Serotonin-releasing agents with reduced off-target effects

Materials

The synthesis of test drug stereoisomers is described in detail in the Supplementary Information. Tritiated 5-HT ([3H]5-HT, 28.3 µCi per mM) and tritiated 1-methyl-4-phenylpyridinium ([3H]MPP+, 80–85 µCi per mM) were obtained from Perkin Elmer (Boston, MA, USA) and American Radiolabeled Chemicals (St. Louis, MO, USA), respectively. Fluoxetine hydrochloride was from Tokyo Chemical Industry Co., Ltd. (TCI America, Portland, OR, USA). All other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. To monitor 5-HT release in vivo with fiber photometric recordings, we utilized genetically encoded G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) [26] (AAV9-hSyn-5-HT2h, WZ Biosciences Inc., Columbia, MD, USA) and DA sensors [27] (GRABDA; AAV-hSyn-DA2m (serotype 9) (Vigene Biosciences, Inc., Rockville, MD USA)). HTR2B-Tango was a gift from Bryan Roth (Addgene plasmid # 66410; http://n2t.net/addgene:66410; RRID:Addgene_66410). pGP-CMV-GCaMP6s was a gift from Douglas Kim & the GENIE Project (Addgene plasmid # 40753; http://n2t.net/addgene:40753; RRID:Addgene_40753). 5-HT2A and 5-HT2C receptor constructs were a gift from Prof. Herrick-Davis (Center for Neuropharmacology and Neuroscience, MC-136 P4000, Albany Medical College, New York, 12208-3479). pcDNA4-TO, pcDNA6-TR, pcDNA3.1 were obtained from Invitrogen (Thermo Fisher, Waltham, MA, USA).

Animals

All in vivo experiments were performed in adult mice (>6 weeks old). For the photometry and microdialysis experiments, which were carried out in Florida, male C57BL/6J mice from The Jackson Laboratory (Bar Harbor, ME, USA) were group-housed in polycarbonate cages (GM500, Tecniplast, Buguggiate, Italy) with lights on from 7 a.m. to 7 p.m. Food and water were provided ad libitum. Photometry and microdialysis experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Florida Atlantic University. For the behavioral experiments, which were carried out in Vienna, C57BL/6N mice from Charles River (Sulzfeld, Germany) were group-housed in polycarbonate cages with lights on from 7 a.m. to 7 p.m. Food and water were provided ad libitum. Behavioral experiments were performed in accordance with a protocol approved by the Austrian national ethical committee on animal care and use (Bundesministerium für Wissenschaft und Forschung: BMWFW-66.009/0016-WF/V/3b/2015). All animal procedures were conducted in agreement with the ARRIVE guidelines and the U.K. Animal (Scientific Procedures) Act, 1986, and associated guidelines, EU Directive 2010/63/EU for animal experiments. Mice were given at least 1 week to acclimate before being used in any experiments.

Behavior

For behavioral assays, animals were assigned to treatment groups and injected with test drugs by an unblinded observer.

Forced-swim test (FST)

The FST was performed as previously described [28]. In brief, mice were placed for 6 min in a beaker (diameter: 19 cm, height: 23 cm), halfway filled with water (22–23 °C), and the immobility time was quantified using the software Videotrack (Viewpoint, Champagne au Mont d’Or, France). Immobility was defined as the absence of any movement, except for those required to keep the head above the water. The time spent immobile during the final 4 min of the test was used as a measure of behavioral despair. Animal sample size was confirmed using a post hoc analysis of the achieved power using G*Power3.1 [29]. Using a significance level (α) of 0.05 and an effect size f = 2.5 and a sample size of 6 animals/group we achieved the commonly accepted statistical power (1−β) of 0.8.

Open-field test

Mice were placed into an open field arena (27.3 × 27.3 × 27.3 cm) surrounded by infrared beams to track movements. The total distance traveled over 1 h was recorded using Activity Monitor (Med Associates Inc., St. Albans, VT, USA) and interpreted as a measure of the psychostimulant effects. Animal sample size was confirmed using a post hoc analysis of the achieved power using G*Power3.1 [29]. Using a significance level (α) of 0.05 and an effect size f = 1.92 and a sample size of 7.5 animals (5–10)/group we achieved the commonly accepted statistical power (1−β) of 0.8.

Cell culture

Human embryonic kidney 293 (HEK293) cells stably expressing the human isoforms of either DAT or SERT were maintained in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal calf serum at a subconfluent state in humidified atmosphere (5% CO2, 37 °C).

