PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Biol Psychiatry. Author manuscript; available in PMC 2010 July 27.
Published in final edited form as:
PMCID: PMC2910402
NIHMSID: NIHMS205860

Positive Allosteric Modulation of Metabotropic Glutamate 5 (mGlu5) Receptors Reverses N-Methyl-D-Aspartate Antagonist-Induced Alteration of Neuronal Firing in Prefrontal Cortex

Abstract

Background

Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptor hypofunction may be associated with schizophrenia. Activation of metabotropic glutamate 5 (mGlu5) receptors enhances NMDA receptor mediated currents in vitro, implying that allosteric modulation of mGlu5 receptors may have therapeutic efficacy for schizophrenia. The aim of this study was to determine if positive allosteric modulators of mGlu5 receptors are effective in reversing two cellular effects of NMDA receptor antagonists that are relevant to schizophrenia: increases in corticolimbic dopamine neurotransmission and disruption of neuronal activity in the prefrontal cortex (PFC).

Methods

In freely moving rats, we measured the effects of the positive modulator of mGlu5 receptor 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) alone or in combination with the NMDA antagonist MK801 on 1) spontaneous firing and bursting of medial PFC (mPFC) neurons, and 2) dopamine release as measured by microdialysis in the mPFC and nucleus accumbens (NAc).

Results

The predominant effect of CDPPB on mPFC neurons was excitatory, leading to an overall excitatory population response. Pretreatment with CDPPB prevented MK801-induced excessive firing and reduced spontaneous bursting. In contrast, CDPPB had no significant effect on basal dopamine release as compared with control rats and did not alter MK801-induced activation of dopamine release in the mPFC and NAc.

Conclusions

These results show that positive modulation of mGlu5 receptors reverses the effects of noncompetitive NMDA antagonists on cortical neuronal firing without affecting dopamine neurotransmission. Thus, these compounds may be effective in ameliorating PFC mediated behavioral abnormalities that results from NMDA receptor hypofunction.

Keywords: Antipsychotic drugs, cognition, dopamine, ensemble recording, schizophrenia

Dopamine D2 receptor antagonists have been used effectively to treat psychosis in patients with schizophrenia (Davis et al. 1991; Delay and Deniker 1955; Miyamoto et al. 2005; Snyder et al. 1974). Chronic exposure to these drugs, however, is associated with profound side effects such as dysphoria, secondary negative symptoms (Carpenter et al. 1985), motor deficits (Llorca et al. 2002), weight gain (Schwartz et al. 2004), hyperprolactinemia (Hummer and Huber 2004), and diabetes (Sathyaprakash and Henry 2004). Moreover, D2 antagonists are generally ineffective in treating negative symptoms and cognitive deficits associated with schizophrenia (Miyamoto et al. 2005). Hence, there is an acute need to develop alternative treatments for schizophrenia that have fewer side effects and are more effective in treating cognitive and negative symptoms of the disorder.

Design of novel therapeutic approaches for schizophrenia is contingent on a better understanding of the pathophysiology of the disease. Although the antipsychotic efficacy of D2 receptor antagonists supports the idea of a hyperactive dopamine system in schizophrenia (Carlsson 1978; Seeman 1987), limited efficacy of these drugs for treating cognitive deficits has prompted efforts to investigate the role of nondopaminergic systems in influencing these deficits. One example is the N-methyl-D-aspartate (NMDA) receptor channel (Coyle et al. 2002; Javitt 2002; Moghaddam 2003). N-methyl-D-aspartate receptors have been implicated in the etiology and pathophysiology of schizophrenia, primarily because NMDA receptor antagonists produce schizophrenia-like symptoms in healthy individuals (Honey et al. 2005; Javitt and Zukin 1991; Krystal et al. 1994; Luby et al. 1959; Malhotra et al. 1996; Newcomer et al. 1999; Olney and Farber 1995) and exacerbate preexisting symptoms in individuals with schizophrenia (Lahti et al. 1995; Malhotra et al. 1997). Postmortem and genetic linkage studies (Harrison and Weinberger 2005; Kristiansen et al. 2006; Moghaddam 2003), as well as a preliminary imaging study (Pilowsky et al. 2006), also support a role for NMDA receptor dysfunction in schizophrenia. N-methyl-D-aspartate receptor antagonists such as dizocilpine (MK801), phencyclidine (PCP), or low doses of ketamine are routinely used in preclinical studies to model schizophrenia. Systemic administration of these drugs to rodents and nonhuman primates produces behavioral deficits that parallel some positive and negative symptoms, as well as the cognitive deficits of schizophrenia (Bakshi and Geyer 1998; Hauber and Andersen 1993; Jentsch et al. 1997; Stefani and Moghaddam 2005; Verma and Moghaddam 1996). At a cellular level, NMDA antagonist administration increases dopamine release in the prefrontal cortex (PFC) of rodents (Verma and Moghaddam 1996) and humans (Aalto et al. 2005), suggesting that they work, in part, by disrupting dopamine neurotransmission.

