Evidence for the Role of Metabotropic Glutamate (mGlu)2 Not mGlu3 Receptors in the Preclinical Antipsychotic Pharmacology of the mGlu2/3 Receptor Agonist (–)-(1R,4S,5S,6S)-4-Amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic Acid (LY404039)

Schizophrenia is a lifelong and devastating psychiatric disorder characterized by positive, negative, and cognitive symptoms (Andreasen and Carpenter, 1993; Ross et al., 2006). Although several neurotransmitter systems (dopamine, serotonin, and glutamate) have been implicated in the pathophysiology of schizophrenia, dopamine D₂ receptors are the primary target of all antipsychotic drugs. Although these medications reduce the severity of positive symptoms, they have only a modest effect on the negative or cognitive impairments present in the disorder. Due to these shortcomings, there is a clear need for alternative strategies for the treatment of schizophrenia. Recent clinical data suggest that a group II metabotropic glutamate (mGlu) receptor agonist LY2140023 (prodrug of LY404039) has antipsychotic properties and may represent an alternative, nondopaminergic treatment for schizophrenia (Patil et al., 2007).

Glutamate is the principle excitatory neurotransmitter in the mammalian central nervous system and mediates it's actions through “ionotropic” glutamate receptors that are ligand-gated ion channels [N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate subtypes) and G protein-coupled “metabotropic” (mGlu) receptors (mGlu1–8 subtypes) (Schoepp, 2001). The group II mGlu (mGlu2 and mGlu3) receptors are structurally related, and they function to inhibit neurotransmitter release as autoreceptors on glutamatergic terminals, presynaptic heteroreceptors (Cartmell and Schoepp, 2000; Schoepp, 2001), or via modulatory actions on glia (Conn and Pin, 1997; Bruno et al., 1998). Several lines of evidence have implicated glutamate neurotransmission, specifically aberrant NMDA receptor function, as a key element in the pathophysiology of the schizophrenia (Kim et al., 1980; Javitt and Zukin, 1991; Harrison and Owen, 2003). According to the glutamate hypothesis of schizophrenia, a drug that can correct or modulate dysfunctional glutamatergic neurotransmission may be an effective therapeutic agent for schizophrenia.

The amino acid analog LY404039 (Monn et al., 2007) is a potent and highly selective agonist for group II mGlu receptors, and it has no appreciable affinity for group I or group III mGlu receptors, ionotropic glutamate receptors, glutamate transporters, or other receptors such as dopamine and serotonin (Rorick-Kehn et al., 2007a). Like atypical antipsychotics (e.g., clozapine and olanzapine), LY404039 displays antipsychotic-like effects in animal models of psychosis. LY404039 reverses phencyclidine (PCP)-induced hyperlocomotion in rats in a dose-dependent manner (Monn et al., 2007; Rorick-Kehn et al., 2007b). Although LY404039 has no affinity at dopamine receptors, it attenuates the locomotor-stimulating effects of d-amphetamine (AMP). In a recent double-blind, phase II proof-of-concept trial in which olanzapine was used as an active control, LY2140023 (prodrug of LY404039) was shown to be efficacious, safe, and well tolerated. LY2140023-treated patients showed statistically significant improvement in all efficacy measures [PANSS total, PANSS positive, PANSS negative, and clinical global impression scale scores) compared with the placebo group, as observed as early as week 1 and maintained through the end of the 4-week study (Patil et al., 2007). It is important to note that LY2140023-treated patients did not experience treatment-emergent adverse events such as prolactin elevation, extrapyramidal symptoms, or weight gain. These results suggest that the activation of group II mGlu receptors may be a therapeutically advantageous approach for the treatment of schizophrenia (Patil et al., 2007).

