Cell Culture and Membrane Preparations.

Cell culture, transfections, and preparation of membranes from cell culture and tissue material for radioligand binding experiments were conducted as previously described (Lindemann et al., 2011).

Radioligand Binding and Selectivity Profiling.

Radioligand binding for mGlu5 and GABA_A receptors was performed as described previously (Lindemann et al., 2011).

Ca²⁺ Mobilization Assays.

Ca²⁺ mobilization assays were performed essentially as described previously (Lindemann et al., 2011). In brief, the cells were seeded at a density of 5 × 10⁴ cells/well in poly-d-lysine–treated, 96-well, black/clear-bottomed plates. After 24 hours, the cells were loaded for 1 hour at 37°C with 2.5 µM (2S)-2-amino-4-phosphonobutanoic acid (Fluo-4 AM) in loading buffer (1× Hanks’ balanced salt solution, 20 mM HEPES). The cells were washed 5 times with loading buffer to remove excess dye, and the intracellular calcium mobilization [Ca²⁺]_i was measured using FLIPR384 (Molecular Devices, Sunnyvale, CA) and FDSS7000 (Hamamatsu, Hamamatsu City, Japan) instruments.

The potency of basimglurant was studied in the presence of an agonist (mGlu1 and mGlu2: glutamate; mGlu5: quisqualate; mGlu4, mGlu8: (2S)-2-amino-4-phosphonobutanoic acid) at a concentration triggering 60%–80% of the maximal agonist response, which was determined daily in a separate experiment. The antagonists were applied in a serial dilution with 10 different concentrations 30 minutes before the application of agonists; the potential agonist activities of basimglurant were monitored online during the 30-minute preincubation period. The responses were measured as the peak increase in fluorescence recorded after the addition of basimglurant and the agonist (testing for antagonist activity), minus the basal value (i.e., fluorescence without addition of agonist), normalized to the maximal stimulatory effect induced by a saturating concentration of the agonist measured on the same plate. Inhibition curves were fitted using XLfit software (IDBS, Surrey, UK) according to the Hill equation: y = 100/(1+(x/IC₅₀)nH), where nH is the slope factor.

Inositol Phosphate Accumulation Assay.

Inositol phosphate (IP) accumulation assays were performed as described previously (Lindemann et al., 2011).

cAMP Accumulation Assays.

The cAMP accumulation assays using a cell line stably transfected with a cDNA encoding human mGlu7 were performed as described previously (Lindemann et al., 2011).

Selectivity Screening.

The selectivity profiling on approximately 100 targets comprised in the Broad Diversity Profile at a single concentration of 10 µM, and follow-up profiling in concentration-response on CB₁ and CB₂ cannabinoid receptors and on I₂ imidazoline receptor were conducted at CEREP (Celle l’Evescault, France). Selectivity profiling on recombinant human mGlu3 and mGlu6 receptors were conducted in concentration-response using a cAMP assay (Bassoni et al., 2012) at DiscoverX (Fremont, CA). Profiling on mGlu1, -2, -4, -7, -8 (Lindemann et al., 2011), monoamine reuptake transporters, and GABA_A (Lindemann et al., 2011) were conducted at F. Hoffmann-La Roche AG (Basel, Switzerland). Key experimental conditions for the selectivity profiling of basimglurant are summarized in Supplemental Table 1.

Monoamine Reuptake Transporter Assays.

Monoamine reuptake experiments were performed essentially as described previously (Hysek et al., 2012). In brief, HEK293 cells stably transfected with plasmids encoding human norepinephrine, 5-hydroxytryptamine (serotonin), or dopamine reuptake transporters were seeded at 3 × 10⁴ cells/well in 96-well opaque white assay plates. After 24 hours at 37°C, the cells were washed once with 100 µl of assay buffer (Krebs-Ringer bicarbonate buffer; Sigma-Aldrich, Buchs, Switzerland) for 10 minutes while shaking. Plates were snap-inverted, and 50 µl of fresh assay buffer was added followed by 50 µl of basimglurant or indatraline [(1R,3S)-3-(3,4-dichlorophenyl)-N-methyl-2,3-dihydro-1H-inden-1-amine] (positive control) diluted in assay buffer. Cells were incubated at 37°C for 30 minutes, and 50 µl of the radioligand solution was added ([³H]norepinephrine from Anawa Trading SA; [³H]serotonin and [³H]dopamine from PerkinElmer, Waltham, MA). After incubation at 37°C for 15 minutes, the assay was terminated by washing the cells twice with assay buffer using a cell washer. The remaining assay buffer was decanted, 250 µl of Microsynth 40 (PerkinElmer) was added per well, and the radioactivity was counted on a Topcount microplate scintillation counter (PerkinElmer). The results were expressed as a percentage of the radioactivity detected in the absence of drugs, and the IC₅₀ values were determined using XLfit software (IDBS), as described previously for radioligand competition binding.

