Abstract
Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca2+ assays (EC50 = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 μM) and competed for binding of [3H]2-methyl-6-(phenylethynyl)pyridine (Ki = 4.3 μM), but not [3H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.
The metabotropic glutamate receptor (mGlu) receptor family includes eight G-protein-coupled receptor (GPCR) subtypes classified on the basis of structural homology, mechanism of signaling transduction, and pharmacological properties. Stimulation of group I receptor (subtypes 1 and 5) leads to activation of phospholipase C, increased phosphoinositide hydrolysis, and mobilization of intracellular calcium (Conn and Pin, 1997).
Positive allosteric modulators (PAMs) of mGlu receptors offer an attractive alternative to the direct activation of mGlu receptors by orthosteric competitive agonists. PAMs have been discovered for mGlu1, mGlu2, and mGlu5 receptors (Knoflach et al., 2001; Johnson et al., 2003; O'Brien et al., 2003). These molecules offer the potential to increase the efficiency of normal glutamate transmission without the risk of inappropriate stimulation. Furthermore, such compounds are more likely to achieve high receptor subtype selectivity by targeting regions of the receptor that are different from those affected by the endogenous ligand. PAMs of mGlu5 include 3,3′-difluorobenzaldazine, CPPHA (O'Brien et al., 2003a, 2004), and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (CDPPB) (Lindsley et al., 2004; Kinney et al., 2005). These compounds have little or no direct agonist activity but act as selective PAMs of competitive agonists of human and rat mGlu5; however, they suffer from relative weak in vitro activity, such as poor potency and efficacy (DFB and CPPHA) (O'Brien et al., 2003, 2004) and solubility (CD-PPB) (Kinney et al., 2005).
Recently, ADX47273 (de Paulis et al., 2006) was identified as a novel selective mGlu5 PAM at the 5th International Metabotropic Glutamate Receptors Meeting (Le Poul et al., 2005). Up to 60 μM ADX47273 showed no agonist, antagonist, or allosteric modulator activity at other rat and/or human family III GPCRs (mGlu1–8 and GABA-B). In addition, ADX47273 (10 μM) failed to displace radioligand binding to 56 GPCRs, transporters, enzymes, and ion channels (Le Poul et al., 2005). ADX47273 is reported to have a brain/plasma ratio of 1.6 and a 2-h half-life in the mouse (Poli et al., 2005). In general, it appears that ADX47273 has pharmacological properties, such as potency, efficacy, and blood-brain barrier penetrability, which make it an improved tool for profiling the in vivo effects of modulating mGlu5 receptor activity.
Several lines of evidence suggest that impaired NMDA receptor-mediated neurotransmission is a major component of the pathophysiology of schizophrenia (Lindsley et al., 2006). Activation of mGlu5 has been suggested as one of the approaches by which NMDA receptor function can be augmented (Marino and Conn, 2002). For example, activation of mGlu5 enhances NMDA receptor-mediated currents in slices from rat hippocampus (Doherty et al., 1997) and subthalamic nucleus (Awad et al., 2000). Conversely, the mGlu5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) potentiates the effects of NMDA receptor antagonists on spontaneous burst and spike activity of cortical neurons (Homayoun and Moghaddam, 2006) and enhances the effect of NMDA antagonists on behavior, such as prepulse inhibition, locomotion, and working memory impairments (Campbell et al., 2004; Homayoun et al., 2004). The ability of mGlu5 antagonists to potentiate the detrimental effects of NMDA receptor antagonists suggests that activation of mGlu5 may represent an approach toward ameliorating symptoms of schizophrenia (Marino and Conn, 2002). As predicted, CDPPB exhibits antipsychotic-like activity on amphetamine-induced hyperlocomotion and prepulse inhibition deficits (Kinney et al., 2005). More recently, CDPPB prevents NMDA receptor antagonist MK-801-induced excessive firing in the prefrontal cortex (Lecourtier et al., 2007). Thus, mGlu5 PAMs may also be effective in treating the cognitive deficits in schizophrenic patients by ameliorating the NMDA receptor hypofunction thought to underlie these cognitive deficits.
The effects of mGlu5 PAMs on the molecular mechanisms associated with learning and memory processes also suggest efficacy in cognitive enhancement. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and the transcription factor cAMP-responsive element-binding (CREB) protein play a major role in synaptic plasticity and cognition (Thomas and Huganir, 2004; Carlezon et al., 2005). Previously, we demonstrated that CPPHA potentiated the response to a subthreshold concentration of the nonselective group 1 agonist (S)-3,5-dihydroxyphenylglycine on ERK and CREB phosphorylation in cortical and hippocampal slices (Liu et al., 2006), suggesting that modulation of mGlu5 by a PAM can regulate these major signaling molecules important in learning and memory.
The present set of studies used ADX47273 to further explore the potential of a mGlu5 PAM as a therapeutic approach for the treatment of positive symptoms and cognitive deficits associated with schizophrenia. ADX47273 was evaluated in models predictive of antipsychotic efficacy [apomorphine-induced climbing/stereotypy, conditioned avoidance responding, and hyperactivity induced by phencyclidine (PCP), apomorphine, and amphetamine] and cognition (novel objective recognition and the five-choice serial reaction time task).
Materials and Methods
Subjects
Male CF-1 mice (20–28 g; Charles River Laboratories, Inc., Wilmington, MA) were used in the antagonism of apomorphine-induced behaviors and locomotor hyperactivity studies. Male Sprague-Dawley rats were used for the conditioned avoidance test (350–450 g; Charles River Laboratories, Inc.). Male Long-Evans rats (350–450 g; Charles River Laboratories, Inc.) were used for the novel object recognition and five-choice serial reaction time (5-CSRT) tasks. All animals were group-housed (except for conditioned avoidance response, novel object recognition, and 5-CSRT subjects, which were housed singly) in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility that was maintained on a 12-h light/dark cycle (lights on at 6:00 AM). Food and water were available ad libitum, except where noted. All studies were approved by the Institutional Animal Care and Use Committee and were performed in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the National Institutes of Health (Institute of Laboratory Animal Resources, 1996).