Pharmacological approaches that may reduce the impact of NMDA receptor deficiency on behavior have primarily focused on stimulating the (co-agonist) glycine site on the NMDA receptors (Javitt 2002; Tsai et al. 1998). Another approach is to target various subtypes of metabotropic glutamate receptor to indirectly augment NMDA receptor function (Gasparini et al. 2002; Marino and Conn 2002; Moghaddam 2004). For example, in most of the cortical and subcortical areas of the central nervous system associated with schizophrenia pathology, NMDA receptors have synergistic relationship with the metabotropic glutamate 5 (mGlu5) receptors at the postsynaptic level (Alagarsamy et al. 2002). Agonists of mGlu5 receptor enhance NMDA receptor-mediated currents in hippocampal (Doherty et al. 1997) and subthalamic nucleus (Awad et al. 2000) slices in the rat. Conversely, the mGlu5 receptor 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). Behavioral studies also show that MPEP enhances the effects of NMDA antagonist blockade on prepulse inhibition, locomotion, working memory, and instrumental learning impairments (Campbell et al. 2004; Homayoun et al. 2004; Kinney et al. 2005a; Spooren et al. 2000). The worsening of the detrimental effects of NMDA receptor antagonists by mGlu5 receptor antagonist suggests that activation of mGlu5 receptors may represent a plausible approach for ameliorating symptoms of schizophrenia (Marino and Conn 2002; Moghaddam 2004). Recent behavioral studies using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), a positive allosteric modulator of the mGlu5 receptor (Kinney et al. 2005b; O’Brien et al. 2004), show that pretreatment with this compound reverses amphetamine-induced hyperlocomotion and prepulse inhibition deficit, two tests which serve to model some aspects of schizophrenia in rodents (Kinney et al. 2005a).

This study sought to characterize the cellular interaction of the mGlu5 receptor positive modulator CDPPB with an NMDA antagonist in freely moving rats. Two measures that may subserve the disruptive behavioral effects of NMDA antagonists are increased dopamine release in corticolimbic regions (Verma and Moghaddam 1996) and increased random firing of prefrontal cortex (PFC) neurons (Homayoun et al. 2005; Homayoun and Moghaddam 2006; Jackson et al. 2004). Thus, we determined the effects of CDPPB administration alone or in combination with the NMDA antagonist MK801 on spontaneous firing and bursting of medial prefrontal cortex (mPFC) neurons and dopamine release in the mPFC and nucleus accumbens (NAc).

Methods and Materials

Animals

A total of 76 adult male Sprague-Dawley rats (Harlan, Somerville, New Jersey), weighing 250 to 420 g were used in this study. Animals were housed on a 12-hour light/12-hour dark cycle (lights on at 07:00 AM) for at least 1 week after their arrival in the animal facilities before surgery. Microdialysis and electrophysiological recordings were performed during the light phase on awake and freely moving animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (http://grants.nih.gov/grants/olaw/olaw.htm).

Drugs

All drugs and vehicles were administered intraperitoneally (IP). The compound CDPPB was synthesized in-house (Lindsley et al. 2004) and dissolved in a vehicle composed of 10% dimethyl sulfoxide (DMSO) + 90% ethylene glycol. The MK801 (Sigma-RBI, St. Louis, Missouri) was dissolved in saline (.9% sodium chloride [NaCl]). The route of administration (IP) and doses of CDPPB (3 and 10 mg/kg) were chosen according to Kinney et al. (2005a).

Electrophysiology Procedure

Extracellular activity of PFC single units was studied in awake unrestricted rats (n = 9). Microelectrode arrays (NB Labs, Denison, Texas) consisting of eight 50-μm diameter Teflon-insulated, stainless steel wires were chronically implanted under halothane anesthesia in the mPFC (target coordinates in millimeters for the center of the array at anterioposterior (AP), +3.0 from bregma; mediolateral (ML), .7; and dorsoventral (DV), 3.5) (according to the atlas of Paxinos and Watson 1998). Arrays were arranged in a 2 × 4 pattern measuring approximately .25 × .7 mm. Animals were allowed 5 to 7 days of recovery and 2 days of habituation to recording chamber before first recording session. Habituation also included sham injections. Each animal received two to four acute intraperitoneal injections based on a pseudo-random order with 7–10 washout period. Each recording session included 30 min of baseline activity and a minimum of 2 hours of postinjection recording. All recordings were performed in a standard transparent rodent housing cage with a modified open top. Animals were connected to a field-effect transistor headstage (NB Labs) using lightweight cabling that passed through a commutator and allowed the animal unrestricted movement during recording. Unit activity was recorded using multiple channel amplifiers with 500× gain and 220 to 5900 Hz band pass filters (Plexon Inc., Dallas, Texas). The amplified signal from each electrode was digitized (30 kHz sampling rate) on computer hard disk for offline spike sorting. Spike sorting was performed with Off-Line Sorter software (Plexon Inc.) using a combination of automatic and manual sorting techniques previously described (Homayoun and Moghaddam 2006). Only units with well-isolated waveform clusters that had stable waveforms during the length of the recording session were accepted as single unit. Data reported here are based on regular firing neurons with a baseline firing rate less than 10 Hz (putative pyramidal neurons). The paucity of recorded fast firing single units (>10 Hz, putative interneurons) did not allow analysis of their responses.

Microdialysis Procedure

Microdialysis probes were implanted under halothane anesthesia (Adams and Moghaddam 1998). Probes (outer diameter, 330 μm; exposed tip, 3.0 mm for mPFC, 2.0 mm for NAc) were gently lowered to the mPFC (AP, +3–4 from bregma; ML, .8; DV, 5.8) and NAc (AP, +1.7 from bregma; ML, .8; DV, 8.5) and secured to the skull and skull screws using dental cement. Immediately after the surgery, rats were placed in a standard rodent housing cage (44 × 22 × 42 cm) with a modified open top and probes connected to a liquid swivel-balance arm assembly. Animals were allowed to recover for 24 hours prior to the start of experiment. Animals had ad libitum access to food and water during the recovery period but not during the experiment. Probes were perfused with Ringer’s solution (in mmol/L: 145 NaCl, 2.7 potassium chloride [KCl], 1.0 magnesium chloride [MgCl2], and 1.2 calcium chloride [CaCl2]) at a flow rate of 1.4 μL/min during the recovery period and 2.0 μL/min during sample collection. Dialysis samples were collected every 20 min and injected immediately onto a high-performance liquid chromatography (HPLC) system with electrochemical detection for the analysis of dopamine as previously described (Adams and Moghaddam 1998).

