Abstract Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ 1 ) receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ 1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ 1 receptor antagonists (BD1063 and BD1047) were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP) 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ 1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ 1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a K i concentration of dextromethorphan reduces both the K d and the B max of [3H](+)-pentazocine binding to σ 1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ 1 receptors.

Citation: Nguyen L, Robson MJ, Healy JR, Scandinaro AL, Matsumoto RR (2014) Involvement of Sigma-1 Receptors in the Antidepressant-like Effects of Dextromethorphan. PLoS ONE 9(2): e89985. https://doi.org/10.1371/journal.pone.0089985 Editor: Allan Siegel, University of Medicine & Dentistry of NJ - New Jersey Medical School, United States of America Received: November 21, 2013; Accepted: January 25, 2014; Published: February 28, 2014 Copyright: © 2014 Nguyen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study and personnel were funded by Avanir Pharmaceuticals, West Virginia University, and the National Institutes of Health (DA013978, DA013583). Avanir Pharmaceuticals provided editorial suggestions to improve the clarity of the manuscript prior to submission. The funding entities had no further role in the design of the study; in the collection, analysis and interpretation of the data; in writing the report; and in deciding to submit the paper for publication. Competing interests: Dr. Matsumoto reports having received consultant fees and research funding from Avanir Pharmaceuticals, Inc. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. The other authors report no financial or conflicts of interest.

Introduction Depression affects up to one fifth of the world population, stands as the second leading cause of disability worldwide, and imposes a substantial economic burden [1], [2]. In addition, the available pharmaceutical agents for treating depression are not effective in approximately a third of patients [3] and have a delayed clinical efficacy of several weeks to months [4]. Consequently, there is still a great need for faster acting and more effective treatments for depression. Recently, a hypothesis was offered that dextromethorphan may have fast-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) antagonist ketamine [5], a drug repeatedly shown in human populations to display rapid antidepressant effects but whose use is severely limited by the need for intravenous administration and the presence of notable adverse effects (e.g., hallucinations and dissociations) [6], [7], [8]. Similar to ketamine, dextromethorphan binds to NMDA receptors and can modulate glutamatergic signaling [5]. Dextromethorphan also has higher affinity than ketamine for serotonin transporters (SERT) [9] and several other protein targets, including sigma-1 (σ 1 ) receptors [5], [9] which have been proposed as therapeutic targets for antidepressant drugs [10]. Unlike ketamine, however, dextromethorphan has a high margin of safety; it has been used as a nonprescription antitussive over the past 40 years and thus may serve as a safer alternative to ketamine. In addition, it readily undergoes first-pass metabolism by cytochrome P450 (CYP) 2D6 to its major active metabolite dextrorphan [11]. Dextromethorphan in combination with quinidine, which raises the plasma concentration and bioavailability of dextromethorphan through the inhibition of CYP2D6 metabolism [12], is approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of pseudobulbar affect and is thought to produce part of its therapeutic effects through σ 1 receptors [13]. σ 1 Receptors are highly conserved 223 amino acid proteins expressed on the mitochondrial-associated endoplasmic reticulum membrane (MAM) and can translocate between different cellular compartments in response to ligand binding [14]. In addition, σ 1 receptors appear to operate primarily via protein-protein interactions to modulate the activity of various ion channels and signaling molecules, including inositol triphosphates, protein kinases, and calcium [14], [15]. Previous reports implicate σ 1 receptors as protein targets for existing and novel antidepressant drugs [10]. Currently marketed antidepressant drugs, such as tricyclic antidepressants, monoamine oxidase inhibitors, selective serotonin reuptake inhibitors (SSRIs), and newer generations of antidepressant drugs, bind to these receptors [10]. Earlier studies also demonstrate that σ 1 receptor agonists can modulate the activities of neurotransmitter systems, signaling pathways and brain regions implicated in the pathophysiology of depression [10] and that σ 1 receptor knockout mice exhibit a depressive-like phenotype [16]. The potential clinical relevance of these observations is further supported by reports that σ 1 receptor agonists produce antidepressant effects in experimental animals and humans [17], [18], [19], [20], [21], [22]. Notably, the σ 1 receptor agonist igmesine