The relative contribution of mGlu2 versus mGlu3 receptors in the antipsychotic pharmacology of mGlu2/3 receptor agonists has yet to be fully elucidated. Efforts have been hampered by the lack of selective pharmacological tools due to high homology at the mGlu2 and mGlu3 receptor binding site. At present, orthosteric agonists with selectivity for mGluR2 versus mGluR3 have not been identified. Studies with positive allosteric modulators of mGlu2 receptors indicate that the mGlu2 receptor may be primarily responsible for the behavioral effects of mGlu2/3 agonists (Galici et al., 2005; Johnson et al., 2005). For example, the mGlu2 potentiator CBiPES induces behavioral effects similar to those of the mGlu2/3 agonists in animal models predictive of antipsychotic efficacy (Johnson et al., 2005). The role of mGlu2 receptors in the antipsychotic actions of mGlu2/3 receptor agonists is further supported by the observation that the racemate of the mGlu2/3 receptor agonist LY354740 reversed PCP-evoked locomotor activation in wild-type but not mGlu2 receptor-deficient mice (Spooren et al., 2000). Whether mGlu3 receptor activation further contributes to the antipsychotic efficacy of group II agonists remains to be determined. In this study, we use mGlu receptor-deficient mice to investigate the relative roles of mGlu2 and mGlu3 receptors in mediating the antipsychotic-like actions of LY404039 in the PCP and AMP models of psychosis. To further explore the mechanism of action of LY404039, we compared the drugs' ability to block PCP-induced hyperlocomotion to that of the atypical antipsychotics clozapine and risperidone in wild-type and mice lacking mGlu2/3 receptors.

Materials and Methods

Generation of Separate mGlu2, mGlu3, and mGlu2/3 Receptor Knockout Mice. Separate mGlu2 and mGlu3 receptor knockout mice (Taconic Farms, Germantown, NY) were generated by homologous recombination as described in detail by Linden et al. (2005). The targeting construct was injected into the R1 line of mouse embryonic stem cells. The recombinant embryonic stem cells were injected into murine C57BL/6 blastocysts, and chimeric males were mated with ICR(CD-1) females. Male offspring carrying the null allele were backcrossed for three generations (N3) with ICR(CD-1) females. N3 offspring were then interbred, and siblings homozygous for the null allele were used as founders that provided all mGlu2 receptor knockout mice and mGlu3 receptor knockout mice used here. Males homozygous for the null allele of mGlu3 were intercrossed with females homozygous for the null allele of mGlu2 to produce an intercross colony heterozygous for both mGlu2 and mGlu3 null alleles. Breeding of siblings from the intercross colony produced an intracross colony, and progeny were genotyped to identify homozygotes for both the mGlu2 and mGlu3 null alleles. Founder animals were selected to generate colonies of double-knockout mice and wild-type mice that were used in all subsequent experiments. Phenotypically, mGlu2/3 receptor double-knockout mice did not exhibit any gross differences compared with their wild-type littermates. No differences were observed in body weights or in autonomic function and sensorimotor responses as assessed by the Irwin test (Irwin, 1968), including eye and ear reflexes, respiratory rate, body temperature, salivation, urination, defecation, skin color, irritability, stance, limb strength, placing, grasping, righting, tail pinch, and tail-flick latency. Male mice (mGlu2, mGlu3, mGlu2/3 knockouts, and wild type) weighing approximately 25 to 30 g (Taconic Farms) were tested in PCP or AMP-induced hyperlocomotion experiments. Mice were housed in groups by genotype under a 12:12 light/dark cycle (lights on at 6:00 AM, lights off at 6:00 PM), with food and water freely available. All animal use protocols were approved by Lilly Research Laboratories Animal Care and Use Committee and were in accordance with the Institute of Laboratory Animal Resources (1996). All efforts were made to minimize the discomfort and number of animals used.

Activity Assessment. Behaviors were monitored in transparent, plastic shoebox cages with dimensions of 45 × 25 × 20 cm, with a 1-cm depth of wood chips as bedding, and a plastic cage top. These were placed in a rectangular frame containing a grid of 12 photocell beams in an 8 × 4 configuration (Kinder Scientific, Poway, CA) that was positioned 2.5 cm from the floor of the cage for the detection of body movements (ambulations) and recorded by computer for analysis. Mice were initially acclimated to a plastic shoebox cage for 15 min before being given an i.p. injection of LY404039, clozapine, risperidone, or sterile water. After an additional 30 min, the mice were administered an i.p. injection of sterile water or PCP. Motor activity was monitored for 60 min after the injection of PCP. The same test paradigm was followed for AMP treatment. Software analysis of beam breaks, under the definitions of Hamilton Kinder, resulted in the measurement of three different parameters: ambulations (pattern of beam breaks indicating that the animal has relocated its entire body); distance moved (cm^–1); and time at rest (total seconds in a 60-min session in which no new beams were broken, measured at 1-s intervals).

Materials. PCP and d-amphetamine sulfate (Sigma-Aldrich, St. Louis, MO) were dissolved in 0.9% saline. Risperidone (Toronto Research Chemicals Inc., North York, Canada) and clozapine (Sigma-Aldrich) were dissolved in a few drops of an 11.5% solution of lactic acid and diluted in 0.9% saline. LY404039 (synthesized at Lilly Research Laboratories, Indianapolis, IN; see Monn et al., 2007) was dissolved in 0.9% saline, and the pH was adjusted to 6.0 with 1 N NaOH. All drugs were mixed fresh before use and administered i.p. Mice were dosed with a volume of 10 ml/kg.

