Complex effects of mGluR5 antagonism on sociability and stereotypic behaviors in mice: Possible implications for the pharmacotherapy of autism spectrum disorders

Brain Research Bulletin 86 (2011) 152–158

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Complex effects of mGluR5 antagonism on sociability and stereotypic behaviors in mice: Possible implications for the pharmacotherapy of autism spectrum disorders Jessica A. Burket, Amy L. Herndon, Erin E. Winebarger, Luis F. Jacome, Stephen I. Deutsch ∗ Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, Norfolk, VA, United States

a r t i c l e

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Article history: Received 20 July 2011 Accepted 1 August 2011 Available online 5 August 2011 Keywords: Balb/c mice Autism spectrum disorders Sociability Stereotypies Metabotropic glutamate receptors NMDA receptor MPEP

a b s t r a c t Balb/c mice display deficits of sociability; for example, they show reduced locomotor activity in the presence of an enclosed or freely-moving social stimulus mouse. Transgenic mice with defective or diminished expression of NMDA receptors manifest impaired sociability, while a partial and full agonist of the obligatory glycine co-agonist binding site on the NMDA receptor improved sociability in the Balb/c mouse strain. Because 2-methyl-6-(phenylethynyl)-pyridine (MPEP), an antagonist of the mGluR5 metabotropic glutamate receptor (mGluR), reduced self-grooming behavior in BTBR T+tfJ (BTBR) mice, another inbred genetic mouse model of autism spectrum disorders (ASDs), and mGluR5 antagonism is emerging as an experimental treatment for the ‘fragile X syndrome,” which has a high prevalence of co-morbid ASDs, we examined the effects of MPEP on sociability and stereotypic behaviors in Balb/c and Swiss Webster mice in a standard paradigm. MPEP had complex effects on sociability, impairing some measures of sociability in both strains, while it reduced the intensity of some spontaneous measures of stereotypic behaviors emerging during free social interaction in Swiss Webster mice. Conceivably, mGluR5 antagonism exacerbates diminished endogenous tone of NMDA receptor-mediated neurotransmission in neural circuits relevant to at least some measures of sociability in Balb/c mice; the mGluR5 receptor contributes to regulation of the phosphorylation status of the NMDA receptor. In any event, although stereotypies are an important therapeutic target in ASDs, medication strategies to attenuate their severity via antagonism of mGluR5 receptors must be pursued cautiously because of their potential to worsen at least some measures of sociability. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Currently, there are no approved medications that target the symptom domain of “qualitative impairment in social interaction,” which contributes significantly to the functional disability of persons with autism spectrum disorders (ASDs) [1]. Importantly, defective or diminished expression of the NMDA receptor is associated with quantitative impairments of sociability in transgenic mice [22,25]; also, d-cycloserine and d-serine, a partial and full agonist of the obligatory glycine co-agonist binding site on the NMDA receptor, respectively, improved measures of sociability in mice without known defects of NMDA receptor expression [15,16,24,28]. For example, d-cycloserine improved measures of sociability in

∗ Corresponding author at: Ann Robinson Endowed Chair in Psychiatry, Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, Virginia 23507-1912, United States. Tel.: +1 757 446 5888; fax: +1 757 446 5918. E-mail address: [email protected] (S.I. Deutsch). 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.08.001

both resident and intruder mice in the “resident mouse-intruder mouse model of social interaction [28].” Further, d-cycloserine and d-serine improved quantitative measures of impaired sociability in the genetically-inbred Balb/c mouse that is behaviorally hypersensitive to MK-801 (dizocilpine) [15,16,24], a noncompetitive NMDA receptor antagonist, although its immunoreactive protein content for six of the eight identified splice variant isoforms of the NR1 subunit, and NR2A and NR2B subunits in cerebral cortex and hippocampus did not differ from an outbred comparator strain [31]. Examples of the behavioral hypersensitivity of the Balb/c strain to MK-801 include greater sensitivity to the elicitation of irregular episodes of intense jumping behavior, referred to as “popping;” antagonism of electrically precipitated seizures; and induction of circling behavior, compared to other inbred and outbred comparator strains; these data suggest that the endogenous tone of NMDA receptor-mediated neurotransmission is altered in Balb/c mice, which may be causally related to its impaired sociability [4,6,18,17]. However, the reason for the altered tone may not be at the level of expression of the NMDA receptor itself, but rather due to abnormalities within circuits that utilize NMDA receptors for