hydrochloride proved to be as effective an antidepressant as the well-established SSRI fluoxetine in some clinical trials, though not in all cases [10], [22]. Compared to existing medications, σ 1 receptor agonists may facilitate a more rapid onset of antidepressant efficacy [23]. Consistent with this, σ 1 receptor agonists such as (+)-pentazocine and SA 4503 can enhance serotonergic neuronal firing in the dorsal raphe nucleus after only two days of treatment, compared to the two weeks of treatment that is typically required of conventional antidepressant drugs [24], [25]. In the studies herein, we test the hypothesis that dextromethorphan can exert antidepressant-like actions at least in part through σ 1 receptors. This activity may convey additional therapeutic advantages over ketamine under clinically relevant conditions since compared to ketamine which has micromolar affinity for σ 1 receptors [26], dextromethorphan exhibits nanomolar binding affinity for these receptors [9], [27], [28]. First, the ability of dextromethorphan to cause antidepressant-like effects was examined in the forced swim test. The forced swim test is the most validated behavioral assay for predicting antidepressant efficacy [3], [29], [30] and thus provides a rational format for the initial evaluation of the antidepressant potential of dextromethorphan. In addition, the effect of dextromethorphan on locomotor activity was measured to determine whether stimulant effects could account for its apparent antidepressant-like actions. The antidepressant drugs imipramine and fluoxetine were used as reference ligands for these behavioral tests. Second, to evaluate the potential involvement of σ 1 receptors in the in vivo antidepressant-like actions of dextromethorphan, pharmacological antagonists targeting σ 1 receptors were examined for their ability to prevent the antidepressant-like effects of dextromethorphan. Third, since dextromethorphan undergoes extensive first-pass metabolism by CYP2D6 to its major active metabolite dextrorphan [11], the CYP2D6 inhibitor quinidine was administered concomitantly with dextromethorphan to raise the plasma concentration and bioavailability of dextromethorphan [12] and determine whether the metabolism of dextromethorphan affects its antidepressant efficacy. Finally, to define the manner in which dextromethorphan binds to σ 1 receptors (competitive and/or non-competitive), saturation binding studies were conducted.

Materials and Methods Animals Male, Swiss Webster mice (24–28 g; Harlan, Frederick, MD) were housed with food and water ad libitum, with a 12:12 h light–dark cycle. Animals were housed in groups of five for at least one week prior to initiation of experiments. All procedures were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at West Virginia University (Morgantown, WV), and all efforts were made to minimize suffering. Drugs and Chemicals Dextromethorphan hydrobromide and quinidine sulfate were provided by Avanir Pharmaceuticals, Inc. (Aliso Viejo, CA; for the behavioral studies) or purchased from Sigma-Aldrich (St. Louis, MO; for the binding assays). Imipramine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Fluoxetine hydrochloride, BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride), and BD1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide) were obtained from Tocris (Ellisville, MO). [3H](+)-Pentazocine (34.8 Ci/mmol) was procured from Perkin Elmer (Hopkington, MA). All other chemicals and reagents were purchased from standard commercial suppliers (Sigma-Aldrich, St. Louis, MO). Drug Treatments Mice (N = 5–15/group) received intraperitoneal (i.p.) injections with the following treatments: (1) Saline; (2) Imipramine (10–20 mg/kg); (3) Fluoxetine (10–30 mg/kg); (4) Dextromethorphan (1–30 mg/kg); (5) BD1063 (3–30 mg/kg); (6) BD1047 (10–30 mg/kg); (7) BD1063 (10 mg/kg) + Imipramine (20 mg/kg); (8) BD1063 (10 mg/kg) + Dextromethorphan (10–50 mg/kg); (9) BD1047 (10–20 mg/kg) + Dextromethorphan (30 mg/kg); (10) Quinidine (30 mg/kg) + Saline; and (11) Quinidine (30 mg/kg) + Dextromethorphan (3–30 mg/kg). Treatment with quinidine was administered concurrently with saline or dextromethorphan. Treatment with a σ 1 receptor antagonist (BD1063 or BD1047) was administered 15 min prior to the second drug (imipramine or dextromethorphan). Locomotor Activity Locomotor activity was measured utilizing an automated activity monitoring system (San Diego Instruments, San Diego, CA). Prior to locomotor activity measurements, animals were acclimated to the testing facility for at least 30 min and habituated to the testing chambers for an additional 30 min. Each testing chamber consisted of a Plexiglas housing and a 16×16 photobeam array to detect lateral (ambulatory and fine) movements, with a separate 16 photobeam array to detect rearing activity. Subsequent to the acclimation period, animals were treated and placed back in their respective chambers. Ambulatory, fine and rearing movements were quantified and summated as a measure of total locomotor activity for the next 30 min. Forced Swim Test Immediately after the locomotor