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Positive Allosteric Modulation of mGluR5 Receptors Facilitates Extinction of a Cocaine Contextual Memory

  • Justin T. Gass
    Affiliations
    Center for Drug and Alcohol Programs, Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina
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  • M. Foster Olive
    Correspondence
    Address reprint requests to M. Foster Olive, Ph.D., Center for Drug and Alcohol Programs, Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, 67 President Street, MSC 861, Charleston, SC 29425
    Affiliations
    Center for Drug and Alcohol Programs, Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina
    Search for articles by this author
Published:December 22, 2008DOI:https://doi.org/10.1016/j.biopsych.2008.11.001

      Background

      The perseverance of the motivational salience of drug-associated memories is an obstacle to the successful treatment of drug addiction and is often a causative factor in triggering relapse.

      Methods

      This study was intended to determine whether potentiation of type 5 metabotropic glutamate receptors (mGluR5), which are biochemically and structurally coupled to N-methyl-D-aspartate (NMDA) receptors, would facilitate the extinction of a cocaine-associated contextual memory as assessed by the conditioned place preference (CPP) paradigm in rats. Following the establishment of a cocaine CPP, rats were treated with the mGluR5 positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB; 0.3, 3 and 30 mg/kg) before extinction test sessions. Additional groups of animals received 30 mg/kg CDPPB in combination with the mGluR5 antagonist 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP, 1 mg/kg) or the NMDA receptor antagonist MK-801 (.1 mg/kg).

      Results

      CDPPB dose-dependently facilitated the extinction of cocaine CPP, and these effects were not observed when animals were coadministered MTEP or MK-801. CDPPB failed to produce any evidence of neurotoxicity as assessed by FluoroJade C staining.

      Conclusions

      Positive allosteric modulation of mGluR5 function facilitates the extinction of a cocaine-associated contextual memory, which may represent a novel approach toward enhancing extinction learning in the context of drug addiction.

      Key Words

      The neural substrates of drug addiction have considerable overlap with those that underlie normal learning and memory processes (
      • Kelley A.E.
      Memory and addiction: shared neural circuitry and molecular mechanisms.
      ,
      • Robbins T.W.
      • Ersche K.D.
      • Everitt B.J.
      Drug addiction and the memory systems of the brain.
      ). During the course of addiction, associations between drugs and specific environmental contexts become overly salient, and these drug–environment associations often lead to drug craving and relapse (
      • Childress A.R.
      • McLellan A.T.
      • Ehrman R.
      • O'Brien C.P.
      Classically conditioned responses in opioid and cocaine dependence: A role in relapse?.
      ). Attempts at extinguishing the salience of drug conditioned cues by exposure therapy have been met with limited success primarily because of the context specificity of extinction (
      • Conklin C.A.
      • Tiffany S.T.
      Applying extinction research and theory to cue-exposure addiction treatments.
      ,
      • Bouton M.E.
      • Westbrook R.F.
      • Corcoran K.A.
      • Maren S.
      Contextual and temporal modulation of extinction: Behavioral and biological mechanisms.
      ). Because extinction is an active learning process, it involves many of the neurobiological substrates that subserve normal learning and memory, including increased transmission and plasticity at central glutamatergic synapses. Pharmacologic enhancement of glutamate transmission—for example, with the N-methyl-D-aspartate (NMDA) receptor partial agonist D-cycloserine—facilitates the extinction of conditioned fear as well as drug-associated memories (
      • Botreau F.
      • Paolone G.
      • Stewart J.
      d-Cycloserine facilitates extinction of a cocaine-induced conditioned place preference.
      ,
      • Davis M.
      • Ressler K.
      • Rothbaum B.O.
      • Richardson R.
      Effects of D-cycloserine on extinction: Translation from preclinical to clinical work.
      ). Because type 5 metabotropic glutamate receptors (mGluR5) are structurally and biochemically linked to NMDA receptors and their activation increases NMDA receptor activity (
      • Marino M.J.
      • Conn P.J.
      Direct and indirect modulation of the N-methyl D-aspartate receptor: Potential for the development of novel antipsychotic therapies.
      ), we sought to determine whether potentiation of mGluR5 receptor function by the systemically active mGluR5 positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) (
      • Lindsley C.W.
      • Wisnoski D.D.
      • Leister W.H.
      • O'Brien J.A.
      • Lemaire W.
      • Williams Jr., D.L.
      • 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.
      ) would facilitate extinction of a cocaine contextual memory as measured by the conditioned place preference (CPP) paradigm.