Drugs
All drugs and vehicles were administrated intraperitoneally. The compound ADX47273 was synthesized by Wyeth Research. PCP hydrochloride, apomorphine hydrochloride, d-amphetamine sulfate, and carboxymethyl-cellulose were obtained from Sigma-Aldrich (St. Louis, MO). MPEP hydrochloride and MTEP hydrochloride were purchased from Tocris Bioscience (Bristol, UK). Drugs were dissolved in saline (PCP and d-amphetamine) or suspended in 0.2% Tween 80/water (apomorphine) or 2% Tween 80 + 0.5% carboxymethyl-cellulose (ADX47273, MPEP, and MTEP). Solutions were administered at a volume of 10 ml/kg to mice and 1 ml/kg to rats, unless otherwise noted. The dose administered for each compound refers to the amount of the active component, rather than the salt. All other materials were analytical grade and were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Sigma-Aldrich.
Procedures
Fluorometric Imaging Plate Reader. Ca2+ influx measurements were made using the fluorometric imaging plate reader (FLIPR) 384 fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA). HEK 293 cells expressing rat mGlu5 or mGlu1 receptor were plated in clear-bottomed 384-well plates in glutamate/glutamine-free media and loaded the next day with calcium-sensitive fluorescent dye Calcium 3, and placed in FLIPR384. HEK 293 cells expressing the cloned rat mGlu5 and mGlu1 receptor show concentration-dependent increases in Fluo-3 fluorescence in response to glutamate, with an EC50 value of 300 nM and 2 μM, respectively. A range of concentrations of ADX47273 alone or together with 50 nM glutamate (EC10 concentration) was added to the cells, and the Ca2+ response was measured by FLIPR384. For MPEP experiments, MPEP (10 μM) was added 30 min before the FLIPR assay as a pretreatment. For primary astrocyte cultures, astrocytes were preloaded for 50 min at 37°C/5% CO2 using the FLIPR calcium three-assay kit (containing 3 U/ml glutamate pyruvate transaminase, 3 mM pyruvate, and 2.5 mM probenecid) according to the manufacturer's instructions (Molecular Devices). Cells were left to equilibrate at room temperature for 15 min before basal fluorescence (t = 10 s) was determined using FLIPR384 (Molecular Devices). Allosteric modulators were added 5 min before the addition of 50 nM (EC10 concentration) glutamate. For the astrocytes, the EC20 of glutamate was determined as 300 nM and was subsequently used for PAM experiments. Data were normalized to the calcium signal produced by maximal concentrations of glutamate on each plate.
Primary Astrocyte Cultures. This protocol was adapted from (Marriot et al., 1995). Mixed glia cultures were prepared from rat cortex using 2-day-old neonates. Cultures were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated, dialyzed fetal bovine serum, l-glutamine, and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). After 10 days, in vitro G-5 (1×) supplement (Invitrogen) was added to the cultures, and astrocytes were purified by overnight shaking (120 rpm). The following day, astrocytes were lifted and plated into poly (d-lysine)-coated 384-well plates at a density of 10,000 cell/well 48 h before experimentation. Using this protocol, the final preparation contained ∼90% glial fibrillary acidic protein-positive astrocytes (data not shown).
Radioligand Binding Assays.[3H]MPEP binding. [3H]MPEP was used to evaluate the interaction of compounds with the receptor. Membranes were prepared from HEK 293 cells expressing rat mGlu5 receptor. Aliquots of these membranes were added to tubes containing ADX47273 (0.4% DMSO final concentration) or vehicle and [3H]MPEP (2 nM final concentration in 50 mM Tris/0.9% NaCl, pH 7.4). The tubes were incubated for 60 min at room temperature with shaking, and the membrane-bound ligand was separated from the free ligand by filtration onto glass fiber filters presoaked with 20 mM HEPES, 2 mM CaCl2, and MgCl2, pH 7.2. Membrane bound radioactivity was determined by scintillation counting of the filters. Competition binding data were analyzed, and Ki was determined using Prism 4.0 (GraphPad Software Inc., San Diego, CA).
[3H]Quisqualate binding. Membranes from CHO cells expressing rat mGlu5 were resuspended in ice-cold assay buffer (20 mM HEPES-NaOH, pH 7.2, containing 2 mM MgCl2 and 2 mM CaCl2) with 20 nM [3H]quisqualate either in the absence or presence of 10 μM ADX47273 for 1 h at room temperature. Nonspecific binding was defined in the presence of 1 mM glutamate. At the end of the incubation, the suspension was filtered onto Whatman GF/C glass fiber filters and washed rapidly three times with 1 ml of cold binding buffer. The radioactivity trapped on the filters was measured by liquid scintillation in a Tri-Carb model 2500 TR counter (Canberra Industries, Meriden, CT).
Functional Calcium Mobilization Assay for mGlu2. Recombinant CHO cell lines coexpressing human mGlu2 and GqGi3 were plated at a seeding density of 0.5 × 105 cells/well in clear-bottomed, non-poly-d-lysine-coated 96-well plates. Cells were incubated in glutamate/glutamine-free medium overnight at 37°C in an atmosphere of 95% O2/5% CO2. Cells were loaded with calcium indicator dye (Calcium 3 Assay Kit) containing 3 U/ml glutamic pyruvic transaminase, 3 mM sodium pyruvate, and 2.8 mM probenecid at 37°C for 1 h. At this stage, cells were used for the calcium mobilization assay. ADX47273 was dissolved to a stock solution of 10 mM in 100% DMSO and then half-log diluted into H2O. The stock solution was added to the assay plate to a final DMSO concentration of 0.4%. Agonist activity was determined by adding ADX47273 (0.1 nM–10 μM) to the well. Antagonist activity was assessed by pretreating cells with ADX47273 (0.1 nM–10 μM) for 5 min followed by a submaximal concentration (30 μM) of glutamate (EC80). PAM activity was evaluated by performing an l-glutamate concentration response curve (3 nM–100 μM) in the absence or presence of 10 μM ADX47273. Calcium mobilization was measured using the FLEXstation II (Molecular Devices).
Functional Cyclase Inhibition Assay for mGlu4. CHO cells expressing human mGlu4 were plated in poly-d-lysine-coated 96-well plates 1 day before assay. Cells were washed with Hanks' balanced salt solution for 10 min at room temperature. Compounds were half-log diluted in 4% DMSO. The assay procedure according to the DiscoverX HitHunter cAMP XP Assay Kit was followed. To measure agonist activity, cells were incubated with forskolin (10 μM), ADX47273 (0.1 nM–10 μM), and cAMP XP antibody supplemented with 500 μM 3-isobutyl-1-methylxanthine, 3 U/ml glutamic pyruvic transaminase, and 3 mM sodium pyruvate for 30 min at 37°C. Antagonist activity was evaluated by treating cells with ADX47273 (0.1 nM–10 μM), 10 μM forskolin, and 1 μM l-AP4 (EC80). PAM activity was assessed by performing an l-AP4 concentration-response curve (0.1 nM–10 μM) in the absence or presence of 10 μM ADX47273. cAMP was measured using the luminescent reader of the Packard TopCount.