Pharmacological Treatments

Electrophysiology Experiment

In the first experiment, the effects of CDPPB (3 and 10 mg/kg) were compared to vehicle. Animals received a single injection 30 min after the start of recording. In the second experiment, rats were treated with CDPPB (10 mg/kg) or vehicle, followed by MK801 (.1 mg/kg) 30 min later. Animals received four to six treatments in random order with a 7–10 day washout period.

Microdialysis Experiment

After 60 to 80 min of baseline collections, rats received a first injection of either CDPPB (10 mg/kg) or vehicle. Twenty minutes later, each rat received a second injection of either MK801 (.1 mg/kg) or saline.

Data Analysis

Electrophysiology Experiment

Electrophysiological data were analyzed using NeuroExplorer (Plexon Inc.) and Matlab (MathWorks, Natick, Massachusetts). Firing rate statistics were calculated using firing rate histograms with 5 min bins normalized to the mean baseline firing (30 min) of individual units. K-means cluster analysis (SPSS, Inc., Chicago, Illinois) was used to detect the predominant patterns of responses to treatment. Clustering used the normalized rate histogram bins of each unit as variables and was performed separately for neurons in single and double injection sessions. The number of clusters used for K-means analysis was initially set at 2 and was incremented to 10 at steps of 1. The clusters isolated at each step were visualized by plotting the average firing rates of all neurons allocated to each cluster and comparing their response patterns. Whenever neurons with similar response patterns had been clustered separately based on a small difference in response magnitude, the clusters were merged. The minimum number of clusters that could explain at least 95% of variability of response patterns was determined as the number of main clusters of responses. The final grouping was verified by obtaining a significant analysis of variance (ANOVA) with time as repeated measure and cluster membership as factor. The distribution of different types of responses between groups was compared using the Pearson chi-square (χ2) test. The temporal profile of normalized firing rates of all neurons in each group was plotted against time and was compared between groups using two-way ANOVA with time as repeated measure. The spontaneous bursting activity, defined as periods of non-Poisson high-frequency firing, was detected in each spike train using Poisson surprise method of Legendy and Salcman (1985) as implemented in NeuroExplorer. For each neuron, the relative change in the percentage of spikes in bursts during the postdrug period (minutes 30 to 150 in single injection groups and minutes 60 to 150 in double injection groups) versus baseline was calculated. One-way ANOVA with Bonferroni post hoc test was used to compare average bursting between groups (p < .05 as the significance criteria). A total of nine rats was used for the study. The number of recorded neurons in each treatment group was as follows: vehicle, n = 123; CDPPB (3 mg/kg), n = 109; CDPPB (10 mg/kg), n = 85; vehicle + MK801, n = 123; CDPPB (10 mg/kg) + MK801, n = 160. The number of rats in each treatment group was as follows: vehicle, n = 4; CDPPB (3 mg/kg), n = 4; CDPPB (10 mg/kg), n = 5; vehicle + MK801, n = 4; CDPPB (10 mg/kg) + MK801, n = 6.

Microdialysis Experiment

Statistical difference between groups was analyzed by two-way analysis of variance. When there was a significant group effect, post hoc pairwise comparison with the Bonferroni test was performed.

Histology

After the termination of the electrophysiology and microdialysis experiments, animals were anesthetized with chloral hydrate (Sigma-RBI) and intracardially perfused with 9% saline followed by 10% buffered formalin. Brains were removed and stored in 10% buffered formalin. The 250-μm serial sections of the fixed brains were stained with cresyl violet acetate (Sigma-RBI) and electrode and microdialysis probe placements were verified under a microscope. Only rats showing correct placements were included in the statistical analysis (Figure 1).

Figure 1
Placements of electrode arrays and microdialysis probes. Placement of electrode arrays are depicted by black circles [mPFC: (A) 2.7 mm from bregma; (B) 3.2 mm from bregma]. Location of the active surface of microdialysis probes is depicted by straight ...

Results

Electrophysiology Experiments

Effects of CDPPB Treatment Alone

Single neurons showed different firing rate responses to CDPPB treatment (Figure 2A). K-means clustering identified five patterns of firing rate responses in single injection groups (CDPPB or vehicle) that included: 1) an early increase, 2) a sustained increase, 3) an early decrease, 4) a sustained decrease, and 5) no change in firing rate (Figure 2A, black lines). The distribution of these response types was significantly different between vehicle and CDPPB groups (Figure 2B, χ2 = 104.55, p < .001). The effect of CDPPB on distribution of response types was dose-dependent, with the higher dose causing more sustained responses (both excitatory and inhibitory) than the lower dose (between two doses, χ2 = 24.35, p < .001). The predominant effect of CDPPB at both doses was to increase excitatory responses in PFC neurons. The CDPPB also dose-dependently increased the average firing rate of PFC neurons (Figure 2C, average normalized rate of all units). Two-way ANOVA revealed significant effects for treatment [F(2,314) = 14.45, p < .000], time [F(29,9106) = 11.57, p < .000], and treatment × time interaction [F(58,9106) = 8.14, p < .000]. In addition, CDPPB changed the pattern of firing of PFC units (Figure 2D) by significantly increasing the percentage of spikes in bursts compared with vehicle [one-way ANOVA, F(2,314) = 3.22, p < .05, post hoc test, p < .05).