      Methods and Materials

      Animals

      All procedures were conducted with the approval of an Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (200–275 g, Harlan, Indianapolis, Indiana) were maintained on a 12 hour light-dark cycle (lights off at 7:00 am), and all experimentation was conducted during the dark phase. Rats were given ad libitum access to food and water throughout all procedures except during behavioral testing.

      Apparatus

      The CPP apparatus (Med Associates, St. Albans, Vermont) consisted of two adjacent conditioning compartments (20 × 16 × 21 cm) separated by a manual guillotine-type door. One of the conditioning compartments was equipped with vertical striped acrylic walls and a steel mesh floor, and the other was equipped with plain acrylic walls and a wire rod floor. Infrared photobeams monitored the animal's position in the apparatus and provided a measure of motor activity.

      CPP Procedures

      Animals were initially habituated to the testing apparatus during a single 20-min session with free access to both conditioning compartments. Animals were then subjected to a 20-min preconditioning test (Pre) to determine any initial preference for one of the conditioning compartments. Each animal was then assigned to receive cocaine (10 mg/kg) in the initially nonpreferred compartment, and saline in the initially preferred compartment. Next, animals underwent place conditioning in twice-daily 20-min conditioning sessions. Saline-conditioning sessions were conducted in the morning, and cocaine-conditioning sessions were conducted in the afternoon. Following 4 sequential conditioning days, animals were tested for place preference by allowing free access to both conditioning compartments for 20 min (Post). Next, animals underwent extinction testing, whereby they were administered vehicle or CDPPB (.3, 3, or 30 mg/kg) 20 min before being placed in the CPP apparatus and allowed free access to both conditioning compartments for 20 min. Additional groups of animals received CDPPB in combination with the mGluR5 antagonist MTEP (1 mg/kg) or the NMDA receptor antagonist MK-801 (.1 mg/kg), which were administered 10 min before CDPPB. Extinction tests were conducted daily for 5 consecutive days (i.e., E1, E2, E3, etc.).

      Assessment of Neurotoxicity

      Because stimulation of mGluR5 receptors enhances NMDA receptor function, and excessive NMDA receptor activation can lead to neurotoxicity, we assessed potential effects of repeated administration of CDPPB on neurotoxicity and neurodegeneration using FluoroJade C staining (
      • Schmued L.C.
      • Stowers C.C.
      • Scallet A.C.
      • Xu L.
      Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons.
      ). Rats were treated daily with CDPPB at a dose of 30 mg/kg for 5 consecutive days to mimic the treatment regimen during extinction. As a positive control, separate animals received a single administration of kainic acid (10 mg/kg). One hour following the final administration of CDPPB or 2 days following kainic acid administration, rats were anesthetized and perfused transcardially with saline followed by 4% w/v paraformaldehyde. Following postfixation, coronal 25-μm brain sections through the ventromedial prefrontal cortex and CA3 region of the hippocampus were cut on a cryostat and mounted onto gelatin coated slides. Sections were stained with FluoroJade C (Chemicon, Temecula, California) as described elsewhere (
      • Schmued L.C.
      • Stowers C.C.
      • Scallet A.C.
      • Xu L.
      Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons.
      ). Sections were viewed under epifluorescence microscopy at 488-nm excitation using a Leica (Bannockburn, Illinois) DMLB microscope equipped with a digital camera.

      Drugs

      3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) was custom synthesized by IQsynthesis (St. Louis, Missouri; according to ref. 9). CDPPB was suspended in a vehicle consisting of 20% w/v 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, St. Louis, Missouri). 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP, Alexis Biochemicals, San Diego, California) was dissolved in 20% w/v 2-hydroxypropyl-β-cyclodextrin. MK-801 (Tocris Biosciences, Ellisville, Missouri), kainic acid (Tocris), and cocaine hydrochloride (Sigma-Aldrich) were dissolved in sterile .9% saline. CDPPB and its corresponding vehicle were administered via the subcutaneous route; all other compounds were given via the intraperitoneal route. Injections were given in a volume of 1 mL/kg.

      Data Analysis

      Time spent in each conditioning compartment on each test day was converted to percent time spent in the initial nonpreferred compartment as determined in the Pre test for each animal. The CPP and locomotor data were analyzed by a mixed multivariate analysis of variance. Effects of treatment group were assessed using the Wilks Lambda test statistic, and within-subjects effects across test sessions were assessed by repeated-measures analysis of variance. For CPP data, multiple pairwise comparisons with were performed for each treatment group at each extinction test session against Pre values. For locomotor data, multiple pairwise comparisons were performed for each treatment group at each extinction test against vehicle-treated animals during the same test session. All multiple pairwise comparisons used Sidak adjustments. Cocaine CPP was considered to be extinguished when the mean percent time spent in the nonpreferred compartment for a particular treatment group during an extinction test was not statistically different from that observed during the Pre test. Data are presented as mean ± SEM, and the significance level was set at p < .05.