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neurotransmission. Also, d-cycloserine promoted pro-socialization “partner preference” in female prairie voles that socially bond with males in this socially monogamous species [29]. Fragile X syndrome (FXS) is associated with a high prevalence of co-morbid ASDs; excessive protein synthesis in basilar dendrites due to absent expression of the “fragile X mental retardation protein (FMRP)” is implicated in the pathophysiology of this neurodevelopmental disorder [3,19,37]. FMRP inhibits translation by complexing with mRNA associated with polyribosomes in basilar dendrites, leading to unopposed stimulation of protein synthesis, mediated by the mGluR5 subtype of group 1 metabotropic receptors, a Gq -protein linked receptor positively coupled to phospholipase C [9,14,35,36]. The ability of 2-methyl6-(phenylethynyl)-pyridine (MPEP), a relatively selective, potent (IC50 value in the 30 nM range), blood–brain barrier permeable noncompetitive mGluR5 antagonist, to dampen protein synthesis in basilar dendrites is given as the explanation of its beneficial effects on the behavioral phenotype and histopathology of the Fmr1 knockout mouse, a transgenic mouse model of FXS deficient in expression of FMRP [20,37,38]. The G protein signaling pathway initiated by stimulation of group 1 mGluRs may also be involved in “cross talk” with NMDA receptors, regulating the sensitivity of NMDA receptors by influencing the phosphorylation status of the S890 serine residue in the intracellular C-terminal domain of the NR1 subunit [2,10,11,26,27,32,35]. Conceivably, MPEP’s ability to affect NMDA receptor activation may also contribute to its beneficial effects in the Fmr1 knockout mouse. MPEP binds to a site in the seven transmembranous hydrophobic domain of the mGluR5 receptor, which is a site that also binds positive allosteric modulators (PAMs) of this metabotropic glutamate receptor [11]; PAMs may potentiate NMDA receptor-mediated neurotransmission. Because MPEP improves the phenotype of transgenic mouse models of FXS [38], and FXS and ASDs are frequent comorbid disorders, MPEP was studied in BTBR T+tf/J (BTBR) mice, a genetically-inbred mouse strain with phenotypic similarities to all of the prominent symptom domains in ASDs, including qualitative impairments in social interaction and communication and “restricted repetitive and stereotyped patterns of behavior” [34]. MPEP significantly reduced repetitive self-grooming behavior in BTBR mice at doses that did not affect locomotor activity in an open field or measures of sociability. The current study explored the effect of MPEP (30 mg/kg, intraperitoneally) on sociability and spontaneously occurring stereotypic behaviors in the inbred Balb/c strain, another mouse model of ASDs, and the outbred comparator Swiss Webster strain [5,7,15,16,24,33] in a standard paradigm; spontaneous stereotypic behaviors were measured while Balb/c and Swiss Webster mice were allowed to interact freely with a social stimulus mouse in this paradigm. Importantly, stereotypies emerging spontaneously during social interaction can be a disabling symptom in persons with ASDs, competing or interfering with the salience of social stimuli [8,21]. The data confirm a beneficial effect of MPEP on spontaneously occurring stereotypic behaviors in the context of a social interaction; however, there were complex effects on sociability in the two test strains, including worsening of at least some measures of sociability. 2. Methods 2.1. Subjects Experimentally-naïve, 8-week old male, outbred Swiss Webster and genetically inbred Balb/c “test” mice (Harlan Laboratories, Frederick, MD) were housed 2 per cage, in hanging clear Plexiglas cages with free access to food and water, and maintained on a 12 h light/dark cycle. The “stimulus” mice were 4-week old male ICR mice, housed 4 per cage. Housing conditions were adopted from prior literature [33]. All animal procedures were approved by the Eastern Virginia Medical School Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Group sizes in each of the