Figure 2
Effect of CDPPB on spontaneous firing rate of PFC single units. (A) Different types of PFC single unit responses to CDPPB. Neuronal responses to CDPPB (3 or 10 mg/kg) and vehicle were classified as one of five patterns using K-means clustering (see Methods ...

Effects of the Combined Administration of CDPPB and MK801

K-means clustering identified four patterns of firing rate responses in double injection groups that included: 1) an early increase, 2) a sustained increase, 3) a sustained decrease, and 4) no change in firing rate (Figure 3A). The majority of neurons in the vehicle pretreatment group responded to MK801 with a sustained increase in firing rate (Figure 3B). In contrast, CDPPB pretreatment decreased the proportion of this response type and instead increased the proportion of neurons with either early transient increase or sustained decrease responses (χ2 = 121.85, p < .001). As a result, the robust excitatory effect of MK801 on the average firing activity of PFC neurons was blocked by CDPPB pretreatment (Figure 3C). Two-way ANOVA showed significant effects for both treatment [F(1,281) = 74.66, p < .000] and time [F(29,8149) = 55.03, p < .000], as well as for treatment × time interaction [F(29,8149) = 86.41, p < .000]. In vehicle pretreated group, MK801 decreased the percentage of spikes in bursts in the majority of neurons (compared with single vehicle group, Figure 3D). This effect was blocked by CDPPB pretreatment [one-way ANOVA including the single vehicle group, F(2,403) = 4.48, p < .05, post hoc test, p < .05, t test between double injection groups, p < .05].

Figure 3
Blockade of MK801-induced effects on PFC firing rate by CDPPB. (A) Different types of PFC single unit responses to CDPPB (10 mg/kg, or vehicle) plus MK801 (.1 mg/kg) treatment. Neuronal responses were classified as one of four patterns using K-means clustering. ...

Microdialysis Experiments

Effects of the Administration of CDPPB Alone

In the mPFC, a small increase in dopamine release was observed after injection of CDPPB (n = 8) and the corresponding vehicle (n = 7). The two-way ANOVA with group as factor and sample as repeated measure showed a significant effect of sample [F(12,156) = 12.85, p < .0001]. There was no between-group differences in the amplitude of the response, as shown by a lack of effect of group [F(1,13) = 1.38, p > .05] and of sample × group interaction [F(12,156) = .63, p > .5] (Figure 4A).

Figure 4
Effects of the administration of CDPPB, alone or in combination with MK801, on dopamine release in mPFC (A) and NAc (B). Results (dopamine release, mean ± SEM) are shown as percentage of baseline value. Each sample represents a 20-minute period. ...

Similar to the results in the mPFC, injection of CDPPB (n = 10) or vehicle (n = 8) produced a small increase in dopamine release in the NAc. Two-way ANOVA with group as factor and sample as repeated measure indicated an effect of sample [F(12,192) = 4.74, p < .0001], no significant effect of group [F(1,16) = .006, p > .05], and no significant sample × group interaction [F(12,192) = .30, p > .05] (Figure 4B).

Effects of the Combined Administration of CDPPB and MK801

In the mPFC, injection of MK801 produced the expected large and prolonged increase in dopamine release (vehicle + MK801, n = 6). Comparison of vehicle + saline, vehicle + MK801, and CDPPB + MK801 groups, by means of a two-way ANOVA with sample as repeated measure, showed a significant effect of group [F(2,18) = 3.77, p < .05] compared with vehicle + saline group. Pretreatment with CDPPB (CDPPB + MK801, n = 8) produced a similar magnitude of increase on dopamine release. Comparison between both vehicle + MK801 and CDPPB + MK801 groups with a two-way ANOVA with group as factor and sample as repeated measure showed no difference [no effect of group: F(1,12) = .63, p > .05; a significant effect of sample: F(12,144) = 21.83, p < .001; no significant sample × group interaction: F(12,144) = .43, p > .05], demonstrating that CDPPB lacks in reversing MK801 effects on dopamine release (Figure 4A).

Similar results were obtained in the NAc in that vehicle + MK801 (n = 8) and CDPPB + MK801 (n = 12) produced similar increases in dopamine release that were significantly larger than the increase observed after vehicle + saline. Comparison of vehicle + saline, vehicle + MK801, and CDPPB + MK801 groups, by means of a two-way ANOVA with sample as repeated measure, showed a significant effect of group [F(2,25) = 3.86, p < .05], a significant effect of sample [F(12,300) = 16.04, p < .0001], and a significant sample × group interaction [F(24,300) = 2.92, p < .0001] (Figure 4B).

Discussion

Positive allosteric modulation of mGlu5 receptors by CDPPB produced a predominately excitatory influence on population response and burst activity of mPFC neurons in awake rats. In contrast, CDPPB inhibited the profound excitatory effects of the NMDA antagonist MK801 on spontaneous activity of mPFC neurons. The CDPPB also normalized MK801-induced disruptions in burst activity. These effects of CDPPB were not related to changes in dopamine neurotransmission, as CDPPB alone did not change dopamine release in the mPFC or NAc as compared with control rats and did not modify the magnitude of MK801-induced dopamine release in either region.