Assessment for the Potential to Form Covalent Protein Adducts.

Assessment of the potential to form covalent protein adducts with hepatic proteins was essentially performed as described previously elsewhere (Fitch et al., 2010). In brief, [¹⁴C]-labeled basimglurant (49.2 µCi/µmol) was incubated with microsomal preparations, and radioactivity irreversibly bound to proteins after washing was measured. The incubation media (600 µl) consisted of 100 mM sodium phosphate buffer (pH 7.4), 1 mg/ml microsomal protein (human liver microsomes; Becton Dickinson Bioscience, Allschwil, Switzerland) 10 µM radiolabeled basimglurant, and 1 mM NADPH. The control incubations lacked NADPH. Additional experiments were conducted in the presence of reduced glutathione. The incubations were started by the addition of NADPH, after which the mixture was incubated at 37°C for 30 minutes. Experiments were conducted in triplicate.

The incubation was quenched by transferring 500 µl of the mixture into 650 µl of cold acetonitrile on a Multiscreen deep-well filter plate (Solvinert hydrophilic PTFE with prefilter; Millipore, Billerica, MA) to afford protein precipitation. Recovery of proteins was achieved by centrifugation at 1000g for 20 minutes at room temperature. The protein pellet was washed repeatedly with 750 µl of cold methanol containing H₂SO₄ (0.1%, v/v) followed by centrifugation at 1000g for 10 minutes at room temperature until background radioactivity levels were reached in the supernatants (typically 6 to 8 times). The remaining protein was dissolved in 500 µl of aqueous 1 M NaOH containing 1% (w/v) SDS at 60°C for 1 hour.

A 100-µl aliquot of the protein solution was used for protein determination (DC protein assay; Bio-Rad Laboratories, Hercules, CA), and the remaining solution was used for measuring the amount of incorporated radioactivity by liquid scintillation counting. Covalent protein binding was expressed as pmol equivalents of [¹⁴C]-labeled material bound per milligram of microsomal protein. Covalent binding to proteins was considered statistically significant if an increase as compared with the control incubation (lacking NADPH) was greater than 5-fold and exceeded background radioactivity levels. Covalent binding of >100 pmol [¹⁴C]/mg protein was considered as alert for metabolic activation under the assay conditions.

Human Ether-à-Go-Go-Related Gene Recordings.

Recording of human ether-à-go-go-related gene (hERG) currents on the recombinant human hERG channel was performed essentially as described previously (Fleury et al., 2011). In brief, the outward K⁺ currents were recorded in a Chinese hamster ovary cell line stably expressing recombinant hERG channels cloned from human heart (F. Hoffmann-La Roche Ltd., New York, NY) using a whole-cell configuration of the patch-voltage-clamp technique at 35–37°C using an EPC-10 triple amplifier (HEKA Elektronik GmbH, Lambrecht, Germany) and associated Patch MasterPro software (HEKA Elektronik GmbH). Cells were held at a resting voltage of −80 mV and were stimulated by a voltage pattern to activate hERG channels and to conduct outward IK_hERG current (500-millisecond prepulse to +20 mV followed by a 500-millisecond test pulse to −40 mV) at a stimulation frequency of 0.1 Hz (6 beats per minute).

After the cells had stabilized for a few minutes and the currents were steady, the amplitude and kinetics of IK_hERG were recorded under control conditions (vehicle control) for 3 minutes. Thereafter, basimglurant was applied at ascending concentrations for 3 minutes, each followed by a 100 nM solution of the standard IK_hERG blocker E-4031 (N-[4-[1-[2-(6-methylpyridin-2-yl)ethyl]piperidine-4-carbonyl]phenyl]), which completely blocks IK_hERG and is used as the positive control.