Statistical Analysis. Statistical analyses of behaviors were carried out using the GraphPad Prism statistical program (GraphPad Software Inc., San Diego, CA). Mean and S.E.M. were calculated for animals in each dose group (n = 8/group). Data were analyzed by one-way analysis of variance, and then post hoc comparisons were made by a Bonferroni-corrected t test.

Results

Effects on PCP-Induced Motor Activity. PCP evoked dose-dependent increases in ambulations in wild-type mice at doses between 5 and 10 mg/kg [Fig. 1a; F(5,42) = 39.23, p < 0.001]. Maximal effects of PCP on ambulatory activity were observed at 10 mg/kg. At this dose, PCP increased ambulations 11-fold compared with the vehicle-treated group (p < 0.05). Furthermore, PCP increased distance traveled over the range of 5 to 10 mg/kg [F(5,42) = 34.01, p < 0.001], with a maximal effect at 10 mg/kg (13-fold increase compared with the vehicle-treated group). PCP (5–10 mg/kg) significantly reduced time spent at rest [F(5,42) = 50.41, p < 0.001] with a maximal effect observed at 10 mg/kg (60% reduction compared with the vehicle control group; p < 0.01). The lower doses of PCP (1 and 3 mg/kg) were without statistically significant effects on ambulations, distance traveled, or time at rest (Fig. 1, a–c).

Intraperitoneal administration of LY404039 (10 mg/kg) to wild-type mice significantly inhibited increases evoked by PCP (7.5 mg/kg) in ambulations [F(5,42) = 11.31, p < 0.001], distance traveled [F(5,42) = 10.88, p < 0.001], and reduced time spent at rest [Fig. 2c; F(5,42) = 15.95, p < 0.001]. LY404039 (10 mg/kg) inhibited PCP-induced ambulations by 56% (p < 0.05), distance moved by 62% (p < 0.05), and increased time at rest by 112% (p < 0.05). LY404039 was not effective at blocking PCP-evoked behaviors at any of the lower doses (0.3–3.0 mg/kg) tested.

To examine the mechanism of LY404039-mediated inhibition of PCP-evoked behaviors, LY404039 (10 mg/kg) was tested for its ability to reverse behaviors evoked by PCP (7.5 mg/kg) in wild-type and mGlu receptor-deficient mice (mGlu2, mGlu3, and mGlu2/3). Basal locomotor activity and behavioral activation induced by PCP (7.5 mg/kg) did not show statistically significant differences between the various genotypes treated with vehicle (Fig. 3). Whereas LY404039 (10 mg/kg) produced a highly significant reversal of ambulations induced by PCP (7.5 mg/kg), distance traveled, and decreased time at rest in wild-type and mGlu3 knockout mice (p < 0.05 in all cases), the compound was unable to block PCP-evoked increases in ambulations, distance traveled, and decreased time at rest in either mGlu2 or mGlu2/3 receptor-deficient mice (Fig. 3). In another experiment, the atypical antipsychotic drugs clozapine (10 mg/kg) and risperidone (1 mg/kg) were tested for the ability to reverse PCP-induced behaviors in wild-type and mGlu2/3 receptor knockout mice. Similar to the mGlu2/3 receptor agonist LY404039, both clozapine and risperidone reduced PCP-evoked ambulations [Fig. 4a; F(3,28) = 24.28, p < 0.001] and distance traveled [Fig. 4b; F(3,28) = 23.29, p < 0.001] in wild-type mice. Clozapine and risperidone attenuated PCP-evoked reductions in time at rest approximately 5-fold [Fig. 4c; F(3,28) = 33.26, p < 0.001]. In marked contrast to LY404039, the effects of clozapine (10 mg/kg) and risperidone (1 mg/kg) on PCP-evoked behaviors were not attenuated in mGlu2/3 receptor knockout mice. Both clozapine and risperidone significantly reduced PCP-evoked ambulations [Fig. 4a; F(3,28) = 66.74, p < 0.001] and distance traveled [Fig. 4b; F(3,28) = 67.49, p < 0.001] in mGlu2/3 receptor-deficient mice. Furthermore, PCP-evoked reductions in time at rest were significantly reversed by treatment with both clozapine and risperidone [Fig. 4c; F(3,28) = 77.00, p < 0.001].