Immunoblot Analysis. Drugs were administered intraperitoneally to six male Long-Evans rats per dose level. Thirty minutes later, animals were sacrificed (decapitation), and hippocampus and prefrontal cortex were dissected and homogenized by sonication in 1% SDS/50 mM NaF buffer. Equivalent amounts of protein from each individual sample were resolved in 4 to 12% SDS-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in Tris-buffered saline containing Tween 20 and then incubated with the phosphospecific antibody of interest (phospho-ERK antibody, 1:1000; phospho-CREB Ser133 antibody, 1:1000; Cell Signaling Technology Inc., Danvers, MA) overnight at 4°C followed by incubation with horseradish peroxidase-linked goat anti-rabbit IgG (1:10,000) and developed using enhanced chemiluminescence (Promega, Madison, WI). The blots then were incubated in stripping buffer (62 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β-mercaptoethanol). The stripped blots were incubated with antibody directed against total levels for the respective protein overnight at 4°C (ERK, antibody, 1:1000; CREB antibody, 1:1000; Cell Signaling Technology Inc.). Densitometric analysis of phospho-immunoreactivity and total immunoreactivity for each protein was conducted using the Bio-Rad GS-710 Calibrated Imaging Densitometer and quantified using Quantity One version 4.1.0. Phosphorylated immunoreactivity was normalized to total immunoreactivity. Data were statistically analyzed by Student's t test (unpaired) using Microsoft Excel (Microsoft, Redmond, WA).
Conditioned Avoidance Responding. Rats were maintained on a food-restricted schedule (15 g of standard rodent feed each day after training/testing). Four shuttle box test chambers (MED Associates, St. Albans, VT) were used (divided into two compartments by an archway). Each chamber floor half was composed of 13 3/16-inch-diameter stainless steel grid rods placed on 1/2-inch centers wired for the presentation of a scrambled electric foot shock (0.5 mA). In addition, each side of the chamber was equipped with a stimulus light and tone (Sonalert, ENV-223AM; Med Associates, Inc., St. Albans, VT), and two infrared beam source/detectors were used to locate the rat within the chamber. Rats trained to avoid the foot shock were placed in the experimental chambers for a 4-min habituation period followed by 50 trials presented on a 15-s variable interval schedule (range = 7.5–22.5 s). Each trial consisted of a 10-s warning tone and stimulus light (conditioned stimulus) followed by a 10-s shock (unconditioned stimulus), presented through the grid floor on the side where the rat was located, in the presence of the tone and light. If during the initial 10 s of the trial, an animal crossed through the archway that divides the shuttle box, thereby breaking the infrared beam location 13 cm from the center archway, the tone and light were terminated, and the response was considered an avoidance response. If an animal crossed through the archway that divides the shuttle box after a foot shock was initiated, the tone, light, and shock were terminated, and the response was considered an escape response. If a response was made during an intertrial interval, the response was punished with a 0.5-s shock (0.5 mA). A MED Associates computer with MedState Notation software controlled the test session and counted the number of trials in which the animal avoided shock, escaped shock, and did not respond. On test days, ADX47273 was administered intraperitoneally 30 min before testing. In the reversal experiments, MPEP (10 mg/kg i.p.) or MTEP (1 mg/kg i.p.) was administered 45 min before testing, whereas ADX47273 was administered 30 min before testing. The dose of MPEP or MTEP was chosen based on literature and in-house data demonstrating anxiolytic and/or antidepressant-like effects (Palucha and Pilc, 2007). The same eight animals, part of a colony of trained subjects used for antipsychotic screening, received each treatment with at least 3 days intervening. Demonstration of baseline performance (greater than 90% avoidance responses) was a criterion for subsequent testing. The order of treatments was ADX47273 at 0, 10, 30, and then 100 mg/kg followed by MPEP (10 mg/kg pretreatment) plus ADX47273 (100 mg/kg). In a second group of eight subjects, the effects of ADX47273 (100 mg/kg i.p.) were replicated followed by an evaluation of the effect of MTEP (1 mg/kg i.p.) pretreatment. Yet, separate groups of subjects were used to test MPEP (10 mg/kg i.p.) or MTEP (1 mg/kg i.p.) alone (n = 8). Avoidance response and response failure data were analyzed by repeated-measures analyses of variance with post hoc least significant difference tests (p < 0.05).
Antagonism of Apomorphine-Induced Climbing and Stereotypy. Drugs were administered intraperitoneally to 6 to 18 mice per dose level. A control group, run simultaneously with drug-treated groups, received saline at equal volumes. Thirty minutes later, experimental and control animals were challenged with 1 mg/kg s.c. apomorphine. Five minutes after the apomorphine injection, the sniffing-licking gnawing (0, absent; 1, present) syndrome (stereotyped behavior) and climbing behavior (0, all four feet on ground; 1, two feet up on wire cage; 2, all four feet on wire cage) induced by apomorphine were scored and recorded for each animal. Readings were repeated every 5 min during a 30-min test session. Scores for each animal were totaled over the 30-min test session for each syndrome (stereotyped behavior and climbing). Mean climbing and stereotypy scores were then expressed as a percentage of control values observed in vehicle-treated mice that received apomorphine. One-way analysis of variance (ANOVA) followed by the least significance difference test was used to determine the minimum effective dose (MED). In the reversal experiments, MPEP (10 mg/kg i.p.) or MTEP (10 mg/kg i.p.) was administered 45 min followed by ADX47273 (300 mg/kg i.p.) 30 min before apomorphine. Data in the reversal experiments were analyzed with a two-way analysis of variance followed by a least significant difference test (p < 0.05).