DAT and SERT uptake inhibition assays in HEK293 cells

Uptake inhibition assays were performed as described earlier [30]. Briefly, HEK293 cells stably expressing human SERT or DAT were seeded onto poly- D-lysine (PDL) coated 96-well plates (40,000 cells well−1) the day before an experiment. Prior to the uptake inhibition assay, DMEM was replaced with Krebs-HEPES buffer (KHB, 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, and 5 mM D-glucose, pH 7.3) and the cells were preincubated with test drugs for 5 min at room temperature. Uptake assays were initiated by adding 20 nM [3H]MPP+ to DAT cells or 100 nM [3H]5-HT to SERT cells. After 180 s (DAT) or 60 s (SERT), the tritiated substrate was removed by aspiration. Cells were washed once with ice-cold KHB, then lysed in 1% sodium dodecyl sulfate (SDS) and subjected to liquid scintillation counting to quantify tritium accumulated in each sample. Non-specific uptake was determined in the presence of mazindol (1 µM) for DAT or paroxetine (1 µM) for SERT and subtracted throughout.

[3H]5-HT release assays in HEK293 cells

HEK293 cells stably expressing SERT were seeded onto PDL-coated glass coverslips (5 mm diameter) that were placed into 96-well plates at a density of 40,000 cells per well the day before the experiment. Cells were preloaded with [3H]5-HT (0.4 µM, 20 min at 37 °C). Subsequently, the cells were transferred into small chambers with a total volume of 200 µl and superfused with KHB at a flow rate of 0.7 ml per min for 40 min to establish a stable baseline before the collection of 2-min fractions was initiated. For each experiment, three basal fractions were collected before the cells were exposed to various concentrations (given in the figure legends) of the test drugs for five fractions. Subsequently, the cells were superfused with 1% SDS for three final fractions that served to determine the total amount of radioactivity present within the cells at the end of each experimental run. The amount of tritium within each fraction was determined by liquid scintillation counting, and release was expressed as percentage of radioactivity released in relation to the total radioactivity present at the beginning of that fraction [30]. Observations from superfusion experiments were excluded in case of premature exposure to SDS.

VMAT uptake inhibition assays in PC12 cells

VMAT uptake inhibition assays were conducted as previously described, with some modifications [31]. In brief, 107 PC12 cells per well were seeded in 24-well plates the day before experimentation. The cells were pretreated with 100 nM nisoxetine and mazindol to inhibit non-specific binding. PC12 cells were then preincubated with 200 µM digitonin to permeabilize membranes and were kept at room temperature on a plate shaker at low velocity for 30 min. Cells were treated with test drugs and shaken for another 10 min. Tritiated substrate (0.05 µM [3H]5-HT) was added, and the cells incubated on the plate shaker for 30 min. To terminate uptake, cells were transferred into 1.5 ml Eppendorf tubes, centrifuged at 5000 rpm for 5 min. The supernatant was aspirated, cells were washed with cold KHB, recentrifuged and lysed with 200 µl SDS. The solutions were transferred into vials containing 2 ml scintillation cocktail and radioactivity was determined via beta-scintillation counting (Perkin Elmer, Waltham, MA, USA). For analysis, inhibitory potency of test drugs was normalized to uptake inhibition caused by the potent VMAT inhibitor reserpine.

5-HT receptor-dependent induction of GCamP6 fluorescence in HEK293 cellsCloning of expression plasmids

All cloning steps were performed using NEB Builder (New England Biolabs, Frankfurt/Main, Germany). GCamP6s was cloned into pcDNA3.1. GCaMP is a genetically encoded calcium indicator which allows measurement of calcium flux. To allow for visual inspection of co-expressed 5-HT2 receptors, the enhanced cyan fluorescent protein (eCFP) was added to the C-Terminus of the 5-HT2 receptors. The resulting constructs were then cloned into pcDNA4-TO to allow tetracycline dependent expression.

Establishment of stable cell lines

In a first step, HEK293 cells were transfected with pcDNA6-TR and cells stably expressing the Tet-repressor were selected by the addition of blasticidin (60 µg ml−1). In a second step, one of the resulting clones was transfected with one of the 5-HT2 receptor constructs and stable clones were selected by the addition of zeocin (150 µg ml−1). Suitable clones were selected by visual inspection of the eCFP signal at the plasma membrane. In a last step, the eCFP positive clones were transfected with GCamp6s, and stable clones were selected using G418 (250 µg ml−1). The resulting clones were sorted via FACS to isolate GCaMP6s (green fluorescent protein) positive cells and the resulting polyclonal cell lines were used for further experiments. Cells were maintained in DMEM supplemented with 2% fetal bovine serum, blasticidin, zeocin, and G418 at concentrations described above. To induce receptor expression, tetracycline (1 µg ml−1) was added to the medium 18–24 h prior to the experiment.