The excitatory effects of CDPPB, when given alone, are consistent with previous findings demonstrating that the mGlu5 antagonist MPEP decreases firing rate and number of bursts in the mPFC of awake rats (Homayoun and Moghaddam 2006) and inhibits bicuculine-induced persistent spontaneous bursting in rat hippocampal slices (Stoop et al. 2003). These effects of CDPPB may be direct, as the activation of mGlu5 receptors produces excitatory postsynaptic potentials (EPSP) in vitro (Fitzjohn et al. 1999; Sharifullina et al. 2004; Wisniewski and Car 2002), or indirect through increasing the efficacy of endogenous glutamate neurotransmission at NMDA receptors. For example, positive allosteric modulation of mGlu5 receptors has been shown to enhance phosphorylation of the NR1 subunit of the NMDA receptors and the expression of signaling molecules such as cyclic adenosine monophosphate (cAMP) response element-binding (CREB) and extracellular signal-regulated kinase (ERK), all of which are critical for glutamate-mediated signal transduction mechanisms (Liu et al. 2006).

The significant effect of CDPPB was long lasting in that the firing rate remained elevated above baseline for over an hour after injection, suggesting that this drug does not lead to rapid desensitization of mGlu5 receptors. This is interesting given that agonist stimulation of mGlu1/5 receptors leads to rapid desensitization (Contractor et al. 1998; Gereau and Heinemann 1998). Another reason for the sustained effects of CDPPB may be that functional mGlu5 receptors have been localized on the nuclear membrane of cortical and midbrain neurons, where their activation increases intranuclear calcium release (Jong et al. 2005; O’Malley et al. 2003). Such an increase may stimulate signaling pathways and favor changes in gene expression, leading to sustained firing rate activation.

Pretreatment with CDPPB blocked the MK801-induced disruption of spontaneous firing and bursting activity. Several lines of evidence suggest a synergistic interaction between mGlu5 and NMDA receptors, whereby stimulation of one receptor may increase ligand affinity for, or signal transduction mechanism mediated by, the other receptor (Marino and Conn 2006). Systemic administration of NMDA antagonists is thought to initiate a disinhibition process by inhibiting NMDA-mediated firing rate of gamma-aminobutyric acid (GABA) interneurons, resulting in increased firing rate of pyramidal neurons and glutamate release in the PFC (Jackson et al. 2004; Moghaddam et al. 1997; Olney 1990). The origin of glutamatergic projections on GABA neurons that are disinhibited by NMDA antagonist is not clear but may involve afferents from the hippocampus (Greene 2001). Pretreatment with CDPPB may reduce the extent of this disinhibition by “recruiting” more NMDA receptors that are activated by endogenous glutamate. This mechanism is consistent with our previous findings showing that pretreatment with the mGlu5 receptor antagonist MPEP exaggerates the effects of MK801 on firing rate of mPFC neurons (Homayoun and Moghaddam 2006). One caveat with the present finding is that MK801 (and psychotomimetic NMDA antagonists such as ketamine or phencyclidine) are noncompetitive use-dependent antagonists; therefore, increased activity of NMDA receptors before administration of this class of antagonists should potentiate their effect. Thus, pretreatment with mGlu5 receptors positive modulators, which are thought to increase the efficacy of endogenous glutamate neurotransmission at NMDA receptors (Gasparini et al. 2002; Liu et al. 2006; Marino and Conn 2002; Moghaddam 2004), would be expected to exacerbate the effects of MK801. This was not the case with our findings, suggesting that other mechanisms such as mGlu5 receptor-mediated activation of independent pathways that balance or counteract the effects of MK801 on cortical firing may play a role in systemic effects of CDPPB.

Antagonists of NMDA receptor such as ketamine or MK801 produce a behavioral syndrome in humans and laboratory animals that resemble some aspects of schizophrenia (Coyle 1996; Javitt and Zukin 1991; Krystal et al. 2003; Moghaddam 2003; Olney and Farber 1995). In particular, these compounds disrupt PFC-dependent cognitive functions, such as working memory and set shifting, in a manner similar to schizophrenia (Adler et al. 1999; Aultman and Moghaddam 2001; Krystal et al. 2000; Stefani et al. 2003). Our finding that CDPPB reduces the disruptive effects of MK801 on PFC neurons suggests that this compound may ameliorate some of the adverse cognitive effects of NMDA receptor hypofunction (Darrah et al., unpublished data, 2007).

Systemic administration of CDPPB did not alter baseline and MK801-induced increases in dopamine release in either mPFC or NAc. Given that the mGlu5 receptor antagonist MPEP enhances dopamine release in mPFC (Homayoun et al. 2004), we had expected that administration of the positive modulator of mGlu5 receptor would decrease dopamine release in this region. One concern with the microdialysis study is that the vehicle used to dissolve CDPPB, a noxious mixture of DMSO and ethylene glycol, also increased the release of dopamine in mPFC and NAc. This finding is consistent with the literature showing that mild stressors increase dopamine neurotransmission in both structures (Abercrombie et al. 1989; Thierry et al. 1976). Therefore, the stress-induced increases in dopamine release may have masked a small decrease in dopamine release by CDPPB. Nonetheless, the lack of a significant difference in dopamine release between CDPPB-injected and vehicle-injected animals suggests that reversal of MK801-induced alterations on spontaneous firing and bursting of mPFC neurons occurs independently of dopamine-mediated mechanisms. This finding also suggests that CDPPB may be devoid of effects on mood and motivation that usually confounds the use of therapeutic ligands that affect dopamine neurotransmission.