PCP-, Apomorphine-, and Amphetamine-Induced Locomotor Hyperactivity. Mice (10 mice/treatment group) were weighed and placed in the locomotor chambers and allowed to habituate for 90 min. After habituation, the mice were dosed with vehicle or ADX47273 and activity was measured for another 30 min, at which time the mice were dosed with PCP (3 mg/kg i.p.), apomorphine (1 mg/kg s.c.), amphetamine (1 mg/kg s.c.), or vehicle. Locomotor activity was measured for an additional 30 min. Locomotor activity data were recorded under room light using AccuScan infrared beam activity monitors with enclosed Plexiglas chambers (8 × 8 inches; AccuScan Instruments, Inc., Columbus, OH). AccuScan Versamax and Versadat software (Columbus, OH) was used to convert the infrared beam breaks into centimeters traveled in each 10-min bin. Data were analyzed with two-way repeated measures ANOVA, followed by a least significant difference post hoc test, comparing the treatment groups across time during the 30-min pretreatment period and during the 30 min after stimulant challenge (p < 0.05). Separate analyses were conducted for PCP-, apomorphine-, amphetamine-, and vehicle-challenged groups.
Cataleptogenic Potential in Mice. ADX47273 (0, 10, 30, 100, or 300 mg/kg i.p.) was administered to six mice per treatment group. Every 30 min for 2 h after dosing, the animal's forelegs were draped over a thin horizontal rod 1 3/4 inches high. The amount of time (in seconds) for which the animal maintained this awkward position was recorded (60-s maximum). Maintenance of this position was considered catalepsy. Mean seconds spent in the catalepsy position for each dose at each time point were calculated. The time point at which the peak catalepsy was exhibited was analyzed with a one-way analysis of variance with a post hoc least significant difference test (p < 0.05) and expressed graphically.
Forty-Eight Hour Delay Novel Object Recognition. The test arena consisted of a circular field (diameter, ∼70 cm; 30 cm high) constructed of plastic and containing bedding. The novel object recognition task was divided into three sessions: habituation, trial 1, and trial 2. Rats were placed in the arena with two identical assemblies of Lego blocks and were allowed to explore freely for a total of 10 min to allow habituation on day 1. On day 2, 24 h after habituation, rats were treated 30 min before trial 1. Rats were then placed in the arena with a different set of two identical Lego objects and allowed to explore freely for a total of 5 min. Time spent sniffing each object was recorded by an observer and totaled for an overall object exploration time for each rat. On day 4, 48 h after trial 1 and without further drug treatment, rats were placed back in the arena with one novel and one familiar object and allowed to explore freely for a total of 5 min. Time (seconds) spent sniffing each of the objects was recorded. Seconds spent on each object were analyzed by repeated measures ANOVA with main effects of trial and treatment, followed by a post hoc least significant difference test comparing novel versus familiar object exploration time for each treatment (p < 0.05).
5-CSRT Task. The test apparatus consisted of 10 25- × 25-cm operant chambers (MED Associates). The rear wall of each chamber was concavely curved and contained five accessible apertures, each 2.5 cm square and 4 cm deep and set 2 cm above floor level. A standard 3-W light-emitting diode located at the rear of each aperture provided illumination of each hole. The 10 chambers were individually housed within sound-attenuating cabinets and were ventilated by low-level noise fans, which also served to mask extraneous background noise. Each chamber was illuminated by a 3-W house light mounted near the ceiling in the center of the front wall alongside a small general-purpose loud speaker. The program controlling the software was developed by Conclusive Solutions (Harlow, UK).
Before drug treatments, rats were trained to discriminate a brief visual stimulus presented randomly in one of the five spatial locations. At the beginning of each test session, the house light was illuminated, and free delivery of a single food pellet to the magazine was made. Trial initiation was triggered when the rat opened the magazine to collect this pellet. After a fixed 5-s intertrial interval (ITI), the light at the rear of one of the five openings was illuminated for a 500-ms stimulus duration (SD). A nose poke in this opening during illumination and for a limited hold period of 5 s afterward was reinforced by the delivery of a food pellet, and a correct response was recorded. A response in a nonilluminated opening during the signal period including the 5 s immediately afterward (incorrect response) and failures to respond within the limited hold period (missed trial) were followed by a “time-out” period of darkness for 5 s, where no food pellet was delivered. Premature responses, those nose pokes into apertures before illumination, were also followed by the timeout period and reset the ITI. The consequence of a premature response was that the ITI further delayed the aperture illumination. Once animals had been trained to a baseline performance of 75% correct responding on the standard procedure (500-ms light SD/5 s ITI), they began testing.
To increase impulsivity and decrease attention during test sessions, animals were exposed to light stimuli (500-ms SD) presented on a variable ITI schedule (ITI lengths of 10, 7, 5, and 4 s). Equal numbers of each of the four ITIs were randomly presented during each of 100 trial test sessions. All subjects received drug treatments and testing on the variable ITI schedule on Tuesday and Friday of each week, giving a minimum of a 2-day washout period. Interspersed with those treatment days were standard training days (500-ms SD/5 s ITI) reinstating baseline performance of >75% correct responding. A within-subject design was employed such that all animals received all treatments in a fully counterbalanced regimen.
The various performance measures were analyzed with a mixed linear model because a standard linear model does not allow correlation of observations or nonconstant variability across different levels of certain factors. The fixed factors included in the model were dose, ITI, and interactions of dose and ITI. The correlation of observations taken from the same animal was modeled by covariance structure “compound symmetry,” which assumed any two different observations from the same animal were correlated to the same degree. After fitting the data, the residuals, the observed values minus the predicted values, were plotted against the predicted values. A tool to accommodate possible unequal variability in different levels of dose or ITI is to group covariance structure, e.g., grouping by ITI would use different sets of covariance parameters for different levels of ITI. The selection of grouping was aided by values of Akaike's Information Criterion (Akaike, 1974).
In Vivo Microdialysis.Animal housing and surgery. Male Sprague-Dawley rats weighed between 280 and 325 g at the time of surgery. After induction of anesthesia with 3% isoflurane, rats were secured in a stereotaxic frame with ear and incisor bars (David Kopf Instruments, Tujunga, CA). A microdialysis probe guide cannula (CMA/12; CMA/Microdialysis, Solna, Sweden) was implanted into the striatum (anteroposterior, +0.2 mm; lateral, -3.0 mm; ventral, -3.2 mm) or nucleus accumbens (anteroposterior, +2.2 mm; lateral, -1.2 mm; ventral, -6.0 mm). Coordinates were taken according to Paxinos et al. (1985) reference points taken from bregma and vertical from dura using a flat skull position. Guide cannulae were fixed to the skull with two stainless steel screws (Small Parts, Inc., Miramar, FL) and dental acrylic (Plastics One, Roanoke, VA). After surgery, animals were individually housed in Plexiglas cages (45 cm square) for approximately 24 h, where they had access to food and water ad libitum.