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experimental treatment conditions were 20 animals per group with the exception of the saline-treated Swiss Webster mice, whose group size was equal to 22 animals. 2.2. Drugs MPEP (2-methyl-6-(phenylethynyl)-pyridine; Sigma–Aldrich Co., St. Louis, MO) was dissolved in 0.9% saline and prepared each day of the experiment. MPEP (30 mg/kg or the saline vehicle) was injected intraperitoneally in a volume of 0.01 ml/g of body weight 20 min prior to testing sociability. The dose of MPEP was selected based on its ability to reduce repetitive self-grooming in BTBR mice and modulate the stress-induced reduction of the antiseizure efficacy of MK-801 (dizocilpine) in Balb/c mice [15], [34]). 2.3. Apparatus The three-compartment testing apparatus consisted of a black Plexiglas rectangular box (52.07 cm × 25.40 cm × 22.86 cm), without a top or bottom. The center compartment was slightly smaller (12.07 cm × 25.40 cm) than the two end compartments that were of equal size (19.05 cm × 25.40 cm). Inverted wire cups (Galaxy Cup, Kitchen Plus, http://www.kitchen-plus.com) were placed in each side of the end compartments during sessions I and II (discussed below) and housed the stimulus mouse. 500 ml glass bottles were placed on top of the inverted wire cups to prevent climbing during testing. After each test mouse was studied in the sociability paradigm, the apparatus and wire cups were thoroughly cleaned with Quatricide PV solution. 2.4. Sociability procedure The laboratory adopted an established mouse behavioral procedure for the quantitative assessment of sociability [5,7,12,13,15,16,24,30,33,34]. Briefly, in the first session, a test mouse is placed in the middle compartment and allowed to acclimate to the sociability apparatus for 5 min. In the second 5-min session, a stimulus mouse is enclosed in an inverted wire cup in the side designated as the social compartment, and an empty inverted wire cup is placed in the side designated as the nonsocial compartment. The side designated for the location of the enclosed stimulus mouse is randomly assigned in a counterbalanced fashion throughout the experiment. In the third 5-min session, the stimulus mouse is released from the inverted wire cup, and the test and stimulus mice are allowed to interact freely with each other. All sessions were conducted in dim lighting and videotaped using a Panasonic SDR-S26 SD Video Camera (Panasonic Corp., Osaka, Japan) for future viewing and data collection. The amount of time test mice spend in the social and nonsocial compartments, the amount of time test mice explore (sniffing) within a 2-cm vicinity of the social and nonsocial inverted cups and locomotor activity (i.e., transitions between compartments) is measured in the second 5-min session. The following measures of sociability, stereotypic behaviors and locomotor activity were reliably obtained in the third 5-min session of free interaction between test and stimulus mice and analysed in this report: discrete episodes of social approach; time spent engaged in social pursuit; discrete episodes of rearing; discrete episodes of wall climbing and time spent engaged in self-grooming [23,34]. Social approach is defined as a discrete episode of initiation of sniffing the social stimulus mouse by the test mouse within at least a two-cm vicinity of each other. Social pursuit is defined as the amount of time the test mouse is engaged in following or chasing the social stimulus mouse from initiation of the encounter until both mice separate by a distance of at least two cm. Rearing is defined as a discrete episode of raising forelimbs and standing on hindlimbs. Wall climbing is defined as a discrete episode of raising forelimbs and placing front paws on walls of the sociability apparatus. Grooming is defined as the amount of time the test mouse is engaged in licking and rubbing of fur with forelimbs during the 5-min period of free social interaction (i.e., third 5-min session). A transition between compartments is defined as the number of times all four extremities cross between compartments. 2.5. Statistics A two-way ANOVA was used to examine effects of strain (Balb/c vs. Swiss Webster), treatment condition (MPEP vs. saline), and their interaction in discrete episodes of social approach; discrete episodes of rearing; discrete episodes of wall climbing; and time spent engaged in self-grooming. Fisher’s LSD MultipleComparison post-hoc tests were applied, where appropriate. An inferential statistical technique (e.g., ANOVA) was not used to analyse the data obtained for time engaged in social pursuit due to a floor effect for this dependent variable in one of the cells (i.e., for Balb/c mice treated with MPEP, the time they engaged in social pursuit was 0 s for all cases). Thus, a Pearson Chi-Square test was used to compare the proportion of mice that spent either 0 s or greater than or equal to 5 s engaged in social pursuit as a function of treatment condition (i.e., saline and MPEP). Paired t tests were used for within strain comparisons examining the ability of MPEP to influence the salience of a social stimulus, relative to saline-treated test mice. Independent Samples t-tests were used to conduct exploratory analyses of differences in the salience of the social stimulus between Balb/c and Swiss Web-