Effects on AMP-Induced Motor Activity. AMP produced a clear dose-related biphasic effect on ambulations. A dose of 5 mg/kg AMP maximally increased ambulations by 482% of basal activity (p < 0.05). The higher doses of amphetamine (7.5 and 10 mg/kg) produced significant but smaller increases in ambulations compared with the 5 mg/kg dose (p < 0.05 in both cases). In addition to ambulations, AMP also increased distance traveled in a biphasic manner with a maximal effect again seen at 5 mg/kg (521% of basal activity; p < 0.05) and lesser increases at the higher doses. AMP produced a significant increase in distance traveled at a dose of 3 mg/kg. Time at rest was dose dependently reduced by AMP at 5 to 10 mg/kg, with 5 mg/kg again producing the greatest effect with an approximately 50% reduction compared with the vehicle-treated group (p < 0.05). In general, the maximal increases in AMP-evoked behaviors were lower than those seen in response to PCP.

LY404039 produced a dose-dependent reduction of increases evoked by AMP (5 mg/kg) in ambulations [Fig. 5; F(5,40) = 16.29, p < 0.001], distance traveled [F(5,40) = 16.77, p < 0.001], and reduced time spent at rest [F(5,40) = 19.57, p < 0.001] in wild-type mice (Fig. 6a–c). AMP-evoked ambulations were significantly reduced at 3 to 30 mg/kg with maximal effects seen at 10 and 30 mg/kg (an approximately 70% reduction). AMP-evoked increases in distance traveled were maximally reduced by LY404039 at 30 mg/kg by 76% (p < 0.05), whereas 10 mg/kg produced a 72% reduction in AMP-evoked distance traveled (p < 0.05). Although LY404039 (1 mg/kg) induced reductions in AMP-evoked ambulations and distance traveled did not reach statistical significance, this dose of LY404039 did produce a statistically significant increase in AMP-evoked time at rest (p < 0.05). AMP-evoked time at rest was increased over the range of 1 to 30 mg/kg after LY404039, with maximal effect seen at 30 mg/kg (224% increase in time at rest). Overall, LY404039 produced effects on AMP-evoked behaviors at lower doses than those needed to block the effects of PCP. LY404039 significantly reduced AMP-evoked behaviors at doses as low as 1 mg/kg, a dose that was ineffective at blocking all PCP-evoked behavioral effects.

When studying the effects of AMP in the various genotypes, we found overall a weaker behavioral activation in the mGlu2/3 double knock-out animals compared with the other groups (Fig. 7). Thus, in wild-type animals, AMP increased ambulations approximately 10-fold compared with the vehicle control group, whereas in mGlu2 and mGlu2/3 receptor-deficient mice, the AMP-evoked increase in ambulations were approximately 7-fold and 2-fold greater than the vehicle control groups, respectively. The effects of LY404039 on AMP-evoked behaviors were clearly reduced in mGlu2 and even more so in the mGlu2/3 receptor-deficient animals compared with the wild-type and mGlu3-deficient mice (Fig. 7).

Discussion

Recent clinical data published by Patil et al. (2007) offer compelling new evidence for the role of glutamate modulation in treating psychosis, specifically for mGlu2/3 receptor activation as a viable therapeutic approach to treat schizophrenia. Group II mGlu receptors are highly expressed in the forebrain, including limbic regions associated with schizophrenia such as hippocampus, amygdala, striatum, nucleus accumbens, and the prefrontal cortex (Ohishi et al., 1993; Swanson et al., 2005). In general, the group II mGlu receptors negatively modulate glutamate excitation by reducing the synaptic release of glutamate and decreasing postsynaptic excitability to glutamate. Group II mGlu receptors are highly homologous (∼70% at the amino acid level), and, at present, there are no orthosteric agonists that show selectivity between mGlu2 and mGlu3 receptors. Given the positive clinical findings with the mGlu2/3 agonist prodrug LY2140023, it is increasingly important to delineate the relative contributions of mGlu2 and mGlu3 receptors in the antipsychotic pharmacology of mGlu2/3 agonists. In this study, using transgenic mice lacking mGlu2, mGlu3, or mGlu2/3 receptors, we show that the antipsychotic-like effects of the mGlu2/3 receptor agonist LY404039 in the PCP and AMP models of psychosis are mediated through the activation of mGlu2 and not mGlu3 receptors. Furthermore, we provide evidence that the antipsychotic-like effects of LY404039 are mechanistically different from atypical antipsychotic drugs that primarily target dopaminergic and serotonergic systems.