Acute ADX47273 treatment. Concentric-style microdialysis probes (CMA/12; 20-kDa cut-off) were purchased from CMA/Microdialysis and consisted of a 2-mm active membrane (o.d., 0.5 mm) and a 14-mm stainless steel shaft (o.d., 0.64 mm). Probes were perfused with artificial cerebrospinal fluid (125 mM NaCl, 3 mM KCl, 0.75 mM MgSO4, and 1.2 mM CaCl2, pH 7.4) for at least 18 h according to the manufacturer's specifications. On the morning of the microdialysis experiment, probes were inserted, via the guide cannula, into the nucleus accumbens or striatum and perfused with artificial cerebrospinal fluid at a flow rate of 1 μl/min. After a 3-h stabilization period after probe insertion, dialysis samples were collected every 30 min. Initially, three dialysate samples were taken before drug injection to demonstrate a steady baseline. At the end of baseline, animals received an acute injection of either ADX47273 (175 mg/kg i.p.) or vehicle (2% Tween 80/0.5% methylcellulose). After injections, dialysis samples were collected for the following 4 h. At the end of these experiments, animals were euthanized, and probe placement was verified histologically. Data from animals with incorrect probe placements were discarded from the study.
Neurochemical analysis. Microdialysis samples (30 μl) containing dopamine were separated by high-performance liquid chromatography (C18 ODS3 column, 150 × 3.0 mm; MetaChem Technologies, Inc., Torrance, CA) and detected using an ANTEC electrochemical detector set at a potential of 0.65 V versus an Ag/AgCl reference electrode. Mobile phase (0.15 M NaH2PO4, 0.25 mM EDTA, 1.75 mM 1-octane sulfonic acid, 2% isopropanol, and 4% methanol, pH 4.6) was delivered by a Jasco PU1580 high-performance liquid chromatography pump (Jasco, Tokyo, Japan) at a flow rate of 0.5 ml/min. Neurochemical data were compared with an external standard curve, and all data were acquired using the Atlas software package (Thermo Fisher Scientific, Waltham, MA) for the PC. Neurotransmitter levels (femtomole concentrations) collected during the baseline samples were averaged, and this value was denoted as 100%. Subsequent sample values were expressed as percentage change from this preinjection baseline value. Neurochemical data, excluding preinjection values, were analyzed by two-way ANOVA with repeated measures (time). Post hoc analyses were made using the Bonferroni's/Dunn's adjustment for multiple comparisons (p < 0.05). All statistical calculations were performed using the Statview software application (Abacus Concepts, Berkeley, CA) for the PC.
Results
ADX47273 Potentiates Glutamate Response in HEK 293 Cells Expressing Rat mGlu5 Receptors and Primary Astrocyte Cultures. In FLIPR384 assays, HEK 293 cells expressing rat mGlu5 exhibited concentration-dependent increases in Fluo-3 fluorescence in response to glutamate with an EC50 value 300 nM. A subthreshold concentration of glutamate 50 nM caused approximately 10% of the maximal glutamate response (Fig. 1A). ADX47273 caused a concentration-dependent increase in the response to 50 nM glutamate in HEK 293 cells expressing rat mGlu5 without eliciting a response by itself. The maximal increase in the response was approximately 9-fold, with an EC50 value for potentiation of 0.17 ± 0.03 μM (n = 8) (Fig. 1A). Preincubation with 10 μM MPEP for 30 min completely blocked the effects of ADX47273 on potentiation (Fig. 1A). In primary astrocyte cultures, ADX47273 caused a concentration-dependent increase in the response to 300 nM glutamate, with an EC50 value of 0.23 ± 0.07 μM (n = 12) (Fig. 1B). Increasing concentrations of ADX47273 caused a parallel, leftward shift of the glutamate concentration response curve. In the presence of 0.1 or 1 μM ADX47273, the EC50 for glutamate decreased 4- and 9-fold, respectively, in HEK 293 cells expressing rat mGlu5 (Fig. 2A). Similarly in astrocytes, in the presence of 1 or 3 μM ADX47273, the EC50 for glutamate decreased 4- and 9-fold, respectively (Fig. 2B). The functional cross-selectivity of ADX47273 was evaluated in rat mGlu1, human mGlu2 (group II receptor), and mGlu4 (group III receptor) receptor cell lines. No agonist, antagonist, or allosteric responses were observed with ADX47273 (up to 10 μM) in any of the cell lines (data not shown).
ADX47273 Competes with [3H]MPEP but Not [3H]-Quisqualate Binding. ADX47273 inhibited binding of [3H]MPEP to membranes prepared from HEK 293 cells expressing mGlu5 receptors (Fig. 3) [Ki value, 4.3 ± 0.5 μM (n = 5); Hill slope = 0.833 based on one-site model; R2 = 0.96, p < 0.05). In the in vitro [3H]quisqualate binding assay, ADX47273 did not affect the binding of the radioligand at the glutamate recognition (orthosteric) site of the rat mGlu5 receptor at concentrations up to 10 μM (specific [3H]quisqualate binding, mean ± S.E.M., 1506 ± 66 versus 1546 ± 65 fmol/mg protein in the absence and presence of ADX47273, respectively; n = 3, not significant by Student's t test).
ADX47273 Increases ERK and CREB Phosphorylation in Hippocampus and Cortex. Rats were administered ADX47273 (1 and 10 mg/kg i.p.) and sacrificed 30 min later. The homogenates from prefrontal cortex and hippocampus of these subjects were analyzed by immunoblotting with anti-phospho-ERK and anti-phospho-CREB antibodies. ADX47273 (10 mg/kg i.p.) treatment increased ERK and CREB phosphorylation in both the prefrontal cortex and hippocampus (Fig. 4). Pretreatment with MPEP (10 mg/kg i.p. 60 min before sacrifice) had no significant effects on its own and blocked ADX47273-induced increases in ERK and CREB phosphorylation (Fig. 4).
ADX47273 Produces an Antipsychotic-Like Decrease in Conditioned Avoidance Responding. ADX47273 (10–100 mg/kg i.p.) produced dose-dependent decreases in avoidance responding in rats (Fig. 5A) (p < 0.0001) and increased escapes at doses that did not produce any response failures. These effects of ADX47273 were significantly attenuated by pretreatment with either the mGlu5 receptor antagonist MPEP (10 mg/kg i.p.) or MTEP (1 mg/kg i.p.) 15 min before ADX47273 (Fig. 5, B and C) (p < 0.05). MPEP alone (10 mg/kg i.p.) given to a separate group of animals reduced avoidance responding and increased escape responding by approximately 30% relative to vehicle treatment, whereas MTEP alone (1 mg/kg i.p.) did not significantly affect avoidance responding (data not shown).