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Fig. 1. Effect of MPEP on locomotor activity in sessions II and III. Bars represent means and ±SEM of the number of transitions made between compartments by Balb/c and Swiss-Webster mice in the presence of an enclosed 4 week-old male ICR stimulus mouse (A) and when test and stimulus mice were allowed to interact freely (B) after treatment with saline or MPEP (30 mg/kg). *p < 0.05, **p < 0.01 and ***p < 0.001 compare number of transitions made between compartments within each group and between saline conditions (see Section 3 for details).

ster mice, comparing these test strains in both saline-treated and MPEP-treated conditions.

3. Results ANOVA showed a significant main effect of strain (i.e., Balb/c versus Swiss Webster; F[1,81] = 110.41, p < 0.0001) and treatment condition (i.e., MPEP versus saline; F[1,81] = 28.82, p < 0.0001) on the number of transitions between compartments in the presence of an enclosed social stimulus mouse (Fig. 1, Panel A). Subsequent comparisons using the Fisher’s LSD Multiple Comparison Test showed that MPEP-treated Balb/c mice made a significantly greater number of transitions between compartments in the presence of an enclosed social stimulus mouse (mean = 24.75 ± 2.76 [SEM]) than saline-treated Balb/c mice (mean = 8.45 ± 2.57 [SEM]) (p < 0.001). Moreover, MPEP-treated Swiss Webster mice made a significantly greater number of transitions between compartments in the presence of an enclosed social stimulus mouse (mean = 50.50 ± 3.34 [SEM]) than saline-treated Swiss Webster mice (mean = 38.36 ± 1.83 [SEM]) (p < 0.01). Thus, irrespective of strain, treatment with MPEP was associated with increased locomotor activity in the presence of an enclosed social stimulus mouse, as reflected in the number of transitions made between compartments. Importantly, the data replicate earlier reports that saline-treated Balb/c mice make fewer transitions between compartments than the saline-treated comparator Swiss Webster strain in the presence of an enclosed social stimulus mouse (p < 0.001). ANOVA showed a significant main effect of strain (i.e., Balb/c versus Swiss Webster; F[1,81] = 71.92, p < 0.0001) and treatment condition (i.e., MPEP versus saline; F[1,81] = 6.60, p < 0.05) on the number of transitions between compartments when test mice were allowed to interact freely with stimulus mice (Fig. 1, Panel B). Subsequent comparisons using the Fisher’s LSD Multiple Comparison Test showed that MPEP-treated Swiss Webster mice made a significantly greater number of transitions between compartments (mean = 55.80 ± 5.32 [SEM]) than saline-treated Swiss Webster mice (mean = 44.77 ± 3.06 [SEM]) (p < 0.05). Thus, treatment with MPEP in the Swiss Webster mice was associated with increased locomotor activity during the 5-min session of free interaction, as reflected in the number of transitions made between com-