      Results

      Conditioning with cocaine (10 mg/kg) produced a significant place preference in all groups, as evidenced by a significant increase in the percent time spent in the initial nonpreferred side (Figures 1A and 1C). Cocaine CPP was observed to extinguished during the extinction test sessions as follows: vehicle (E5), .3 mg/kg CDPPB (E4), 3 mg/kg CDPPB (E1), 30 mg/kg CDPPB (E2), 30 mg/kg CDPPB + 1 mg/kg MTEP (E5), 30 mg/kg CDPPB + .1 mg/kg MK-801 (CPP was not extinguished after 5 days of extinction testing). Analysis of locomotor data (Figure 1B and 1D) revealed that only animals pretreated with MK-801 exhibited changes in activity, which was increased on extinction test sessions E1–E5.
      Figure thumbnail gr1
      Figure 1Effects of the type-5 metabotropic glutamate receptor (mGluR5) positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the extinction of cocaine conditioned place preference (CPP) and on locomotor activity. Data are presented as mean ± SEM. (A) CDPPB dose-dependently facilitated extinction of cocaine CPP, as evidenced by fewer extinction test sessions required to reach extinction criteria. A significant interaction between treatment group and test session [F(30,458) = 1.70, p < .05] was observed. Sample sizes are n = 21 for vehicle, .3, and 3.0 mg/kg CDPPB, and n = 20 for 30 mg/kg CDPPB. * p < .05 versus pretest. (B) CPDDB had no significant effects on locomotor activity during extinction of CPP (p > .5) (C) The effects of 30 mg/kg CDPPB were reversed by pretreatment with the mGluR5 antagonist 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) or the N-methyl-D-aspartate antagonist MK-801. Sample sizes are n = 21 for vehicle, n = 22 for 30 mg/kg CDPPB + 1 mg/kg MTEP, and n = 21 for 30 mg/kg CDPPB + 0.1 mg/kg MK-801. The vehicle-treated group in panels A and C are the same but are separated for visual clarity purposes. * p < .05 versus pretest. (D) MK-801 increased locomotor activity during each of the five extinction trials, as evidenced by a significant interaction between treatment group and test session [F(30,462) = 4.82, p < .001]. * p < .05 versus vehicle-treated animals. Locomotor activity was not altered during extinction testing in MTEP pretreated animals. Pre, preconditioning test session; Post, post-conditioning test session; E1–E5, extinction test sessions 1–5.
      The effects of repeated administration of the 30 mg/kg dose of CDPPB on neuronal degeneration, as assessed by FluoroJade C staining, are shown in Figure 2. Neurotoxic effects of CDPPB were not observed in the ventromedial prefrontal cortex, CA3 region of the hippocampus, or other brain regions examined (not shown), as evidenced by a lack of fluorescent staining in these regions. In contrast, fluorescent staining was observed in these regions in positive control animals treated with kainic acid.
      Figure thumbnail gr2
      Figure 2Lack of neurotoxic effects of repeated administration of 30 mg/kg 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) as assessed by FluoroJade C staining in the (A) ventromedial prefrontal cortex (CDPPB-vmPFC) and (B) CA3 region of the hippocampus (CDPPB-Hipp CA3). Animals treated with kainic acid (KA) as a positive control demonstrated significant neuronal degeneration as evidenced by FluoroJade C staining (arrows) in the (C) vmPFC (KA-vmPFC) and (D) CA3 region of the hippocampus (KA-Hipp CA3). Scale bar = 500 μm.