ADX47273 Blocks Apomorphine-Induced Climbing. Under vehicle treatment, the absolute values (mean ± S.E.M.) for apomorphine-induced climbing were 9.67 ± 0.42, and those for apomorphine-induced stereotypy were 6.0 ± 0. ADX47273 (10–300 mg/kg i.p.) produced a dose-dependent decrease in apomorphine-induced climbing (Fig. 6A) (F4,49 = 25.663, p < 0.001; MED = 100 mg/kg) at doses that had negligible effects on apomorphine-induced stereotypy (F4,49 = 4.729, p < 0.001; MED = 300 mg/kg), a profile similar to that produced by the atypical antipsychotic clozapine (Marquis et al., 2007). These effects of ADX47273 in blocking apomorphine-induced climbing were significantly attenuated by pretreatment with MPEP (10 mg/kg i.p.) (F3,20 = 4.767, p < 0.05) or MTEP (10 mg/kg i.p.) (F3,20 = 61.7, p < 0.05) 15 min before ADX47273 (Fig. 6, B and C, respectively). MPEP (1–30 mg/kg i.p.) alone had no significant effect on apomorphine-induced climbing (F4,25 = 0.822, p > 0.05) but did produce a small (8–11% relative to control) but statistically significant decrease in stereotypy (F4,25 = 3.750, p < 0.05) (data not shown). MTEP (10 mg/kg i.p.) did not affect either apomorphine-induced climbing or stereotypy (Fig. 6C).
ADX47273 Blocks PCP, Apomorphine, or Amphetamine-Induced Locomotor Activity. ADX47273 (10–100 mg/kg i.p.) was tested for its effects on PCP, apomorphine, or amphetamine-induced locomotor activity in habituated mice (Fig. 7). When locomotor activity during the pretreatment period was analyzed, there was no significant main effect of ADX47273 pretreatment [F3,31 = 0.73, F3,32 = 0.74, F3,29 = 2.84, in subjects earmarked for PCP (Fig. 7A), apomorphine (Fig. 7B), and amphetamine (Fig. 7C) challenge, respectively, all p > 0.05]. No treatment × time interaction was detected in subjects earmarked for PCP (F6,62 = 0.65, p > 0.05) or apomorphine (F6,64 = 0.47, p > 0.05), whereas those subjects earmarked for amphetamine challenge (F6,58 = 2.72, p < 0.05) tended to show increased activity at 10 mg/kg and decreased activity at 100 mg/kg compared with vehicle in the initial 10-min period after ADX47273. These effects were not observed in the other cohorts earmarked for stimulant challenge or a separate group of subjects pretreated with ADX47273 (100 mg/kg i.p.) followed by vehicle challenge (treatment main effect, F1,16 = 1.49, p > 0.05; treatment × time interaction, F2,32 = 1.18, p > 0.05) (Fig. 7D).
Analysis of locomotor activity after challenge with psychostimulant revealed a significant main effect of ADX47273 [F3,31 = 1.97, F3,32 = 1.69, F3,29 = 12.94, p < 0.05 for PCP-challenged (Fig. 7A), apomorphine-challenged (Fig. 7B), and amphetamine-challenged (Fig. 7C) subjects, respectively] and treatment × time interactions (F6,62 = 2.91, F6,64 = 2.6, F6,58 = 4.73, p < 0.05 for PCP-, apomorphine-, and amphetamine-challenged subjects, respectively). Post hoc analysis revealed that pretreatment with ADX47273 at 100 mg/kg decreased locomotor activity compared with vehicle pretreatment at 20 min after PCP, 30 min after apomorphine, and at all time points after amphetamine challenge. In a separate group of vehicle-challenged mice that were habituated to the locomotor apparatus, ADX47273 (100 mg/kg i.p.) failed to affect motor activity significantly different from vehicle pretreatment over this same time frame (F1,16 = 1.22, p > 0.05) (Fig. 7D). The effect on unhabituated motor activity was not tested.
ADX47273-Induced Catalepsy. Although there was a significant effect of treatment (F4,25 = 5.41, p < 0.05), there was no significant time (F3,75 = 1.5) or treatment × time interaction (F12,75 = 1.59). At 300 mg/kg, mice maintained the cataleptic position for 37 ± 15.7% of the maximal 60-s response at 90 min post-treatment. Catalepsy was detected at this dose at 30 and 60 min post-treatment but was of a lesser magnitude (approximately 25% maximum) (data not shown).
ADX47273 Decreases Dopamine Levels in the Nucleus Accumbens but Not in the Dorsal Striatum. Basal levels of extracellular dopamine in the nucleus accumbens were on average 927 and 1212 fmol/10-μl sample in vehicle- and ADX47273-treated subjects, respectively. In the striatum, dopamine basal levels were 4823 and 4086 fmol/10-μl sample in vehicle- and ADX47273-treated subjects, respectively. Acute administration of ADX47273 (175 mg/kg i.p.) lowered dopamine levels in the rat nucleus accumbens (Fig. 8A) but not in the dorsal striatum relative to vehicle-treated animals (Fig. 8B). The maximal decrease in dopamine in the nucleus accumbens was 66.02 ± 8.35%. A two-way ANOVA with repeated measures (time) revealed a significant decrease in dopamine at 175 mg/kg (F1,14 = 23.91, p < 0.05) and a significant time effect (F7,98 = 3.79, p < 0.05). Post hoc analyses revealed that the effect of ADX47273 on extracellular dopamine levels in the nucleus accumbens was significantly different from vehicle treatment as early as 60 min post-treatment and was sustained for the remainder of the experiment. No significant effect occurred in striatum of rats dosed with ADX47273 (175 mg/kg i.p.) (treatment effect, F1,11.9 = 0.07; time effect, F6,58.3 = 0.58; treatment × time, F6,58.3 = 1.47, p > 0.05).