Experiments were performed in n = 3 replicates. If the drug effect on hERG currents was >20% compared with vehicle control, the concentration-response curve data were fitted using nonlinear regression analysis with FitMaster Pro software (HEKA Elektronik GmbH); if the drug effect was <20% compared with vehicle control, the inhibition was expressed as mean ± S.D.

Ames Mutagenesis Test.

The Ames mutagenesis test was performed essentially as described elsewhere (Muster et al., 2003). In brief, the Salmonella typhimurium strains TA1535, TA97, TA98, TA100, and TA102 were obtained from B. N. Ames (University of California–Berkeley). Fresh S9 rat liver mixtures were prepared for each experiment by mixing 0.1 ml of S9 preparation (Molecular Toxicology, Boone, NC), 0.2 ml of a 165 mM KCl solution, 0.2 ml of a 40 mM MgCl₂ solution, 0.2 ml of 200 mM sodium phosphate-buffered saline, pH 7.4, 3.2 mg of NADP (Roche Diagnostics, Rotkreuz, Switzerland), and 1.53 mg of glucose-6-phosphate (Roche Diagnostics). Bacterial growth media and agar, supplements, and tetracycline were obtained from Sigma-Aldrich.

Cultures of the strains were grown overnight at 37°C in a shaking water bath in a nutrient broth liquid medium to which 0.3 μg/ml of tetracycline was added for strain TA102 to maintain a stable plasmid copy number (Albertini and Gocke, 1988). The bacterial density was checked photometrically, and the cultures were diluted in 0.85% NaCl as needed. The sensitivity of the S. typhimurium strains was verified using the following positive controls: NaN₃ with strains TA1535 and TA100, ICR 191 with strain TA97, 2-nitrofluorene with strain TA98, and MMC with strain TA102. Moreover, 2-aminoanthracene was used with all strains with and without metabolic activation to confirm the activity of the S9 mix.

For testing basimglurant, test tubes containing 2 ml of 0.7% agar medium were autoclaved and kept in a prewarmed water bath at 42–45°C, and the following solutions were added: 1) 0.2 ml of a histidine/biotin mixture corresponding to 21 μg l-histidine and 24.4 μg biotin, 2) 0.1 ml solutions of basimglurant (20–2000 µg/plate) and positive controls, 3) 0.1 ml of bacterial overnight liquid cultures, 4) 0.5 ml of the S9 mixture where metabolic activation was needed, or 0.5 ml of 200 mM sodium phosphate-buffered saline, pH 7.4, where no metabolic activation was needed.

The contents of the tubes were mixed and poured immediately onto Vogel-Bonner minimal agar plates, allowed to solidify, and incubated at 37°C upside down for 2 days. Bacterial colonies were counted electronically using a DOMINO automatic image analysis system (Perceptive Instruments, Haverhill, UK) after inspection of the background lawn for signs of toxicity. The outcome of the test was considered positive for indicating mutagenicity when a dose-dependent increase in the number of colonies was observed that reached at least a 2-fold (strains TA1535, TA98) or 1.5-fold (strains TA97, TA100, TA102) increase over background.

In Vitro Micronucleus Test.

The in vitro micronucleus test was performed as described previously (Kirchner and Zeller, 2010). In brief, L5178Ytk^+/− mouse lymphoma cells (Covance Laboratories Ltd., Harrogate, UK) in exponential growth phase in growth medium (RPMI 1640 supplemented with 10% heat-inactivated horse serum, Glutamax-I, 100 IU/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml kanamycin) were seeded in 24-well cell culture plates. The S9 rat liver mixtures were prepared freshly for each experiment, as described previously (Kirchner and Zeller, 2010), and were added at a final concentration of 2% in the treatment medium.

For the micronucleus test without metabolic activation, 0.3 × 10⁶ cells in 700 µl of growth medium were seeded per well, and basimglurant as well as positive controls were added in 7 µl as dimethylsulfoxide stock solutions; the cells were incubated for 24 hours. For the micronucleus test with metabolic activation, 0.4 × 10⁶ cells in 700 µl of growth medium per well were incubated for 3 hours at 37°C with 140 µl of S9 mix and 8.4 µl of basimglurant and the reference compound dimethylsulfoxide stock solutions. Basimglurant was tested across a range of concentrations of up to 28 µg/ml without and 70 µg/ml with metabolic activation. The cells were then washed twice with growth medium in sterile 1.5-ml Eppendorf cups, resuspended in 700 µl of growth medium, seeded in 24-well plates, and incubated for another 21 hours at 37°C.