      Discussion

      We found that the systemically active mGluR5 positive allosteric modulator CDPPB dose-dependently facilitated the extinction of a cocaine contextual memory, as evidenced by fewer extinction test sessions required to reach extinction criteria. This effect appeared to be mediated by both mGluR5 and NMDA receptors, because the effect of the highest dose of CDPPB tested was reversed by coadministration of MTEP and MK-801, respectively. CDPPB did not influence locomotor activity, nor did it exhibit neurotoxic effects, as animals treated with this compound showed a lack of fluorescent staining with the neurodegeneration marker FluoroJade C (
      • Schmued L.C.
      • Stowers C.C.
      • Scallet A.C.
      • Xu L.
      Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons.
      ). Thus, positive allosteric modulation of mGluR5 receptors may be a novel mechanism by which to facilitate the extinction of drug-associated contextual memories.
      CDPPB potentiates the function of mGluR5 by fourfold to fivefold in the presence of glutamate by acting at an allosteric binding site separate from the orthosteric glutamate binding site (
      • Lindsley C.W.
      • Wisnoski D.D.
      • Leister W.H.
      • O'Brien J.A.
      • Lemaire W.
      • Williams Jr., D.L.
      • 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.
      ,
      • Chen Y.
      • Nong Y.
      • Goudet C.
      • Hemstapat K.
      • de Paulis T.
      • Pin J.P.
      • et al.
      Interaction of novel positive allosteric modulators of metabotropic glutamate receptor 5 with the negative allosteric antagonist site is required for potentiation of receptor responses.
      ). This enhancement of mGluR5 function indirectly increases NMDA receptor function (
      • Marino M.J.
      • Conn P.J.
      Direct and indirect modulation of the N-methyl D-aspartate receptor: Potential for the development of novel antipsychotic therapies.
      ), which may lead to increased synaptic plasticity and facilitated extinction learning, as has been previously shown with the NMDA partial agonist D-cycloserine (
      • Botreau F.
      • Paolone G.
      • Stewart J.
      d-Cycloserine facilitates extinction of a cocaine-induced conditioned place preference.
      ,
      • Davis M.
      • Ressler K.
      • Rothbaum B.O.
      • Richardson R.
      Effects of D-cycloserine on extinction: Translation from preclinical to clinical work.
      ). However, the possibility exists that NMDA receptor antagonism influenced extinction independently of the actions of CDPPB, because animals treated with CDPPB plus MK-801 showed slower rates of extinction as compared with vehicle-treated animals. Other alternative interpretations of these data are that CDPPB inhibits the expression of cocaine CPP or disrupts the reconsolidation of drug-associated memories due to destabilization of contextual memory traces (or both) (
      • Centonze D.
      • Siracusano A.
      • Calabresi P.
      • Bernardi G.
      Removing pathogenic memories: A neurobiology of psychotherapy.
      ,
      • Nader K.
      Memory traces unbound.
      ). However, given that mGluR5 positive allosteric modulators have pro-cognitive effects (
      • Liu F.
      • Grauer S.
      • Kelley C.
      • Navarra R.
      • Graf R.
      • Zhang G.
      • et al.
      ADX47273: A novel metabotropic glutamate receptor 5 selective positive allosteric modulator with preclinical antipsychotic-like and pro-cognitive activities.
      ), enhance synaptic plasticity in the hippocampus (
      • Ayala J.E.
      • Chen Y.
      • Banko J.L.
      • Sheffler D.J.
      • Williams R.
      • Conn P.J.
      mGluR5 positive allosteric modulators facilitate both LTP and LTD induction in the rat hippocampal CA1 region.
      ), and increase performance in spatial learning tasks such as the Morris water maze (
      • Olive M.F.
      • Watson N.L.
      Positive allosteric modulation of mGluR5 receptors improves performance in the Morris water maze.
      ), it is likely that CDPPB facilitates extinction of a cocaine CPP by enhancing extinction learning. The potential neuroanatomic regions that mediate this phenomenon need to be further investigated and likely include the infralimbic prefrontal cortex (
      • Zavala A.R.
      • Weber S.M.
      • Rice H.J.
      • Alleweireldt A.T.
      • Neisewander J.L.
      Role of the prelimbic subregion of the medial prefrontal cortex in acquisition, extinction and reinstatement of cocaine-conditioned place preference.
      ,
      • Hsu E.
      • Packard M.G.
      Medial prefrontal cortex infusions of bupivacaine or AP-5 block extinction of amphetamine conditioned place preference.
      ), the basolateral amygdala (
      • Fuchs R.A.
      • Weber S.M.
      • Rice H.J.
      • Neisewander J.L.
      Effects of excitotoxic lesions of the basolateral amygdala on cocaine-seeking behavior and cocaine conditioned place preference in rats.
      ), and the hippocampus (
      • Meyers R.A.
      • Zavala A.R.
      • Speer C.M.
      • Neisewander J.L.
      Dorsal hippocampus inhibition disrupts acquisition and expression, but not consolidation, of cocaine conditioned place preference.
      ), which mediate extinction learning, stimulus-reward learning, and drug-context associations, respectively.
      We thank Jeffrey Conn and Carrie Jones for technical advice on the use of CDPPB in vivo, and Michael Reynolds, Jr., for technical assistance with the place conditioning studies. Funding for this work was provided by Public Health Service Grant No. DA024355 from the National Institute on Drug Abuse (MFO) and by a T32 Institutional Training Grant No. AA007474 from the National Institute on Alcohol Abuse and Alcoholism (JTG).
      The authors report no biomedical financial interests or potential conflicts of interest.

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