ADX47273 Improves Recall after a 48-h Delay in Novel Object Recognition. ADX47273 (0.1–50 mg/kg i.p.) was evaluated for cognition enhancement in novel object recognition paradigm, which is a task for testing memory performance based on the rat's natural differential exploration of new and familiar objects (Ennaceur and Delacour, 1988; Ennaceur and Meliani, 1992). During the learning trial, there was no difference in the behavior of the animals in the different groups (vehicle- versus ADX47273-treated), and the time spent exploring the objects was very similar (between 60 and 80 s for a 5-min trial) among all groups (F5,64 = 1.110, p > 0.05) (Fig. 9A). In the test trial, ADX47273 dose-dependently improved recall, with the 1 to 50 mg/kg groups exploring the novel object more than the familiar, whereas the vehicle and 0.1 mg/kg groups explored the object equally (F5,64 = 4.45, p < 0.05) (Fig. 9B).
ADX47273 Reduces Impulsivity in 5-CSRT. To investigate the effects ADX47273 on attention and impulsivity, we employed the 5-CSRT test (Robbins, 2002). Varying the length of ITI reduced the percentage of correct responses (F3,143 = 4.5, p < 0.005) (Fig. 10A). A post hoc analysis revealed that there was a decrease in the percentage of correct responses at the 10-s ITI to 78% from the 86% correct responses made at the baseline ITI of 5 s (p < 0.05). ADX47273 did not produce any treatment effects on percentage of correct responses, either as a main effect of dose (F2,143 = 0.120, p > 0.05) or as a dose × ITI interaction (F2,143 = 0.82, p > 0.05). Varying the ITI also produced a significant main effect on the rate of premature responses (F3,14.2 = 68.04, p < 0.0001) (Fig. 10B). According to post hoc analysis, significantly more premature responses were made during the 10-s ITI compared with all other ITIs and decreased as ITI length was decreased. Treatment with ADX47273 resulted in a main effect of drug treatment (F2,29.3 = 4.63, p < 0.05), with post hoc analysis revealing a decrease in the number of premature responses at both the 10 and 30 mg/kg doses (p < 0.05). There was also a significant dose × ITI interaction on premature responding (F6,31.9 = 2.67, p < 0.05), with a post hoc analysis indicating that the 10 mg/kg dose significantly decreased the number of premature responses made at the 10- and 7-s ITIs (p < 0.05). The decrease in premature responding was not because of a global decrease in motor activity because ADX47273 did not increase the number of missed trials (F2,143 = 0.69, p > 0.05) or the latency to collect the reward (F2,143 = 2.12, p > 0.05) (data not shown). Thus, in the 5-CSRT task, ADX47273 had no effect on the modest decrease in percentage of correct responding produced by instituting a variable ITI but was effective in decreasing the premature responding elicited by the 10-s ITI within the variable ITI session.
Discussion
Reversal of NMDA receptor hypofunction has been suggested as a possible strategy for the treatment of schizophrenia (Lindsley et al., 2006). One mechanism for enhancing NMDA activity is through activation of mGlu5 receptors (Gasparini et al., 2002; Marino and Conn, 2002). In this report, the mGlu5 PAM ADX47273 (Le Poul et al., 2005) is used to demonstrate that modulation of mGlu5 can produce antipsychotic-like and procognitive activities in rodent models, thereby extending other reports of antipsychotic-like effects of mGlu5 PAMs (Kinney et al., 2005). In addition, the enhancement of recognition in the novel object recognition assay is the first direct demonstration of the impact of an mGlu5 PAM on cognition. This effect was predicted by the ability of an mGlu5 PAM to enhance NMDA receptor activation (O'Brien et al., 2003b; Kinney et al., 2005) and to effect biochemical endpoints relevant to cognition (Thomas and Huganir, 2004; Carlezon et al., 2005; Liu et al., 2006). These results underscore the value of mGlu5 PAMs as a novel approach to treating the positive symptoms and cognitive deficits of schizophrenia.
ADX47273 behaves as a positive allosteric modulator in vitro as evidenced by its ability to potentiate the effect of a subthreshold concentration of glutamate in the Ca2+ mobilization assay with an EC50 = 170 nM. Because this effect can be blocked in the presence of the mGlu5 receptor antagonist MPEP, it appears to be because of interactions of ADX47273 at the allosteric modulatory site of mGlu5. In addition, ADX47273 (at concentrations up to 10 μM) did not affect the binding of [3H]quisqualate at the extracellular glutamate recognition (orthosteric) site of rat mGluR5 but was shown to inhibit [3H]MPEP binding by as much as 20% at 10 μM. These results suggest that ADX47273 and MPEP share slightly overlapping sites of interaction within the mGlu5 allosteric pocket that is distinct from the [3H]quisqualate orthosteric site (i.e., an allosteric mechanism). This in vitro pharmacological profile is consistent with a previous report (Le Poul et al., 2005).
ADX47273 was evaluated in the conditioned avoidance model, a standard screening model for antipsychotic efficacy (Arnt, 1982). In this model, ADX47273 dose-dependently decreased avoidance responding, without increasing the number of no-response trials, a profile similar to the typical antipsychotic haloperidol and the atypical antipsychotic clozapine (Marquis et al., 2007). The antipsychotic-like effect of ADX47273 is apparently mediated by the mGlu5 because both mGlu5 receptor antagonists, MPEP and MTEP, attenuated the effect of ADX47273, despite the observation that MPEP (but not MTEP) itself affected avoidance responding.
Additionally, ADX47273 selectively reduced apomorphine-induced climbing behavior, a second model predictive of antipsychotic efficacy that is mediated by the mesolimbic dopaminergic pathway (Costall et al., 1980). Both MPEP and MTEP significantly attenuated the effect of ADX47273 on apomorphine-induced climbing, suggesting a role for mGlu5 in this effect of ADX47273. In addition, ADX47273 reduced amphetamine-induced hyperactivity and reduced the extracellular concentration of dopamine in the nucleus accumbens, the projection target of the mesolimbic dopaminergic pathway. The effect of ADX47273 on direct (apomorphine) and indirect (amphetamine) dopamine agonist-induced behaviors may be, in part, explained by the ability of ADX47273 to decrease extracellular dopamine levels in the nucleus accumbens. Another mGlu5 receptor PAM, CDPPB, was reported to reduce amphetamine-induced locomotor activity and reverse amphetamine-induced deficits in prepulse inhibition in rats (Kinney et al., 2005). These data suggest a possible neurochemical explanation for the antipsychotic-like behavioral results of ADX47273 in that reduction of dopamine neurotransmission by blockade of D2 dopamine receptors in mesolimbic brain regions is argued to be a mechanism whereby current antipsychotics exert their efficacy.