partments. Importantly, the data replicate earlier reports that saline-treated Balb/c mice make fewer transitions between compartments than the saline-treated comparator Swiss Webster strain during the 5-min session of free interaction with the stimulus mouse (p < 0.001). Differences in time spent by test mice (i.e., Balb/c and Swiss Webster) in the compartment containing an enclosed social stimulus mouse, compared to time spent in the compartment containing the empty inverted cup (Fig. 2) and differences in time spent exploring (i.e., sniffing) an enclosed social stimulus mouse, compared to time spent exploring (i.e., sniffing) the empty inverted cup (Fig. 3) are measures of the salience of the social stimulus mouse. Paired t-tests were used to determine effects of MPEP on the salience of the enclosed social stimulus mouse within the Balb/c and Swiss Webster mouse strains; specifically, for both the saline and MPEP treatment conditions, within-strain comparisons were made with respect to time spent in the compartment containing the enclosed social stimulus mouse and time spent exploring (i.e., sniffing) the enclosed social stimulus mouse. Time spent in the social versus nonsocial compartment (Fig. 2, Panel A) and time spent exploring (i.e., sniffing) the enclosed social stimulus mouse versus the empty inverted cup (Fig. 3, Panel A) did not differ for the saline-treated Balb/c mice (p > 0.05); these data suggest that the social stimulus mouse lacked social salience for Balb/c mice. However, as expected, saline-treated Swiss Webster mice spent significantly more time in the compartment containing the enclosed social stimulus mouse (mean = 175.23 ± 8.08 [SEM]) than the compartment containing the empty inverted cup (mean = 93.91 ± 8.47 [SEM]) (t = −5.053; p < 0.001; Fig. 2, Panel B) and spent more time exploring (i.e., sniffing) the enclosed social stimulus mouse (mean = 108.55 ± 7.66 [SEM]) than the empty inverted cup (mean = 25.00 ± 3.96 [SEM]) (t = −8.121 p < 0.001; Fig. 3, Panel B). Importantly, treatment of Balb/c mice with MPEP resulted in their spending significantly more time exploring (i.e., sniffing) the enclosed social stimulus mouse (mean = 67.95 ± 8.93 [SEM]) than the empty inverted cup (mean = 27.05 ± 3.54 [SEM]) (t = −3.626; p < 0.01; Fig. 3, Panel A); however, time spent in the social versus nonsocial compartment did not differ for the MPEP-treated Balb/c mice (p > 0.05; Fig. 2, Panel A). Independent Samples t-tests showed that saline-treated Swiss Webster mice spent significantly more time exploring (i.e.,

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Fig. 2. Effect of MPEP on time spent in social and nonsocial compartments in session II. Bars represent means and ±SEM of time spent (sec) in the social and nonsocial compartments after treatment with saline or MPEP (30 mg/kg) in Balb/c (A) and Swiss-Webster (B) mice. *p < 0.05 and ***p < 0.001 compare time spent in social and nonsocial compartments within groups (see Section 3 for details).

sniffing) the enclosed social stimulus mouse (mean = 108.55 ± 7.66 [SEM]) than MPEP-treated Balb/c mice (mean = 67.95 ± 8.93 [SEM]) (t = −3.468; p < 0.01), suggesting that even though MPEP increased exploring/sniffing behavior in the Balb/c mice, the behavior was still significantly less than in saline-treated Swiss Webster mice. Moreover, in exploratory analyses comparing MPEP-treated Swiss Webster mice with saline-treated Swiss Webster mice using Independent Samples t-tests, MPEP-treated Swiss Webster mice did not differ from the saline-treated Swiss Webster mice in the amount of time they spent in the compartment containing the enclosed social stimulus mouse (p > 0.05; Fig. 2, Panel B); however, MPEP-treated Swiss Webster mice spent significantly less time exploring (i.e., sniffing) the enclosed social stimulus mouse (mean = 82.05 ± 6.40 [SEM]) than saline-treated Swiss Webster mice (mean = 108.55 ± 7.66 [SEM]) (t = 2.63, p < 0.05; Fig. 3, Panel B). Compared to saline-treated Balb/c mice (N = 20; mean = 6.30 s in pursuit ± 2.05 [SEM]), none of the Balb/c mice (N = 20) treated with