The noncompetitive NMDA receptor antagonist PCP induces a schizophrenia-like psychosis (including the positive, negative, and cognitive symptoms) in healthy humans and profoundly exacerbates preexisting symptoms in patients with schizophrenia. Thus, the administration of PCP to animals has become an attractive model of schizophrenia (Geyer and Ellenbroek, 2003). Like the atypical antipsychotic drug clozapine, mGlu2/3 receptor agonists have been shown to block PCP hyperactivity in rats (Moghaddam and Adams, 1998; Cartmell et al., 2000a; Monn et al., 2007). In this study, we assessed the ability of LY404039 to block PCP-induced hyperlocomotion in wild-type and mGlu receptor knockout mice. PCP administration increased motor ambulations and distance traveled and decreased the time spent at rest in wild-type and all mGlu receptor-deficient strains, effects that were reversed by the administration of LY404039 (10 mg/kg) in wild-type and mGlu3 receptor-deficient mice. Importantly, the ability of LY404039 to reverse PCP-evoked hyperlocomotion was clearly reduced in both mGlu2 and mGlu2/3 receptor-deficient mice. LY404039 produced a slight reduction in activity induced by PCP (and AMP) in mGlu2R-deficient mice that was not seen in the double mGlu2/3 knockout mice, and it may be indicative of a small but not statistically significant contribution of the mGlu3 receptor. However, further studies with more selective group II mGlu receptor subtype-specific ligands are needed before the contribution of the mGlu3 receptor can be fully clarified.

To compare the antipsychotic mechanism of action of LY404039 with atypical antipsychotic drugs, we investigated the ability of clozapine and risperidone to reverse PCP-evoked hyperlocomotion. Clozapine and risperidone were equally effective at reversing PCP-induced effects in both genotypes. Our findings are in agreement with Patil et al. (2007) who demonstrated that the effects of olanzapine in the mouse PCP model of psychosis are not dependent on the presence of mGlu2 or mGlu3 receptors. Taken together, these data demonstrate that that the mGlu2/3 receptor agonist LY404039 exerts antipsychotic-like effects in the PCP model of psychosis through the selective activation of mGlu2 rather than mGlu3 receptors. Moreover, the effects of LY404039 are mechanistically distinct from that of atypical antipsychotic drugs because they do not directly involve the antagonism of monoamine receptors.

Psychotomimetic drugs such as PCP and the 5-hydroxytryptamine_2A (5HT2A) agonist DOI share increased glutamate release in the prefrontal cortex as a common mechanism by which they may induce psychotic symptoms. The activation of 5HT2A receptors evokes the asynchronous presynaptic release of glutamate from thalamocortical glutamate afferents (Aghajanian and Marek, 2000). Although LY404039 and other mGlu2/3 agonists do not directly antagonize serotonin receptors, functional 5HT2A receptor antagonism within the prefrontal cortex may represent a common pathway shared by mGlu2/3 agonists and clinically effective atypical antipsychotics. In the prefrontal cortex of rats, mGlu2/3 receptors show an overlapping cellular distribution with 5HT2A receptors (Marek et al., 2000), and the in vivo administration of mGlu2/3 receptor agonists (LY354740 and LY379268) and mGlu2 potentiator biphenyl-indanone A (BINA) block the 5HT2A-mediated behavioral and neurochemical effects of psychotomimetic drugs [PCP, DOI, and (–)-DOB] (Gewirtz and Marek, 2000; Zhai et al., 2002; Benneyworth et al., 2007). Recent electrophysiological studies revealed that LY404039 may also potently suppress serotonin-induced l-glutamate release in the prefrontal cortex, an effect that is reversed by the mGlu2/3 antagonist LY341495 (Rorick-Kehn et al., 2007a). Thus, in the prefrontal cortex, mGlu2/3 agonists, like the atypical antipsychotic drugs, block 5HT2A receptor-mediated glutamate release, although this effect is mechanistically distinct from that of the atypical antipsychotics and mediated by activation of mGlu2/3 receptors.