In the same dose range, ADX47273 had minimal effects on apomorphine-induced stereotypy, an endpoint reportedly mediated by the nigrostriatal dopaminergic pathway (Costall et al., 1975) and useful in predicting Parkinson's-like extrapyramidal motor system side effects of current typical antipsychotics (Marquis et al., 2007). Additionally, ADX47273 did not significantly affect striatal dopamine levels, further supporting a mesolimbic selective effect of the compound. When assessed in the catalepsy assay, ADX47273 did induce a modest level of catalepsy at the dose three times the MED for its effect on apomorphine-induced climbing. These effects of ADX47273 in the apomorphine-induced climbing/stereotypy, catalepsy, and microdialysis models are suggestive of an atypical antipsychotic-like profile for ADX47273, potentially mediated via selective reduction of mesolimbic relative to nigrostriatal dopamine, as has been reported with other nondopaminergic mechanisms with preclinical antipsychotic-like activity (Marquis et al., 2007).
It is worth noting that the mGlu5 receptor PAM, ADX47273, also blocks PCP-induced locomotor activity. This result provides direct evidence that activation of mGlu5 receptor can reverse NMDA receptor hypofunction and adds to the emerging evidence that alterations in dopamine-glutamate interactions may contribute to the pathophysiology of schizophrenia (de Bartolomeis et al., 2005). Position emission tomography studies in human showed that the NMDA receptor antagonist PCP induced alterations in striatal dopamine (Kegeles et al., 2002). Preclinical studies also suggest a dopamine-glutamate interaction based on the converging effect of different drugs, such as amphetamine and PCP on dopamine-modulating factors (dopamine and cAMP-regulated phosphoprotein, DARPP-32) (Svenningsson et al., 2003). Furthermore, antipsychotic compounds are shown to regulate postsynaptic density proteins (e.g., Homer proteins) (Polese et al., 2002), which regulate the function of glutamate receptors.
It has been reported previously that the mGlu5 antagonist MPEP enhanced the detrimental effects of the NMDA antagonist MK-801 on cognition in assays dependent on medial prefrontal cortex (Homayoun et al., 2004). It has been shown more recently that the mGlu5 PAM CDPPB prevented MK-801-induced excessive firing and reduced spontaneous bursting in the medial prefrontal cortex (Lecourtier et al., 2007). These observations suggest that mGlu5 receptors play a role in regulating NMDA receptor-dependent functions and that mGlu5 PAMs may be effective in ameliorating cognitive deficits in schizophrenia. We have shown previously that CP-PHA, another mGlu5 PAM, can increase phosphorylation of ERK and CREB in hippocampal slices (Liu et al., 2006), two signaling molecules implicated in learning and memory. In the current study, we found that ADX47273 also increases ERK and CREB phosphorylation in the hippocampus and the prefrontal cortex, suggesting the potential for mGlu5 PAMs to improve cognition, an area of major unmet medical need in schizophrenia.
To evaluate the potential cognitive enhancing activity of mGlu5 PAMs, we used two models, novel object recognition task and 5-CSRT task. In the object recognition memory task, it has been reported that the activation of groups I and II metabotropic glutamate receptors in the perirhinal cortex is necessary for the acquisition, but not consolidation or retrieval, of long-term familiarity discrimination (Barker et al., 2006). We found that in rats, ADX47273 can enhance object recognition, suggesting that positive allosteric modulation of mGlu5 alone is sufficient to improve performance in this particular behavior paradigm. Our data are consistent with what has been reported on the effect of ADX47273 on object recognition performance in mice (Epping-Jordan et al., 2005).
The continuous performance test has been used widely to measure attention performance in humans (Robbins, 2002) and is sensitive in detecting attention deficits across several disorders (Nieuwenstein et al., 2001). Schizophrenic patients show impairments in the task compared with controls (Moeller et al., 2001). The 5-CSRT test, a preclinical analog of continuous performance test monitors attentional function using measures of percentage of correct responding and response latency to the visual stimuli and impulse control by measuring level of premature responding (Robbins, 2002). It has been reported that impairment of glutamatergic transmission after treatment with the NMDA antagonist PCP can induce deficits in attentional functioning and response control in the 5-CSRT task in mice. PCP decreased percentage of correct responding in DBA, but not C57BL6, mice and increased premature responding in both strains, effects normalized in part by treatment with agents that affect glutamatergic neurotransmission such as the mGlu2/3 agonist LY379268 (Greco et al., 2005).
In 5-CSRT, ADX47273 inhibited premature responding (impulsivity) but did not improve percentage of correct responding (attention). These effects occurred in the absence of significant effects on correct response or reward retrieval latencies. In contrast, MPEP is reported to decrease premature responding similar to ADX47273, but with a concomitant increase in response latency, suggesting a disruption in motivation produced by MPEP in this model (Semenova and Markou, 2007). The ability of ADX47273 to specifically decrease premature responding suggests a mGlu5 PAM may be effective in treating the impulsivity observed in schizophrenia (Moeller et al., 2001).
In summary, ADX47273 acts as a selective positive modulator of mGlu5 and produces behavioral effects suggestive of an antipsychotic-like profile. The effects on CREB and ERK phosphorylation paired with effects in novel object recognition and 5-CSRT tasks suggest that ADX47273 may effectively treat the cognitive symptoms of schizophrenia as well. Taken together, these results suggest that a positive allosteric modulator of mGlu5 may be a novel approach to treating symptoms of schizophrenia and broaden the therapeutic response beyond the treatment of psychosis.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136580.
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ABBREVIATIONS: mGlu, metabotropic glutamate receptor; GPCR, G-protein-coupled receptor; PAM, positive allosteric modulator; CPPHA, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide; CDPPB, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; ADX47273, S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone; NMDA, N-methyl-d-aspartate; MPEP, 2-methyl-6-(phenylethynyl)pyridine; MK-801, (5R,10S)-(-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cylc ohepten-5,10-imine; ERK, extracellular signal-regulated kinase; CREB, cAMP-response element binding; PCP, phencyclidine; 5-CSRT, five-choice serial reaction time; MTEP, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine; FLIPR, fluorometric imaging plate reader; HEK, human embryonic kidney; DMSO, dimethyl sulfoxide; l-AP4, l-(+)-2-amino-4-phosphonobutyric acid; ANOVA, analysis of variance; MED, minimal effective dose; ITI, intertrial interval; SD, stimulus duration; LY379268, (-)-2-oxa-4-aminobicyclo[3.1.0.]hexane-4,6-dicarboxylate.
- Received January 16, 2008.
- Accepted August 21, 2008.
- The American Society for Pharmacology and Experimental Therapeutics