Conclusions

Positive allosteric modulators of mGlu5 receptors have promising therapeutic potential for cognitive disorders including schizophrenia (Gasparini et al. 2002; Moghaddam 2003; Marino and Conn 2006). The present study revealed that systemic treatment with CDPPB, a positive allosteric modulator of mGlu5 receptor, increased spontaneous firing and burst activity of PFC neurons in awake rats, suggesting that this class of compounds influences both the rate and pattern of firing of cortical neurons. More importantly, pretreatment with CDPPB inhibited the increased random firing and reduced spontaneous bursting that results from NMDA receptor hypofunction. The CDPPB did not have a significant effect on dopamine neurotransmissions, suggesting that a clinical application of this compound may not be associated with motor side effects, dysphoria, or abuse liability. Although further characterization is necessary to predict the clinical usefulness of this class of compounds, the present results indicate that allosteric modulation of mGlu5 receptors has beneficial physiological effects on the function of PFC neurons in awake animals.

Acknowledgments

This work was supported by the National Institute of Mental Health and Pittsburgh Life Sciences Greenhouse.

We thank Alicia Defrancesco for technical assistance.

References

  • Aalto S, Ihalainen J, Hirvonen J, Kajander J, Scheinin H, Tanila H, et al. Cortical glutamate-dopamine interaction and ketamine-induced psychotic symptoms in man. Psychopharmacology (Berl) 2005;182:375–383. [PubMed]
  • Abercrombie ED, Keefe KA, DiFrischia DS, Zigmond MS. Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex. J Neurochem. 1989;52:1655–1658. [PubMed]
  • Adams B, Moghaddam B. Corticolimbic dopamine neurotransmission is temporally dissociated from the cognitive and locomotor effects of phencyclidine. J Neurosci. 1998;18:5545–5554. [PubMed]
  • Adler CM, Malhotra AK, Elman I, Goldberg T, Egan M, Pickar D, et al. Comparison of ketamine-induced thought disorder in healthy volunteers and thought disorder in schizophrenia. Am J Psychiatry. 1999;156:1646–1649. [PubMed]
  • Alagarsamy S, Rouse ST, Junge C, Hubert GW, Gutman D, Smith Y, et al. NMDA-induced phosphorylation and regulation of mGluR5. Pharmacol Biochem Behav. 2002;73:299–306. [PubMed]
  • Aultman JM, Moghaddam B. Distinct contributions of glutamate and dopamine receptors to temporal aspects of rodent working memory using a clinically relevant task. Psychopharmacology (Berl) 2001;153:353–364. [PubMed]
  • Awad H, Hubert GW, Smith Y, Levey AI, Conn PJ. Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus. J Neurosci. 2000;20:7871–7879. [PubMed]
  • Bakshi VP, Geyer MA. Multiple limbic regions mediate the disruption of prepulse inhibition produced in rats by the noncompetitive NMDA antagonist dizoclipine. J Neurosci. 1998;18:8394–8401. [PubMed]
  • Campbell U, Lalwani K, Hernandez L, Kinney G, Conn P, Bristow L. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) potentiates PCP-induced cognitive deficits in rats. Psychopharmacology (Berl) 2004;175(3):310–318. [PubMed]
  • Carlsson A. Antipsychotic drugs, neurotransmitters, and schizophrenia. Am J Psychiatry. 1978;135:164–173. [PubMed]
  • Carpenter WT, Jr, Heinrichs DW, Alphs LD. Treatment of negative symptoms. Schizophr Bull. 1985;11:440–452. [PubMed]
  • Contractor A, Gereau RW, 4th, Green T, Heinemann SF. Direct effects of metabotropic glutamate receptor compounds on native and recombinant N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A. 1998;95:8969–8974. [PubMed]
  • Coyle J. The glutamatergic dysfunction hypothesis for schizophrenia. Harv Rev Psychiatry. 1996;3:241–253. [PubMed]
  • Coyle JT, Tsai G, Goff DC. Ionotropic glutamate receptors as therapeutic targets in schizophrenia. Curr Drug Targets CNS Neurol Disord. 2002;1:183–189. [PubMed]
  • Davis KL, Kahn RS, Ko G, Davidson M. Dopamine in schizophrenia: A review and reconceptualization. Am J Psychiatry. 1991;148:1474–1486. [PubMed]
  • Delay J, Deniker P. Neuroleptic effects of chlorpromazine in therapeutics of neuropsychiatry. J Clin Exp Psychopathol. 1955;16:104–111. [PubMed]
  • Doherty AJ, Palmer MJ, Henley JM, Collingridge GL, Jane DE. (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5, but no mGlu1, receptors expressed in CHO cells and potentiates NMDA responses in the hippocampus. Neuropharmacology. 1997;36:265–267. [PubMed]
  • Fitzjohn S, Kingston A, Lodge D, Collingridge G. DHPG-induced LTD in area CA1 of juvenile rat hippocampus; characterisation and sensitivity to novel mGlu receptor antagonists. Neuropharmacology. 1999;38:1577–1583. [PubMed]
  • Gasparini F, Kuhn R, Pin JP. Allosteric modulators of group I metabotropic glutamate receptors: Novel subtype-selective ligands and therapeutic perspectives. Curr Opin Pharmacol. 2002;2:43–49. [PubMed]
  • Gereau RW, 4th, Heinemann SF. Role of protein kinase C phosphorylation in rapid desensitization of metabotropic glutamate receptor 5. Neuron. 1998;20:143–151. [PubMed]