Preparation of slides and the fixation and staining of cells were performed as described previously (Kirchner and Zeller, 2010). The cells were inspected using fluorescence and light microscopy with phase contrast for the occurrence of micronucleation events indicative of DNA damage and cell cycle alterations. Results were considered positive for clastogenic/aneugenic if the number of micronucleated cells at any given concentration of test drug was elevated >2-fold compared with the vehicle (i.e., solvent) control.

Animals and Drug Treatment.

All experiments with animals were conducted in accordance with federal and local regulations at the respective study locations.

Rodents were maintained on a 12-hour light/dark cycle with the light period starting between 6:00 AM and 8:00 AM, with free access to chow and tap water unless specified otherwise. The room temperature (21–26°C) and humidity (30%–75%) were kept constant. The rodents used were Sprague-Dawley rats (conditioned emotional response: 350 g, male; Vogel conflict drinking test: 190–210 g, male; fear-potentiated startle: 225–287 g, male; Bennet neuropathic pain procedure: 100–250 g, female; overactive bladder procedure: 200–250 g, female; electroencephalography (EEG) recordings: 275–325 g, male; microdialysis: 250–350 g, male, in vivo receptor occupancy: 160 g, male), Wistar rats (pharmacokinetic profiles: 230–265 g, male; forced-swim test, 100–130 g, female; chronic mild stress-induced anhedonia: 350 g, male; Chung pain model: 276–340 g, male), Fischer F344 rats (functional magnetic resonance imaging [fMRI]: approximately 250 g, male), NMRI mice (stress-induced hyperthermia: ∼22 g, male; formalin-induced pain model: 24–30 g, male), and C57/Bl6J mice (in vivo receptor occupancy: 30 g, male). All experiments were performed with adult animals. The animals were housed in groups of 2 to 4 (rats) or 10 (mice), except for single-housing in the following procedures: stress-induced hyperthermia, chronic mild stress-induced anhedonia, microdialysis, and EEG. The rodents originated from Harlan/RCC (Füllinsdorf, Switzerland), Elevage Janvier (Le Genest-Saint-Isle, France), or Charles River (San Diego, CA/Margate, UK).

For recordings of pharmacokinetic profiles in nonhuman primates, adult male cynomolgus monkeys (Maccaca fascicularis) with a body weight of approximately 7.5 kg were used. The primates were maintained on a 12-hour light/dark cycle with lights on at 6:00 AM and free access to food and water unless specified otherwise.

Basimglurant and reference comparator drugs were formulated as microsuspensions in 0.9% NaCl/0.3% Tween-80, prepared and stored as previously described (Lindemann et al., 2011). They were administrated by oral (p.o.) gavage or by subcutaneous or intraperitoneal injection unless otherwise indicated.

Recording of Pharmacokinetic Profiles.

Pharmacokinetics (PK) in male rats: for intravenous PK, the compound was formulated in N-methylpyrolidone/saline (30%/70%) as the vehicle and was administered at a volume of 2 ml/kg. For p.o. gavage, the compound was administered as a suspension using gelatin/saline (7.5%/0.62% in water) at an administration volume of 4 ml/kg.

PK in male cynomolgus monkeys: For intravenous PK, the compound was formulated in cyclodextrin solution as the vehicle and was administered at a volume of 2 ml/kg. For p.o. gavage, the compound was administered in capsule form (2 mg in size-2 capsules, i.e., ∼0.3 mg/kg) to fasted or fed monkeys in a crossover design.

Processing of the blood samples and preparation of the liver microsomes and hepatocyte cultures was as described previously (Valles et al., 1995). Human liver tissue was obtained from hepatic surgical resections at the Hospital Hautepierre (Strasbourg, France) in accordance with the guidelines of the ethics committee. Hepatocyte cultures were incubated with basimglurant at a concentration of 1–10 µM for 24 hours; hepatocyte microsomes were incubated with basimglurant at a concentration of 1–10 µM for 1 hour.