MPEP spent any time engaged in the social pursuit of the stimulus mouse during their 5-min session of free interaction. Because all of the MPEP-treated Balb/c mice spent 0 s engaged in social pursuit, a Chi-Square analysis was performed looking at the proportion of mice that spent either 0 s or greater than or equal to 5 s engaged in social pursuit as a function of treatment condition (i.e., saline versus MPEP), irrespective of strain. The “2 × 2” Table confirmed the impression that a higher proportion of MPEP-treated mice (33 out of 35) compared to the saline-treated mice (14 out of 30) spent 0 s engaged in this quantitative measure of sociability (p < 0.001), irrespective of strain (Table 1). ANOVA showed a significant main effect of strain (i.e., Balb/c versus Swiss Webster; F[1,81] = 18.50, p < 0.001) on the number of discrete episodes of social approach when test mice were allowed to interact freely with stimulus mice (Fig. 4). Subsequent comparisons using the Fisher’s LSD Multiple Comparison Test showed that MPEP-treated Swiss Webster mice made significantly fewer social approaches (mean = 5.6 ± 0.95 [SEM]) than

Fig. 3. Effect of MPEP on time spent exploring/sniffing social and nonsocial inverted cups in session II. Bars represent means and ±SEM of time spent exploring (sniffing) the social and nonsocial inverted cups after treatment with saline or MPEP (30 mg/kg) in Balb/c (A) and Swiss-Webster (B) mice. *p < 0.05 represents a comparison of time spent exploring (sniffing) social and nonsocial inverted cups between saline-treated and MPEP-treated Swiss Webster mice (see Section 3 for details). **p < 0.01 and ***p < 0.001 compare time spent exploring (sniffing) social and nonsocial inverted cups within groups (see Section 3 for details).

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Table 1 Effect of MPEP on Social Pursuit Two by Two Table of Pearson’s Chi-Square Analyses showing the proportions of mice engaged in social pursuit that is either 0 s or greater than or equal to 5 s as a function of treatment with MPEP or saline, irrespective of strain.

Treatment Total

MPEP SALINE

Pursuing (s) 0s

≥5 s

Total

33 14 47

2 16 18

35 30 65

Fig. 4. Effect of MPEP on social approach in session III. Bars represent means and ±SEM of the number of discrete episodes of social approach made by 8-week-old male Balb/c and Swiss-Webster mice towards a 4 week-old male ICR stimulus mouse when test and stimulus mice were allowed to interact freely after treatment with saline or MPEP (30 mg/kg). *p < 0.05 compare number of discrete episodes of social approach made by test mice during session III within and between groups (see Section 3 for details).

saline-treated Swiss Webster mice (mean = 8.05 ± 0.78) (p < 0.05). Consistent with earlier findings, post-hoc comparisons showed that saline-treated Balb/c mice made significantly fewer discrete episodes of social approach (mean = 3.65 ± 0.71 [SEM]) than salinetreated Swiss Webster mice (p < 0.05). ANOVA showed significant main effects of strain (i.e., Balb/c versus Swiss Webster; F[1,81] = 7.34, p < 0.01) and interaction of strain and treatment condition (F[1,81] = 6.01, p < 0.05) on the number of discrete episodes of rearing when test mice were allowed to interact freely with stimulus mice (Fig. 5, Panel A). Subse-