In vitro Ca2+ Imaging

Imaging experiments were performed at room temperature (22–27 °C). Medium was exchanged for KHB 10 min prior to the experiment and cells were continuously superfused with KHB, using a fast superfusion device (DAD12, ALA Scientific, Farmingdale, NY, USA). Test drugs (0.1, 1, 10 µM) or 5-HT (1 µM) were applied for 30 s, interleaved by KHB superfusion for 300 s. GCamp6s fluorescence was monitored using an inverted microscope (Nikon Eclipse Ti2) equipped with a Nikon ×40 WI (NA 1.25) objective (Nikon Europe, Amsterdam, Netherlands). Fluorescence was excited using a 470 nm LED (Coolled pe4000, Coolled, Andover, NY, USA). Excitation light was filtered through a 480/20 nm bandpass filter and reflected via 505 nm dichroic mirror. Emission light was filtered using a 535/25 nm optical bandpass filter (Nikon Europe, Amsterdam, Netherlands). Images were taken at a frequency of 1 image s−1 with an exposure time of 20–30 ms using an sCMOS camera (Andor Zyla 5.5, Oxford Instruments, Abingdon, UK) and NIS-Elements 5.2 software (Laboratory Imaging, Praha, Czech Republic).

Image analysis

Image Stacks were background corrected by subtracting the mean intensity of one region of interest (ROI) per image from that of a region devoid of cells. For each image the mean fluorescence intensity of 1–5 ROIs consisting of several clustered cells was measured. The change in intensity of the GCamP6s fluorescence was calculated as the intensity at the end of test drug application minus the intensity before substance application, normalized to the increase of fluorescence evoked by the application of 1 µM 5-HT. Data represent 1–5 fields of view from three independent experiments

Confocal microscopy and image analysis

Confocal microscopy and image analysis was conducted as previously described [32]. In brief, cells expressing yellow fluorescent protein(YFP)-tagged human SERT or DAT, respectively, were seeded on PDL-coated 35 mm glass-bottom dishes 24 h before image acquisition. Culture medium was removed and the cells were exposed to the substances of interest (10 µM: S-4-MC, R-4-MC, S-4-MMC, S-4-TFMMC, R-4-TFMMC. 30 µM: R-4-MMC) for 1 h. Subsequently, trypan blue (0.4 %) was added for 10 min. Cells were washed with KHB and kept in buffer during image acquisition. A Nikon A1R+ laser scanning confocal microscope system with a ×60 NA 1.4 oil immersion objective (Nikon, Vienna, Austria) was used for imaging. Trypan blue was excited at 561 nm and the YFP-tagged transporter with a 488 nm laser line. Emitted light was filtered appropriately and detected with a GaAsP PMT detector. Four to five images were taken on 3 separate days. Fiji ImageJ 1.53c was used for image analysis. For each image, two regions of interest were hand-drawn per cell. One encompassed the cell membrane (defined by trypan blue staining) and one the cell interior. Membrane transporter expression levels were calculated as the ratio of membrane versus intracellular mean intensity.

Measurement of SERT-mediated currents

Whole-cell patch clamp in heterologous systems is a tool for measuring the transport of drugs by sodium-dependent transporters and therefore identifying their substrates [33, 34]. Transporter-mediated currents were measured by means of whole-cell patch clamp in HEK293 cells stably expressing SERT. Cells were voltage-clamped (−60 mV) and continuously superfused with a physiological external solution that contained 140 mM NaCl, 2.5 mM CaCl2, 2 mM MgCl2, 20 mM glucose and 10 mM HEPES, pH = 7.4. The pipette solution contained 133 mM potassium gluconate, 6 mM NaCl, 1 mM CaCl2, 0.7 mM MgCl2, 10 mM HEPES, 10 mM EGTA, pH = 7.2. Currents were measured at room temperature (20–24 °C) using an Axopatch 700B amplifier and pClamp 11.2 software (MDS Analytical Technologies, Sunnyvale, CA, USA). Solutions were applied using a DAD-12 superfusion system and an 8-tube perfusion manifold (ALA Scientific Instruments, Farmingdale, NY, USA), which allowed for rapid solution exchange. Current traces were filtered at 1 kHz and digitized at 10 kHz using a Digidata 1550 (MDS Analytical Technologies). Current amplitudes in response to application of test compounds or 5-HT were quantified using Clampfit 10.2 software (Molecular Devices, San Jose, CA, USA). Transporter-mediated currents elicited by test drugs were normalized to the current amplitude elicited by a saturating concentration of 5-HT (10 µM) applied to the same cell, in order to account for differences in cell expression. For analysis, passive holding currents were subtracted, and the traces were filtered using a 100-Hz digital Gaussian low-pass filter.