All samples were analyzed using routine sample preparation techniques followed by liquid chromatography with tandem mass spectrometry analysis, as described previously (Lindemann et al., 2011). PK parameters for all studies were calculated using noncompartmental analysis.

Receptor Occupancy Measurements.

Receptor occupancy measurements were performed using a tritiated version of ABP688 (Ametamey et al., 2006; Hintermann et al., 2007) essentially as described elsewhere (Lindemann et al., 2011). Animals received p.o. doses of either vehicle or basimglurant (0.03–3 mg/kg) 60 minutes before a tail vein injection of [³H]ABP688 (0.3 mCi/kg). After 30 minutes, the animals were sacrificed, and plasma as well as brain samples were collected. Samples were processed further for mice (Lindemann et al., 2011) and rats (Michalon et al., 2014) as described previously.

[³H]Basimglurant in vivo binding was performed by intravenous injection of rats with [³H]basimglurant (0.3 mCi/kg) in the tail vein. Sixty minutes later, rats received either vehicle (0.9% saline/0.3% Tween-80) or an oral dose of 10 mg/kg RO4623831 (C₁₆H₁₁N₃FCl; molecular weight 311.75; see Supplemental Table 2 for the pharmacological properties of RO4623831). At 60 minutes after the dose of RO4623831, the animals were sacrificed, and the brains were processed in the same manner as for the receptor occupancy measurements.

Chronic Mild Stress–Induced Anhedonia Test.

The chronic mild stress (CMS)–induced anhedonia test was performed essentially as described previously (Moreau et al., 1996). In brief, electrodes were implanted unilaterally in the mesolimbic system at the level of the ventral tegmental area of the midbrain (2 mm anterior from lambda, 0.3 mm lateral from the midline suture, and 8.5 mm ventral from the skull surface; electrode tips approximately 0.5 mm apart in the dorsoventral plane). After surgery, the animals were allowed to recover for 5 days, after which they underwent intracranial self-stimulation (ICSS) training. After a consistent ICSS baseline had been established, the rats either were subjected to 6 weeks of unpredictable CMS or were left undisturbed. From days 21 to 42, the stressed animals received once-daily doses (i.p.) of drugs (basimglurant, fluoxetine) or vehicle. The control group of animals that were not undergoing CMS received high-dose basimglurant (3 mg/kg) or vehicle. The anhedonia index was calculated as the percentage of change in the ICSS threshold from baseline.

Forced-Swim Test.

The FST was performed essentially as described previously (Cryan and Lucki, 2000). Rats were placed inside vertical plexiglas cylinders (height: 40 cm; diameter: 17.5 cm) containing 15 cm of water maintained at 23–24°C for 15 minutes. After 24 hours, the rats were again placed in the cylinder, and the total duration of immobility was measured during a 5-minute period. Rats were administered drugs (p.o.) at 24, 16, and 2 hours before the testing period.

Functional MRI in Rats.

Functional MRI imaging and data processing were performed essentially as described by Bruns et al. (2009). In brief, Fischer F344 rats received basimglurant and comparator drugs as single doses (p.o.) approximately 1 hour before the experiment. For fMRI, rats were anesthetized with isoflurane in oxygen and air (1:5) supplied via a face mask to the spontaneously breathing animals. The isoflurane concentration was adjusted between 1.8% and 2.4% to maintain stable respiration rates at 50–60 breaths per minute. The animals were placed in a cradle, and their heads were immobilized in a stereotaxic frame. Their respiratory rate, body temperature, and O₂ and CO₂ levels in the inhaled and exhaled air were continuously monitored on a PowerLab data acquisition system (ADInstruments, Spechbach, Germany). Body temperature was maintained at 37°C with a feedback-regulated electric heating blanket. The total time under anesthesia was 35 minutes.

