probability level of 0.05. Mice were randomized DAergic activity [20]. when allocated to treatment groups balancing for litter, sex, Here we investigate the role of 5-HT2C receptors in and genotype. Investigators were blinded to group alloca- mediating the effects of 5-HTT blockade on basal ganglia tions during testing and scoring. (BG) function and emotional behaviors. We find that 5- HT2C receptor activation plays a selective and pivotal role Drugs in the suppression of the DAergic nigrostriatal pathway associated with impaired motor function during SSRI Fluoxetine (FLX; ANAWA Trading SA, Wangen, Swit- treatment. We directly assess the roles of SNr 5-HT2C zerland), was dissolved in 0.9% NaCl or drinking water to receptors and SNc DAergic activity in regulating motor achieve 2.5, 5, or 10 mg/kg when administered intraper- function, and we investigate the interaction between 5-HTT itoneally (i.p.) or through the drinking water (see supple- blockade and 5-HT2C receptor antagonism in anxiety- and mentary data). The 5-HT2C receptor inverse agonist depression-related behavior. Our findings provide mechan- SB206553 (Tocris, Ellisville, MO), the 5-HT2C receptor istic insight into 5-HTergic modulation of DAergic function antagonist SB242084 (Tocris), the 5-HT2C receptor agonist and highlight 5-HT2C receptor antagonism for augmenta- WAY161503 (Tocris), and the 5-HT3 receptor antagonist tion therapy to improve antidepressant efficacy of SSRI ondansetron (Tocris) were dissolved by sonication in 0.9% treatment and reduce basal ganglia mediated side-effects. NaCl to achieve concentrations between 0.1 and 5 mg/kg when administered i.p. For local infusions, SB242084 was first dissolved in DMSO to 10 mg/ml, and then diluted at Materials and methods 1:100 in saline to obtain a final concentration of 0.1 mg/ml. Transgenic mice In vivo electrophysiology Mice heterozygous for the 5-htt mutation (5-htt+/−,10th– Extracellular recording microelectrodes (2.0 mm OD bor- 12th generation 129S6/SvEv) were crossed to produce 5-htt osilicate glass capillary tubing, ~1 µm tip diameter, impe- +/+, 5-htt+/-, and 5-htt-/- offspring see [21]. Independent dance 5–10 MOhms) were filled with 2 M NaCl containing groups of male subjects weighing 25–45 g were experi- 2% pontamine sky blue dye. The electrode signal was mentally tested at ages 2–13 months. Heterozygous cross- amplified, filtered and discriminated from noise. Mice were ings were also used to generate experimental groups of +/+ anesthetized with isoflurane 1–2% in oxygen via a standard and −/− mice for htr1a [22], htr1b [23], htr2a [24], and veterinary inhalation anesthesia apparatus. Body tempera- htr4 [25]. Offspring resulting from these crosses were ture was monitored by a rectal probe and maintained experimentally tested at approximately 5 months of age and between 36–37 °C using a water circulation pad placed 5-HT2C receptor blockade reverses SSRI-associated basal ganglia dysfunction and potentiates therapeutic. under a clean gauze pad. A hole was drilled through the virus was allowed to express for 3 weeks, after which the skull overlying the brain structures of interest, and the dura animals were euthanized, and 300 µm horizontal brains was then punctured. A glass electrode was lowered with a slices containing ventral midbrain were sliced using vibra- hydraulic microdrive into the SNc and SNr at coordinates: tome (VT1200S, Leica). Electrophysiological recordings anterior/posterior (AP) +4.0 mm, medial/lateral (ML) 1.1– were performed in regular ACSF (in mM: 125 NaCl, 2.5 2.3 mm from Bregma, and dorsal/ventral (DV) 3.5–5.5 mm KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and from brain surface. DAergic and non-DAergic neurons were 25 glucose, pH 7.4) saturated with 95% O2,5%CO2 at identified using established electrophysiological criteria [26, 31–33 °C. Patch pipettes contained the following solution 27] and once isolated, spontaneous activity was recorded for (in mM): 135 Cs+-methane sulfonate (MeSO4), 5 KCl, 2–3 min. The SNc and SNr were systematically investigated 2 MgCl2, 0.1 CaCl2, 10 HEPES, 1 EGTA, 2 ATP and 0.1 by recording from 4–6 tracks within a region per mouse.