measurements, animals were placed in individual cylinders of water (10 cm deep) for a total of 6 min for the forced swim test. The initial 2 min was an acclimation period and not scored. During the remaining 4 min, immobility time was quantified using ANY-Maze Version 4.63 video tracking software (Stoelting Co., Wood Dale, IL). Immobility was defined as no activity other than that required to maintain the animal's head above the surface of the water. ANY-Maze software settings were as follows: accustomization period = 120 s, test duration = 240 s, minimum immobility time = 2000 ms (2 s), and immobility sensitivity = 75%. Saturation Binding Assays To determine K d and B max by saturation binding, assays were performed in the absence (control) and presence of dextromethorphan (400 nM) using methods previously published in detail [31]. The concentration of dextromethorphan used in these assays was based on the reported K i of the drug for σ 1 receptors [27]. Briefly, 15 concentrations of [3H](+)-pentazocine (0.1–100 nM) were used to label σ 1 receptors in P 2 rat brain homogenates (400–500 µg/sample). Non-specific binding was determined in the presence of 10 µM haloperidol. Incubations occurred for 120 min at 25°C and membranes were washed 2–3 times using ice cold 10 mM Tris HCl, pH 8.0. Data Analysis Data from all experiments were analyzed using GraphPad Prism 5.0 (San Diego, CA). The behavioral data were analyzed by one-way analysis of variance (ANOVA) followed when applicable by post-hoc Dunnett's or Tukey's multiple comparison tests. The correlation between locomotor activity and immobility time was analyzed by Pearson's r correlation test. The K d and B max were determined using nonlinear regression and analyzed by unpaired t-tests. For in vivo data, outliers (data points that were at least two standard deviations away from the mean) were excluded from analyses. Data are represented as mean ± S.E.M. P<0.05 was considered statistically significant for all data analyzed.

Discussion This study is the first to show that dextromethorphan has antidepressant-like effects in vivo, in addition to implicating σ 1 receptors as a mechanism contributing to its antidepressant actions. Moreover, concomitant administration of the CYP2D6 reversible inhibitor quinidine potentiated the effects of dextromethorphan in the forced swim test. This demonstrates that the antidepressant-like effects of dextromethorphan do not require conversion to the metabolite dextrorphan, and reveals dextromethorphan itself has antidepressant efficacy. The antidepressant-like effects of dextromethorphan appear to involve σ 1 receptors. In the current study, two well-established σ 1 receptor antagonists (BD1063 and BD1047) reduced the antidepressant-like actions of dextromethorphan in vivo. They are thought to act in a competitive manner since in the presence of BD1063, the dose response curve for dextromethorphan was shifted to the right. An involvement of σ 1 receptors in the antidepressant-like effects of dextromethorphan is consistent with earlier reports that selective σ 1 receptor agonists can on their own reduce immobility time in the forced swim test [17], [20], [21], [32] and produce antidepressant-like effects in other animal models such as the tail suspension test and olfactory bulbectomy [18], [33]. Thus, additional studies involving these and other animal models used in depression research (e.g., sucrose preference test, novelty suppression) [3], [29], [30] will be needed in the future to further evaluate the antidepressant potential of dextromethorphan and the involvement of σ 1 receptors. The ability of dextromethorphan to elicit antidepressant-like actions through σ 1 receptors suggests future studies to evaluate potential fast-acting therapeutic effects are also warranted. σ 1 Receptor agonists can enhance serotonergic neuronal firing in the dorsal raphe nucleus after only two days vs. two weeks of treatment that is typically required of conventional antidepressant drugs [24], [25]. In addition, the fast-acting antidepressant drug ketamine has recently been shown to potentiate nerve growth factor (NGF)-induced neurite outgrowth through a σ 1 -dependent mechanism [26], supporting the emerging importance of σ 1 receptors in modulating neuronal plasticity, which itself is a critical element for conveying both rapid and delayed antidepressant activity. Earlier competition binding studies showed that dextromethorphan has significant affinity for σ 1 receptors (138–652 nM) [13], [27], [28], [34], and thus further characterization of the interaction of dextromethorphan with σ 1 receptors was undertaken in the current study. The saturation binding studies indicate that the interaction of dextromethorphan with σ 1 receptors is complex, with both a change in B max and K d in the binding of [3H](+)-pentazocine in the presence of dextromethorphan. The reduction in the number of σ 1 receptors (B max ) with which [3H](+)-pentazocine binds suggests non-competitive interactions of dextromethorphan with σ 1 receptors. However, there is also a decrease in K d for [3H](+)-pentazocine binding in the presence