Magnetic resonance imaging was performed on a 4.7 T/40 cm Bruker Biospec animal scanner (Bruker BioSpin, Ettlingen, Germany), equipped with a 72-mm bird-cage resonator for excitation and a surface receiver coil (Rapid Biomedical, Rimpar, Germany). On the scout images, the most rostral extension of the corpus callosum was used as a landmark for selection of eight coronal image planes at −10.0, −7.8, −5.3, −2.9, −1.6, −0.3, +1.0, and +2.3 mm from the bregma (Paxinos, 1986). All subsequent images were acquired from these planes, with a field of view of 4 cm × 4 cm and a slice thickness of 1 mm. First, a set of RARE T₂-weighted anatomical images were obtained (TR/TE_eff 1.8 seconds/39 milliseconds, RARE factor 8, matrix 256 × 256) (Hennig et al., 1986). Next, a T1 image series, required to quantitatively calibrate perfusion readouts, was obtained using an inversion-recovery snapshot FLASH sequence with eight inversion times (TR/TE 3.4 seconds/1.4 milliseconds, matrix 128 × 64) (Haase et al., 2011). Finally, perfusion-weighted images were acquired using continuous arterial spin labeling (Williams et al., 1992) with a centered RARE readout (TR/TE 3.75 seconds/5.7 milliseconds, RARE-factor 32, matrix 128 × 64, labeling pulse 2.5 seconds, postlabeling delay 0.4 seconds). Three consecutive volumes of perfusion images were acquired within 12 minutes.

The images were processed and analyzed using in-house developed software written in IDL (RSI, Boulder, CO) and MATLAB (MathWorks, Natick, MA), including the open-source software SPM5 (Wellcome Trust Centre for Neuroimaging, London, UK). For spatial normalization, the anatomical images were coregistered to a rat-brain template by affine and nonlinear transformations, which were then applied to the all functional images. T1 maps were calculated on a voxel-wise basis by fitting a three-parameter exponential to the intensities across the eight inversion times (Deichmann et al., 1999); the maps were then combined with the related continuous arterial spin labeling images to obtain quantitative absolute perfusion maps, as described elsewhere (Alsop and Detre, 1996; Bruns et al., 2009). To account for possible systemic changes affecting global brain perfusion, and to eliminate part of the interindividual variability, perfusion maps of each individual were normalized slice-wise to the brain-mean value, which was set to 100%. The perfusion values were averaged region-wise with reference to an in-house generated digital atlas, with regions of interest (ROIs) adapted from the Paxinos and Watson rat-brain atlas (Paxinos, 1986).

Statistical analysis of perfusion data was performed with JMP (SAS Institute, Cary, NC). Normalized perfusion for each dose group was compared ROI-wise with those of the pertinent control (vehicle) group using Welch’s t test. P < 0.05 was considered statistically significant. To avoid a severe drop in statistical power, the values were not corrected for multiple testing; rather, the false-discovery rate was estimated per dose group. The estimated number of false-positive results was on average 1 ± 1 (mean ± S.D. across dose groups) and never exceeded 3. The data were further characterized in a framework of pro- and antidepressant interventions by calculating the root-mean-square (RMS) of the perfusion changes across all ROIs and the scale-invariant pattern match coefficients (PMC; the dot product between the two neural activity profile vectors, each scaled to unit length) as measures of response strength and similarity between the effect of basimglurant and reference interventions, respectively.

Vogel Conflict Drinking Test.

The Vogel conflict test was performed as described elsewhere (Lindemann et al., 2011) with p.o. administration of drugs and 1 hour pretreatment time.

Stress-Induced Hyperthermia Test.

The stress-induced hyperthermia test was essentially performed as described elsewhere (Spooren et al., 2002). In brief, body temperature was measured in mice twice, 1 minute apart (T1 and T2, respectively), using a rectal probe. Measurement of T1 served as the handling stressor. The difference in body temperature (ΔT = T2 − T1) was determined. Drugs were administrated p.o. with 1 hour pretreatment time before measuring T1.

Conditioned Emotional Response.

The conditioned emotional response (CER) test was performed as described previously by Ballard et al. (2005) with p.o. administration of drugs and 1 hour of pretreatment time. In brief, the animals were tested using a Latin-square design twice weekly, with at least a 48-hour interval between the test sessions. The readout of the test, the so-called suppression ratio, is defined as the ratio of lever presses within the conditioning period to the total lever presses around this time (Lever presses 2 minutes before + Lever presses within the 2-minute conditioning period).

Fear-Potentiated Startle Response Procedure.

The fear-potentiated startle (FPS) response procedure was performed as described previously (Busse et al., 2004), with minor modifications. Rats were conditioned to associate a light stimulus with a mild foot shock (0.25 mA for 0.5 seconds) during two consecutive sessions on 2 consecutive days. Animals received drugs with p.o. administration and 1 hour of pretreatment time before the test session on the third day, in which an acoustic stimulus was either paired with the light stimulus or not. The fear-potentiated startle response was calculated by subtracting the mean startle response (unpaired) from the mean startle response (paired).