  • Greene R. Circuit analysis of NMDAR hypofunction in the hippocampus, in vitro, and psychosis of schizophrenia. Hippocampus. 2001;11:569–577. [PubMed]
  • Harrison P, Weinberger D. Schizophrenia genes, gene expression, and neuropathology: On the matter of their convergence. Mol Psychiatry. 2005;10:40–68. [PubMed]
  • Hauber W, Andersen R. The non-NMDA glutamate receptor antagonist GYKI 52466 counteracts locomotor stimulation and anticataleptic activity induced by the NMDA antagonist dizocilpine. Naunyn Schmiedebergs Arch Pharmacol. 1993;348:486–490. [PubMed]
  • Homayoun H, Jackson ME, Moghaddam B. Activation of metabotropic glutamate 2/3 receptors reverses the effects of NMDA receptor hypofunction on prefrontal cortex unit activity in awake rats. J Neurophysiol. 2005;93:1989–2001. [PubMed]
  • Homayoun H, Moghaddam B. Bursting of prefrontal cortex neurons in awake rats is regulated by metabotropic glutamate 5 (mGlu5) receptors: Rate-dependent influence and interaction with NMDA receptors. Cereb Cortex. 2006;16:93–105. [PubMed]
  • Homayoun H, Stefani MR, Adams BW, Tamagan GD, Moghaddam B. Functional interaction between NMDA and mGlu5 receptors: Effects on working memory, instrumental learning, motor behaviors, and dopamine release. Neuropsychopharmacology. 2004;29:1259–1269. [PubMed]
  • Honey GD, Honey RA, O’Loughlin C, Sharar SR, Kumaran D, Suckling J, et al. Ketamine disrupts frontal and hippocampal contribution to encoding and retrieval of episodic memory: An fMRI study. Cereb Cortex. 2005;15:749–759. [PMC free article] [PubMed]
  • Hummer M, Huber J. Hyperprolactinaemia and antipsychotic therapy in schizophrenia. Curr Med Res Opin. 2004;20:189–197. [PubMed]
  • Jackson M, Homayoun H, Moghaddam B. NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex. Proc Natl Acad Sci U S A. 2004;101:6391–6396. [PubMed]
  • Javitt DC. Glycine modulators in schizophrenia. Curr Opin Investig Drugs. 2002;3:1067–1072. [PubMed]
  • Javitt DC, Zukin SR. Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry. 1991;148:1301–1308. [PubMed]
  • Jentsch JD, Redmond DE, Jr, Elsworth JD, Taylor JR, Youngren KD, Roth RH. Enduring cognitive deficits and cortical dopamine dysfunction in monkeys after long-term administration of phencyclidine. Science. 1997;277:953–955. [PubMed]
  • Jong YJ, Kumar V, Kingston AE, Romano C, O’Malley KL. Functional metabotropic glutamate receptors on nuclei from brain and primary cultured striatal neurons. Role of transporters in delivering ligand. J Biol Chem. 2005;280:30469–30480. [PubMed]
  • Kinney GG, O’Brien JA, Lemaire W, Burno M, Bickel DJ, Clements MK, et al. A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models. J Pharmacol Exp Ther. 2005a;313:199–206. [PubMed]
  • Kinney G, O’brien J, Lemaire W, Burno M, Bickel D, Clements M, et al. A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models. J Pharmacol Exp Ther. 2005b;313(1):199–206. [PubMed]
  • Kristiansen LV, Beneyto M, Haroutunian V, Meador-Woodruff JH. Changes in NMDA receptor subunits and interacting PSD proteins in dorsolateral prefrontal and anterior cingulate cortex indicate abnormal regional expression in schizophrenia. Mol Psychiatry. 2006;11:737–747. 705. [PubMed]
  • Krystal JH, Bennett A, Abi-Saab D, Belger A, Karper LP, D’Souza DC, et al. Dissociation of ketamine effects on rule acquisition and rule implementation: Possible relevance to NMDA receptor contributions to executive cognitive functions. Biol Psychiatry. 2000;47:137–143. [PubMed]
  • Krystal JH, D’Souza DC, Mathalon D, Perry E, Belger A, Hoffman R. NMDA receptor antagonist effects, cortical glutamatergic function, and schizophrenia: Toward a paradigm shift in medication development. Psychopharmacology (Berl) 2003;169:215–233. [PubMed]
  • Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, et al. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans: Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry. 1994;51:199–214. [PubMed]
  • Lahti AC, Holocomb HH, Medoff DR, Tammings CA. Ketamine activates psychosis and alters limbic blood flow in schizophrenia. Neuroreport. 1995;6:869–872. [PubMed]
  • Legendy CR, Salcman M. Bursts and recurrences of bursts in the spike trains of spontaneously active striate cortex neurons. J Neurophysiol. 1985;53:926–939. [PubMed]
  • Lindsley CW, Wisnoski DD, Leister WH, O’Brien JA, Lemaire W, Williams DL, Jr, et al. Discovery of positive allosteric modulators for the metabotropic glutamate receptor subtype 5 from a series of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides that potentiate receptor function in vivo. J Med Chem. 2004;47:5825–5828. [PubMed]
  • Liu F, Zhang G, Hornby G, Vasylyev D, Bowlby M, Park K, et al. The effect of mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices. Eur J Pharmacol. 2006;536:262–268. [PubMed]
  • Llorca PM, Chereau I, Bayle FJ, Lancon C. Tardive dyskinesias and antipsychotics: A review. Eur Psychiatry. 2002;17:129–138. [PubMed]