quent comparisons using the Fisher’s LSD Multiple Comparison Test showed that MPEP-treated Swiss Webster mice reared significantly less (mean = 1.55 ± 0.44 [SEM]) than saline-treated Swiss Webster mice (mean = 4.0 ± 0.78 [SEM]) (p < 0.01). Consistent with earlier findings, post-hoc comparisons showed that saline-treated Balb/c mice reared significantly less (mean = 1.0 ± 0.37 [SEM]) than saline-treated Swiss Webster mice (p < 0.001). ANOVA showed significant main effects of strain (i.e., Balb/c versus Swiss Webster; F[1,81] = 27.24, p < 0.001), treatment condition (i.e., MPEP versus saline; F[1,81] = 5.55, p < 0.05) and their interaction (F[1,81] = 5.16, p < 0.05) on the number of discrete episodes of wall climbing when test mice were allowed to interact freely with the stimulus mice (Fig. 5, Panel B). Subsequent comparisons using the Fisher’s LSD Multiple Comparison Test showed that MPEP-treated Swiss Webster mice made significantly fewer discrete episodes of wall climbing (mean = 8.6 ± 2.06 [SEM]) than saline-treated Swiss Webster mice (mean = 16.68 ± 2.24 [SEM]) (p < 0.01). Consistent with earlier findings, post-hoc comparisons showed that saline-treated Balb/c mice made significantly fewer discrete episodes of wall climbing (mean = 3.6 ± 1.18 [SEM]) than saline-treated Swiss Webster mice (p < 0.001). ANOVA showed a significant main effect of strain (i.e., Balb/c versus Swiss Webster) on the number of seconds spent in selfgrooming during the session of free interaction (F[1,81] = 8.64, p < 0.01) (Fig. 5, Panel C). Subsequent comparisons using the Fisher’s LSD Multiple Comparison Test showed that saline-treated Balb/c mice spent significantly less time engaged in grooming than saline-treated Swiss Webster mice (mean = 1.1 s ± 0.48 [SEM] versus mean = 11.5 s ± 3.74 [SEM], respectively; p < 0.05). These data suggest that self-grooming is not a prominent stereotypic behavior manifested by Balb/c mice. However, a within-strain comparison of Swiss Webster mice treated with saline or MPEP (mean = 4.15 s ± 1.24 [SEM]) showed that MPEP reduced the amount of time Swiss Webster mice spent in self-grooming (p < 0.05).

4. Discussion Very importantly, our data show that MPEP had complex effects on several measures of sociability. For example, MPEP increased one measure of the salience of the social stimulus mouse for the Balb/c strain (i.e., increased time spent sniffing the enclosed social stimulus mouse), but did not affect the amount of time Balb/c mice spent in the compartment containing the enclosed social stimulus mouse. Moreover, there was the suggestion that MPEP could worsen social exploration in the Swiss Webster strain, as reflected

Fig. 5. Effect of MPEP on stereotypic behaviors in session III. Bars represent means and ±SEM of the number of discrete episodes of rearing (A), the number of discrete episodes of wall climbing (B), and the number of seconds engaged in grooming (C) by 8-week-old male Balb/c and Swiss-Webster mice after treatment with saline or MPEP (30 mg/kg), when test and stimulus mice were allowed to interact freely. *p < 0.05, **p < 0.01 and ***p < 0.001 compare number of discrete episodes of stereotypic behavior (i.e., rearing [A], wall climbing [B]) or number of seconds engaged in grooming (C) by test mice during session III within and between groups (see Section 3 for details).