Formalin-Induced Pain (Paw Licking) Test.

The formalin-induced pain (paw licking) test was performed at Porsolt & Partners Pharmacology (Le Genest-Saint-Isle, France) as described previously elsewhere (Lopes et al., 2013), with minor modifications. In brief, mice received an intraplantar injection of 5% formalin (25 μl) into the posterior left paw. In one group of animals, the time spent on licking paws was recorded for 5 minutes, beginning immediately after injection of formalin (early phase). In a second cohort of animals receiving an identical formalin injection, the paw-licking time was recorded beginning 20 minutes after the formalin injection (late phase). NMRI mice received drugs or vehicle (p.o.) 1 hour before the start of recording paw-licking time.

Chung Model of Neuropathic Pain.

The Chung model of neuropathic pain (Kim and Chung, 1992) was performed at Porsolt & Partners Pharmacology as described previously (Basile et al., 2007). In brief, rats were anesthetized with sodium pentobarbital and the left L5 and L6 spinal nerves were ligated, followed by 2 weeks of recovery. For the thermal stimulation, a mobile infrared radiant source was focused under the nonlesioned and lesioned hind paws, and the paw-withdrawal latency was automatically recorded. The rats received drugs (p.o.) with 1 hour of pretreatment time before testing.

Bennet Model of Cold Allodynia (Chronic Sciatic Nerve Constriction Model).

The Bennett model of cold allodynia was performed as described elsewhere (Bennett and Xie, 1988; Hunter et al., 1997), with minor modifications. In brief, rats were anesthetized with isoflurane, and the sciatic nerve of the right hind limb was mildly constricted with four ligatures that were tied circumferentially around the nerve approximately 1 mm apart. After recovery for ≥4–7 days, rats were tested for cold-induced innocuous pain (allodynia) in a testing chamber filled to a depth of 1.5–2.0 cm with water at 2–4°C. After at least 1 hour of resting period, rats received drugs and vehicle subcutaneously with different pretreatment times as follows: basimglurant (0.1–10 mg/kg, 60 minutes), morphine (1 mg/kg, 30 minutes), duloxetine (3 mg/kg, 90 minutes), and vehicle (60 minutes). The inhibition rate was calculated with the following formula: Inhibition rate = (Paw lifts after treatment − Mean paw lifts after vehicle treatment))/(Sham pretreatment − Mean pretreatment vehicle) × 100. The drug-treated group was compared with the vehicle group using a two-sample t test with equal variance assumption. Basimglurant was formulated in H₂O with 10% propylene glycol adjusted to pH 6.0 with 0.1 M HCl; morphine and duloxetine were dissolved in H₂O.

Overactive Bladder: Measuring Threshold Volume in the Volume-Induced Micturition Reflex Model.

The volume-induced micturition reflex was performed in rats as described elsewhere (Hu et al., 2009). In brief, the urinary bladder was cannulated under anesthesia in rats and infused with saline to determine the micturition threshold. After establishing a stable baseline, the drug or vehicle doses were administrated intravenously 5 minutes before the next infusion cycle; thereafter, the change in micturition threshold volume was recorded.

Overactive Bladder: Contraction Frequency in the Isovolume Bladder Contraction Model.

Bladder contraction frequency was measured in rats as described previously (Hu et al., 2009). In brief, the urinary bladder was cannulated under anesthesia in rats and infused with saline to evoke micturition contractions. After stable isovolume bladder contractions were obtained, the infusion rate was lowered to 5 µl/min, and the system was allowed to stabilize for at least 30 minutes. Thereafter, vehicle was administered, followed 10 minutes later with single doses of basimglurant (0.003–0.03 mg/kg i.v.). The frequency of isovolume bladder contractions was recorded for 60 minutes in total.

EEG Recordings.