AMP-induced motor activation is commonly used to model acute psychosis or the positive symptoms of schizophrenia. The behavioral effects of AMP have been attributed to increased mesolimbic dopaminergic activity as well a noradrenergic component (Auclair et al., 2002). In rodents, all clinically effective antipsychotic drugs reverse AMP-induced locomotor activation, an effect that has been linked to the antagonism of dopamine D₂ receptors in mesolimbic dopaminergic pathways (Ellenbroek, 1993). In previous studies by our group, the mGlu2/3 agonists LY379268, LY354740 (Cartmell et al., 1999; Cartmell et al., 2000b), and LY404039 (Rorick-Kehn et al., 2007b) have been demonstrated to reduce AMP-induced behavioral activation. Similar to clozapine but not haloperidol, the mGlu2/3 receptor agonists selectively reduce AMP-induced rearing and ambulations but not fine movements (an indication of stereotypy), and these effects can be selectively reversed by the mGlu2/3 antagonist LY341495 (Cartmell et al., 1999). Consistent with previous studies, we found that administration of AMP induced a bell-shaped, dose-response curve on locomotor activity with reduced stimulation at high doses, probably reflecting an increase in stereotyped behavior (sniffing and rearing). LY404039 dose-dependently (1–30 mg/kg) reduced locomotor activation induced by AMP (5 mg/kg) in wild-type mice, with a maximal response observed at 30 mg/kg. In this study, we extend our previous work to show that the actions of mGlu2/3 agonists in the AMP model of psychosis are mediated by activation of the mGlu2 receptor rather than the mGlu3 receptor. Whereas LY404039 (10 mg/kg) was effective in reducing AMP-induced behavioral activation in wild-type and mGlu3 receptor-deficient mice, these effects were attenuated in mGlu2 and mGlu2/3 receptor-deficient mice. In addition to AMP, increased dopaminergic neurotransmission may contribute to a significant proportion of PCP-induced behaviors. Swanson and Schoepp (2002) showed that the functional depletion of DA and norepinephrine resulted in a 50% or greater reduction in PCP-induced behavioral activation and PCP also increases extracellular dopamine in the nucleus accumbens shell, an effect that is reversed by pretreatment with the mGlu2/3 agonist LY379268 (Swanson and Schoepp, 2003; Greenslade and Mitchell, 2004). Although mGlu2/3 agonists have no affinity for dopamine receptors, glutamatergic neurons are an important regulator of dopaminergic tone in the nucleus accumbens (Taber and Fibiger, 1995). Thus, mGlu2/3 agonists may reduce glutamate release in the nucleus accumbens and indirectly modulate DA neurotransmission by selectively activating mGlu2 receptors in limbic regions with outputs to the nucleus accumbens. Taken together, these preclinical data indicate that the blockade of mesolimbic dopamine may be a common but mechanistically distinct feature of atypical antipsychotic drugs and group II mGlu receptor agonists. These preclinical findings demonstrating the selective modulation of mesolimbic DA by mGlu2/3 agonists are further supported by the lack of observed extrapyramidal side effects, akathisia, and prolactin elevation in patients treated with LY2140023 (Patil et al., 2007).

In previous studies, we have shown that mGlu2/3 receptor agonists including LY404039 and LY544344 decrease exploratory behavior in rodents at higher doses (Rorick-Kehn et al., 2006; Rorick-Kehn et al., 2007b). However, in contrast to most atypical antipsychotics, LY404039 does not induce motor impairment such as rotarod ataxia and escape failures in the conditioned avoidance responding assay (Rorick-Kehn et al., 2007). This atypical profile was confirmed in early clinical testing, where LY2140023 (prodrug of LY404039) also failed to elevate plasma levels prolactin (Patil et al., 2007).

Allelic variations in mGlu3 receptors have been reported to alter glutamate function in humans and increase the risk for schizophrenia (Egan et al., 2004; Corti et al., 2007). Like the mGlu2 and mGlu3 receptor knockout mice (Linden et al., 2006), we report that the mGlu2/3 double receptor knockout mice seem normal with regard to growth and breeding and show no changes in observational assessment of autonomic nervous system and somatomotor responses compared with wild-type mice. During our locomotor activity studies, we found no genotypic differences in basal motor activity.

In summary, recent clinical data offer compelling new evidence that mGlu2/3 receptor activation is a viable therapeutic approach to treat the positive and negative symptoms of schizophrenia. In this study, we show that the mGlu2/3 receptor agonist LY404039 mediates its antipsychotic-like effects in the mouse PCP and AMP models of psychosis through the selective activation of mGlu2 rather than mGlu3 receptors. In addition, we demonstrate that the antipsychotic profile of this mGlu2/3 agonist is mechanistically distinct from that of current atypical antipsychotic drugs such as risperidone or clozapine.