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in less time MPEP-treated Swiss Webster mice spent engaged in sniffing the enclosed social stimulus mouse. When test and stimulus mice interacted freely with each other, relative to the saline-treated condition, MPEP reduced the number of discrete episodes of social approach by Swiss Webster mice in the presence of a freely-behaving stimulus mouse. Remarkably, our data show that none of the Balb/c mice treated with MPEP spent any time socially pursuing a stimulus mouse; moreover, irrespective of strain, MPEP significantly decreased the proportion of mice that spent greater than or equal to 5 s engaged in the pursuit of a stimulus mouse. Importantly, time spent by a test mouse in “social pursuit” of a stimulus mouse during their 5-min session of free interaction is an operationally-defined measure of sociability that can be reliably rated [23]. The data on transitions between compartments in the presence of an enclosed and freely moving social stimulus mouse made by both strains of test mice confirm earlier findings of [34] that MPEP (30 mg/kg, ip) does not cause a reduction of locomotor activity; in fact, in the current study, MPEP increased locomotor activity in both strains in the presence of the enclosed social stimulus mouse, and increased locomotor activity in Swiss Webster mice when they interacted freely with stimulus mice. Thus, MPEP’s reduction of time spent in social pursuit by both strains and reduction of discrete episodes of social approach in Swiss Webster mice during their free interaction with stimulus mice is not likely to be an epiphenomenon of a reduction in locomotor activity, in general, by MPEP. Rather, in spite of MPEP’s ability to increase locomotor activity in the presence of a social stimulus, it did adversely affect two measures of sociability during the 5-min period of free interaction with a social stimulus. Although development of mGluR5 antagonism as a medication strategy for ASDs may be problematic with respect to sociability as the primary target, the data suggest that it does have promise for the treatment of stereotypic behaviors. In the current study, MPEP decreased stereotypic behaviors in Swiss Webster mice, which was not due to a dampening effect on the locomotor activity of this strain. These data could suggest differences in the circuitry mediating MPEP-induced changes in locomotor activity, some dimensions of sociability and stereotypies. In any event, the data support exploration of mGluR5 antagonism for the treatment of stereotypic behaviors, which can be severe and socially disabling in autism [8,21,34]. Although MPEP diminishes self-grooming in some strains of mice [34], including the outbred Swiss Webster comparator strain, its ability to worsen two measures of sociability in mice suggests that a medication strategy designed to antagonize mGluR5 may have undesired effects on this core symptom domain of ASDs. Additionally, our data show that quantitative impairments of sociability can be manifested independently of spontaneous stereotypic behaviors. Specifically, the Balb/c mouse strain shows profound impairments on several discrete quantitative measures of sociability, while the intensity of several spontaneously observed stereotypic behaviors during social interaction is less than that observed in the outbred Swiss Webster comparator strain [7,15,16,23]. The mGluR5 receptor has a close functional relationship with the NMDA receptor; stimulation of mGluR5 may increase the extent to which a specific intracytoplasmic serine residue on the NR1 subunit of the NMDA receptor is phosphorylated [35]. Thus, MPEP, which binds to a specific transmembranous hydrophobic domain of mGluR5 that also binds certain PAMs of this G-protein coupledreceptor (GPCR), may disrupt the functional coupling between these two receptors [2,10,11,26,35]. In fact, because impaired sociability is associated with defective or diminished expression of NMDA receptors, MPEP’s disruption of cross-talk between these metabotropic and ionotropic glutamate receptors, which may be necessary for optimal functioning of NMDA receptors, may be the

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mechanism by which it worsens measures of sociability in both Balb/c and Swiss Webster mice. However, it must be emphasized that the effects of MPEP are complex and inconsistent, improving measures thought to reflect the salience social stimuli have for the test mice in this paradigm. Given the fact that PAMs may potentiate cross-talk between mGluR5 and NMDA receptors and MPEP binds to a site on mGluR5 shared with certain PAMs [2,10,11,26], it may be profitable to explore the effects of PAMs of mGluR5 on sociability in Balb/c mice. In any event, although promising, development of mGluR5 antagonists for the treatment of the domain of “restricted repetitive and stereotyped patterns of behavior” in persons with ASDs must be pursued cautiously.

Acknowledgement The authors acknowledge the generous support that they receive from the Office of the Dean of the Eastern Virginia Medical School.

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