EEG recordings were performed at SRI International, Biosciences Division (Menlo Park, CA) as described previously (Morairty et al., 2008), with minor modifications. In brief, rats were implanted with telemetric devices for continuous recordings of EEG, electromyograph, core body temperature, and locomotor activity (F40-EET; DSI, St. Paul, MN). Animals were acclimated to the handling procedures and given 2 once-daily 1-ml doses of vehicle 7 and 3 days before start of the study. Drugs (basimglurant 0.03–0.3 mg/kg, caffeine 10 mg/kg) were formulated in standard vehicle (0.9% saline/0.3% Tween-80). Using a repeated-measure, counterbalanced design, each rat received five subchronic dosing conditions with 9 days of washout between each subchronic treatment condition. For each condition, daily dosing occurred 2 hours into the active period for 5 consecutive days (at the start of Zeitgeber time [ZT] 14). For the caffeine condition, however, vehicle was administered on days 1–4, and caffeine was only administered on the fifth day. Data were recorded and analyzed only for the fifth day of dosing starting at lights off (2 hours before dosing).

Microdialysis.

Microdialysis was performed at Renasci Ltd. (Nottingham, UK) as described elsewhere (Rowley et al., 2014), with minor modifications. In brief, rats were anesthetized with isoflurane (5% to induce, 2% to maintain) in an O₂/N₂O (1 l/min each) mixture and dual-probed whereby two microdialysis probes were stereotaxically implanted bilaterally into 1) the prefrontal cortex (2 mm tip, coordinates: anterior-posterior +3.2 mm; L (lateral): ±2.5 mm relative to bregma; ventral: −4.0 mm relative to the skull surface) and 2) the nucleus accumbens (2 mm tip, coordinates: anterior-posterior +2.2 mm; L: ±1.5 mm relative to bregma; ventral: −8.0 mm relative to the skull surface). After surgery, the animals were individually housed in circular chambers (dimensions 450 mm internal diameter, 320 mm wall height) with the microdialysis probes connected to a liquid swivel and a counterbalanced arm to allow unrestricted movement.

Rats were allowed a recovery period of at least 16 hours with free access to food and water before the onset of sample collection. The experiments were performed 1 day after surgery. Dialysate samples were collected from freely moving rats at 20-minute intervals, beginning 80 minutes before drug administration and continuing for 4 hours after (4 basal samples and 12 postdrug samples). Animals received p.o. injections of either basimglurant (0.1 or 1 mg/kg), paroxetine (3 or 10 mg/kg), or vehicle.

Detection and subsequent quantification of 5-HT (5-hydroxytryptamine [serotonin]), dopamine (DA), and norepinephrine (NE) in the dialysis samples were based on reverse-phase, ion-pair high-performance liquid chromatography coupled with electrochemical detection and involved the use of an ALEXYS monoamine analyzer (Antec Leyden, The Netherlands). The system consisted of two separate analytic columns that shared a dual-loop autosampler, allowing for one sample to be simultaneously analyzed by two systems optimized for different neurotransmitters. In this instance, one column separated NA (ALF-115, 150 mm × 1 mm internal diameter) while the other separated 5-HT and DA (ALF-105, 50 mm × 1 mm internal diameter). Two solvent delivery pumps (LC 110) were used to circulate the respective mobile phases (5-HT/DA: 50 mM phosphoric acid, 8 mM NaCl, 0.1 mM EDTA, 4.6 mM 1-octane sulfonic acid, 20% methanol, pH 6.0; NA: 50 mM phosphoric acid, 8 mM NaCl, 0.1 mM EDTA, 3 mM 1-octane sulfonic acid, 10% methanol, pH 3.25) at a flow rate of 50 µl/min; an Antec in-line degassing unit was used to remove air. Samples (10 µl) were injected onto the columns via an autosampler (AS 110) with a cooling tray set at 4°C.

Antec DECADE II electrochemical detectors were used with an Antec micro VT 03 cells employing a high-density, glassy carbon working electrode (+0.3 V for 5-HT and DA, +0.59 V for NA) combined with an ISAAC reference electrode. The electrode signal was integrated using Antec’s Clarity data acquisition system. Individual stock solutions of 5-HT, DA, and NA (1.0 mM) were prepared in a mixture of equal quantities of deionized water and 0.1 M perchloric acid (to prevent oxidation) and were stored at 4°C. A working solution containing all three transmitters was prepared daily by dilution in artificial cerebrospinal fluid.

At the end of the study, the rats were sacrificed, and probe placement was visually confirmed. Data were reported only from animals where probe membranes were correctly positioned.