of dextromethorphan, suggesting additional competitive interactions. Together, the data support the presence of at least two distinct sites or modes of interaction with which dextromethorphan binds to the σ 1 receptor, one with which it has competitive interactions, and another with which it has non-competitive interactions. This interpretation would be consistent with other reports of multiple regions for ligand interactions on the σ 1 receptor, some of which have functional ramifications for agonist vs. antagonist activity [35], [36], [37]. The affinity differences of dextromethorphan for its two putative binding sites appear to be similar (<100-fold difference) since competition binding assays of dextromethorphan at σ 1 receptors are consistent with a one-site fit [27]. The antidepressant-like effects of dextromethorphan are thought to be mediated through the competitive binding site since i) there appears to be a rightward shift in its dose response curve in the forced swim test with no apparent change in maximal effect, and ii) (+)-pentazocine, the σ 1 agonist used to label the receptor, has previously also been reported to produce similar antidepressant-like effects [10], [17]. In addition to interacting with σ 1 receptors, dextromethorphan has been reported to alter monoamine reuptake, particularly serotonin and norepinephrine at Ki values of 23 and 240 nM, respectively [38], which have implications for antidepressant effects in humans. The significant affinity of dextromethorphan for SERT (40 nM) [9] would be expected to contribute to antidepressant efficacy in humans, although it would not account for potential fast-acting effects, nor reductions in immobility time herein. Under the experimental parameters used in the current study, the classical SSRI fluoxetine did not produce significant reductions in immobility time in the forced swim test. This is consistent with the reports of others that the forced swim test does not reliably detect the antidepressant potential of SSRIs [39]. Thus, this mechanism, which is a known contributor to antidepressant efficacy in humans, is unlikely to account for the pattern of antidepressant-like effects observed with dextromethorphan herein. In contrast to its high affinity for SERT, dextromethorphan binds much more weakly with NET (>1 µM) [9], but its reported ability to modulate norepinephrine reuptake [38] would be expected to contribute conventional antidepressant effects under clinical conditions. Compared to the ability of BD1063 pretreatment to significantly block the antidepressant-like effects of dextromethorphan, it failed to attenuate that of imipramine, which has an overlapping binding profile with dextromethorphan: SERT (1.3–20 nM) [40], [41], [42], [43], and σ 1 receptors (343 nM) [44]. This indicates that the σ 1 interaction may have a larger role in producing the antidepressant-like effects of dextromethorphan than that of imipramine. This is consistent with the wider range of protein targets through which imipramine, but not dextromethorphan, interacts, which include: serotonin 5-HT 2 , muscarinic, and histamine H 1 receptors [9], [45], [46], [47], [48], [49]. Finally, dextromethorphan elicits stimulant actions which were quantified herein as increases in locomotor activity. Two observations are of note with regard to these actions. First, the stimulant effects cannot account for the antidepressant-like actions of dextromethorphan. Second, quinidine enhances the antidepressant-like effects of dextromethorphan without producing an increase in locomotor activity. This suggests that addition of quinidine to dextromethorphan can be used clinically to optimize therapeutic antidepressant actions, without eliciting unwanted stimulant effects. In conclusion, the data presented here show for the first time that dextromethorphan has antidepressant-like effects in an in vivo model and provide evidence that this effect occurs at least in part through a σ 1 receptor dependent mechanism. This is also the first report of the manner in which dextromethorphan interacts at the σ 1 receptor. Together with earlier studies and the potential of increasing dextromethorphan bioavailiabity by using the FDA- and EMA-approved dextromethorphan/quinidine formulation, these data suggest dextromethorphan should be further explored for translational potential as an antidepressant drug in clinical trials, as it may offer rapid-acting relief of depressive symptoms and the ability to resolve cases of treatment-resistant depression. In addition, further studies to understand the molecular and cellular mechanisms by which these effects occur are necessary and may yield important information about how various receptors, transporters and processes are involved in the ability of dextromethorphan to convey its antidepressant effects.

Acknowledgments We appreciate the technical assistance of Dr. Ying Huang Zhang.

Author Contributions Conceived and designed the experiments: LN MJR RRM. Performed the experiments: LN MJR JRH ALS. Analyzed the data: LN MJR JRH ALS RRM. Wrote the paper: LN RRM. Edited the manuscript: LN MJR JRH ALS RRM.