  • Luby E, Cohen B, Rosenbaum G, Gottlieb J, Kelley R. Study of a new schizophrenomimetic drug-sernyl. Arch Neurol Psychiatry. 1959;81:363–369. [PubMed]
  • Malhotra AK, Pinals DA, Adler CM, Elman I, Clifton A, Pickar D, et al. Ketamine-induced exacerbation of psychotic symptoms and cognitive impairment in neuroleptic-free schizophrenics. Neuropsychopharmacology. 1997;17:141–150. [PubMed]
  • Malhotra AK, Pinals DA, Weingartner H, Sirocco K, Missar CD, Pickar D, et al. NMDA receptor function and human coginition: The effects of ketamine in healthy volunteers. Neuropsychopharmacology. 1996;14:301–307. [PubMed]
  • Marino MJ, Conn PJ. Direct and indirect modulation of the N-methyl D-aspartate receptor. Curr Drug Targets CNS Neurol Disord. 2002;1:1–16. [PubMed]
  • Marino MJ, Conn PJ. Glutamate-based therapeutic approaches: Allosteric modulators of metabotropic glutamate receptors. Curr Opin Pharmacol. 2006;6:98–102. [PubMed]
  • Miyamoto S, Duncan G, Marx C, Lieberman J. Treatments for schizophrenia: A critical review of pharmacology and mechanisms of action of antipsychotic drugs. Mol Psychiatry. 2005;10:79–104. [PubMed]
  • Moghaddam B. Bringing order to the glutamate chaos in schizophrenia. Neuron. 2003;40:881–884. [PubMed]
  • Moghaddam B. Targeting metabotropic glutamate receptors for treatment of the cognitive symptoms of schizophrenia. Psychopharmacology (Berl) 2004;174:39–44. [PubMed]
  • Moghaddam B, Adams B, Verma A, Daly D. Activation of glutamatergic neurotransmission by ketamine: A novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J Neurosci. 1997;17:2921–2927. [PubMed]
  • Newcomer JW, Farber NB, Jevtovic-Todorovic V, Selke G, Melson AK, Hershey T, et al. Ketamine-induced NMDA receptor hypofunction as a model of memory impairment and psychosis. Neuropsychopharmacology. 1999;20:106–118. [PubMed]
  • O’Brien JA, Lemaire W, Wittmann M, Jacobson MA, Ha SN, Wisnoski DD, et al. A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain. J Pharmacol Exp Ther. 2004;309:568–577. [PubMed]
  • Olney J, Farber N. Glutamate receptor dysfunction and schizophrenia. Arch Gen Psychiatry. 1995;52:998–1007. [PubMed]
  • Olney JW. Excitotoxic amino acids and neuropsychiatric disorders. Annu Rev Pharmacol Toxicol. 1990;30:47–71. [PubMed]
  • O’Malley KL, Jong YJ, Gonchar Y, Burkhalter A, Romano C. Activation of metabotropic glutamate receptor mGlu5 on nuclear membranes mediates intranuclear Ca2+ changes in heterologous cell types and neurons. J Biol Chem. 2003;278:28210–28219. [PubMed]
  • Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press; 1998.
  • Pilowsky LS, Bressan RA, Stone JM, Erlandsson K, Mulligan RS, Krystal JH, et al. First in vivo evidence of an NMDA receptor deficit in medication-free schizophrenic patients. Mol Psychiatry. 2006;11(2):118–119. [PubMed]
  • Sathyaprakash R, Henry RR. Hyperglycemia with antipsychotic treatment. Curr Diab Rep. 2004;4:41–45. [PubMed]
  • Schwartz TL, Nihalani N, Virk S, Jindal S, Chilton M. Psychiatric medication-induced obesity: Treatment options. Obes Rev. 2004;5:233–238. [PubMed]
  • Seeman P. Dopamine receptors and the dopamine hypothesis of schizophrenia. Synapse. 1987;1:133–152. [PubMed]
  • Sharifullina E, Ostroumov K, Nistri A. Activation of group I metabotropic glutamate receptors enhances efficacy of glutamatergic inputs to neonatal rat hypoglossal motoneurons in vitro. Eur J Neurosci. 2004;20:1245–1254. [PubMed]
  • Snyder S, Banerjee S, Yamamura H. Drugs, neurotransmitters and schizophrenia. Science. 1974;184:1243–1253. [PubMed]
  • Spooren WP, Vassout A, Neijt HC, Kuhn R, Gasparini F, Roux S, et al. Anxiolytic-like effects of the prototypical metabotropic glutamate receptor 5 antagonist 2-methyl-6-(phenylethynyl)pyridine in rodents. J Pharmacol Exp Ther. 2000;295:1267–1275. [PubMed]
  • Stefani MR, Groth K, Moghaddam B. Glutamate receptors in the rat medial prefrontal cortex regulate set-shifting ability. Behav Neurosci. 2003;117:728–737. [PubMed]
  • Stefani MR, Moghaddam B. Systemic and prefrontal cortical NMDA receptor blockade differentially affect discrimination learning and set-shift ability in rats. Behav Neurosci. 2005;119:420–428. [PubMed]
  • Stoop R, Conquet F, Zuber B, Voronin L, Pralong E. Activation of metabotropic glutamate 5 and NMDA receptors underlies the induction of persistent bursting and associated long-lasting changes in CA3 recurrent connections. J Neurosci. 2003;23:5634–5644. [PubMed]
  • Thierry AM, Tassin JP, Blanc G, Glowinski J. Selective activation of mesocortical DA system by stress. Nature. 1976;263:242–244. [PubMed]
  • Tsai G, Yang P, Chung L, Lange N, Coyle J. D-serine added to antipsychotics for the treatment of schizophrenia. Biol Psychiatry. 1998;44:1081–1089. [PubMed]
  • Verma A, Moghaddam B. NMDA receptor antagonists impair pre-frontal cortex function as assessed via spatial delayed alternation performance in rats: Modulation by dopamine. J Neurosci. 1996;16:373–279. [PubMed]
  • Wisniewski K, Car H. (S)-3,5-DHPG: A review. CNS Drug Rev. 2002;8:101–116. [PubMed]