Stereotaxic surgery

Mice were anesthetized with isoflurane (5% induction, 2% maintenance) and placed into a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA) oriented in the flat skull position. Ophthalmic ointment was applied to prevent drying of the eyes. Ketoprofen (10 mg kg−1) was administered subcutaneously (s.c.), whereas bupivacaine and lidocaine were administered locally (s.c.; 100 µl of sterile saline containing 0.05% bupivacaine and 0.2% lidocaine) to the surgical site atop the head. A midline incision was made through the skin on the dorsal surface of the head to expose the skull. Fascia was removed, coordinates for the NAc were determined relative to bregma: anterior/posterior = 1.54 mm; medial/lateral = 0.7 mm; dorsal/ventral = −4.1 mm. A 1 mm burr hole was drilled through the skull. For the mice used in microdialysis, a 5 mm guide cannula (S-5000, Synaptech Inc., Marquette, MI, USA) was slowly lowered through the burr hole into the NAc and secured with glass ionomer cement. For the mice used for fiber photometry, 4*1012 genome copies of AAV9-hSyn-5-HT2h or 1010 genome copies of AAV-hSyn-DA2m were delivered into the NAc at an infusion rate of 100 nl min−1 (total volume of 1 µl) using a 34-gauge needle attached to a 10 µl Nanofil microsyringe (Hamilton, Reno, NV, USA), as previously described [35]. Following injection of the virus, an optical fiber was lowered through the burr hole and secured with glass ionomer cement. Three 1.6 mm screws (00-96X1/16 39052, Plastics One Inc., Roanoke, VA, USA) were affixed to the skull to support a cement stage that was created to secure the implanted guide cannulae or optical fiber. Mice were singly housed following surgery and allowed 6 days of recovery for the microdialysis experiments or 21 days for the photometry experiments. Food and water were available ad libitum.

In vivo microdialysis

Microdialysis was performed based on a previous publication [36]. After recovery from surgery, mice were placed into a clear cylindrical MTANK W/F enclosure (Instech, Plymouth Meeting, PA, USA) with bedding and water/food available ad libitum. A microdialysis probe with an active membrane length of 1 mm (S-5010, Synaptech Inc., Marquette, MI, USA) was inserted into the guide cannula and perfused with artificial cerebrospinal fluid (aCSF; 149 mM NaCl, 2.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, and 5.4 mM D-glucose, pH 7.2) at a flow rate of 1 µl per min. Dialysate samples were collected on ice and stored at −80 °C until they were analyzed using high-performance liquid chromatography with electrochemical detection. Drug-induced changes in extracellular DA and 5-HT were expressed as fold change compared to basal monoamine levels obtained during the first three pretreatment samples.

Fiber photometry

To monitor light emitted from the G-protein coupled receptor-activation-based 5-HT (GRAB5-HT) and DA (GRABDA) sensors, fiber photometry was performed as described [35]. Briefly, a light emitting diode (465 nm) (CLED_465, Doric Lenses, Quebec, QC, Canada), reflected through a dichroic mirror, was coupled to an optical fiber (200 µm core/225 cladding diameter; Thorlabs, Newton, NJ, USA) that was glued to a metal ferrule (Doric Lenses, Quebec, QC, Canada) and implanted into the NAc as described above. Emitted, band-pass filtered light (500–550 nm, FMC6, Doric Lenses, Quebec,QC, Canada) was detected with a photodetector (Newport Femtowatt silicone PIN; New Focus, San Jose, CA, USA). Data were recorded using a RZ5P lock-in amplifier (Tucker-Davis Technologies, Alachua, FL, USA) controlled with Synapse software. Sinusoidal excitation was delivered at 210 Hz by an LED driver Doric Lenses, LEDD_4) at low power mode. A demodulated signal was low-pass filtered at 6 Hz and digitized at 1017 Hz. Data were analyzed and processed using OriginPro (OriginLab, Northampton, MA, USA), Prism 9 (GraphPad, San Diego, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA). To quantify the effect of test drugs on GRAB5-HT and GRABDA dependent fluorescence, the signal was normalized to the average of the fluorescence as indicated in the corresponding figures, saline controls were fitted with a one phase decay and the traces were corrected accordingly. Mice were placed into a clear cylindrical MTANK W/F chamber (Instech, Plymouth Meeting, PA, USA) and received test drugs via intraperitoneal injection at the timepoints indicated in the figures and figure legends.

Data analysis

All data were analyzed using GraphPad Prism 9 (GraphPad Inc., L Jolla, CA, USA). The statistical tests used are given in each figure legend. Statistical significance was set at p ≤ 0.05. Data are shown as the mean and standard deviation. Homoscedasticity was assessed using Brown–Forsythe test. Detailed sample sizes are given in the Supplementary Information.

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