d -cycloserine improves sociability and spontaneous stereotypic behaviors in 4-week old mice

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Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report D-cycloserine

improves sociability and spontaneous stereotypic behaviors in 4-week old mice

Stephen I. Deutscha,⁎, Gerald J. Pepeb , Jessica A. Burketa , Erin E. Winebargera , Amy L. Herndona , Andrew D. Bensona a

Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, Norfolk Virginia, USA Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk Virginia, USA

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Balb/c mice are a model of impaired sociability and social motivation relevant to autism

Accepted 19 December 2011

spectrum disorders (ASDs). Impaired sociability of 8-week old Balb/c mice is attenuated by

Available online 29 December 2011

agonists of the glycineB site on the NMDA receptor, such as D-cycloserine. Although ASDs are often recognized in toddlerhood, there is interest in earlier identification (e.g., before

Keywords:

6 months) and disease-modifying interventions to improve functional outcomes. Thus,

D-cycloserine

we wondered if D-cycloserine could improve sociability in 4-week old Balb/c mice, similar

NMDA receptor

to its effects in 8-week old mice. D-Cycloserine improved measures of impaired sociability

Sociability

in 4-week old (i.e., one-week post-weanling) Balb/c mice. Moreover, because stereotypies

Stereotypies

can compete with the salience of social stimuli, we compared Balb/c and Swiss Webster

Balb/c mice

mice on several spontaneous stereotypic behaviors emerging during social interaction with a social stimulus mouse. Interestingly, similar to 8-week old mice, spontaneous stereotypic behaviors during social interaction were more intense in the 4-week old Swiss Webster mice; furthermore, D-cycloserine reduced their intensity. Thus, D-cycloserine improves both sociability and stereotypic behaviors, but these effects may lack strainselectivity. A prosocial effect of D-cycloserine was observed at a dose as low as 32.0 mg/kg in Balb/c mice.

D-cycloserine

has the therapeutic properties of a desired medication for

ASDs; specifically, a medication should not improve stereotypic behaviors at the expense of worsening sociability and vice versa. The data suggest that targeting the NMDA receptor can have promising therapeutic effects on two prominent domains of psychopathology in ASDs: impaired sociability and spontaneous stereotypic behaviors. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

The genetically inbred Balb/c mouse has emerged as a model of impaired sociability and social motivation relevant to autism spectrum disorders (ASDs) (Brodkin et al., 2004; Burket et al., 2010b; Deutsch et al., 2011; Jacome et al., 2011a,b,c;

Sankoorikal et al., 2006); moreover, several quantitative measures of its impaired sociability improved in 8-week old young adult mice after treatment with D-cycloserine and Dserine, a partial and full glycineB agonist, respectively, of the strychnine-insensitive glycine binding site on the NMDA receptor (Deutsch et al., 2011; Jacome et al., 2011a,b). The data

⁎ Corresponding author at: Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507-1912, USA. Fax: +1 757 446 5918. E-mail address: [email protected] (S.I. Deutsch). 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.12.040

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on D-cycloserine and D-serine improving the impaired sociability of Balb/c mice (Deutsch et al., 2011; Jacome et al., 2011a,b) are consistent with prior work in transgenic mice that showed an association between impaired sociability and both diminished expression of the NR1 subunit and expression of an NR1 subunit with fivefold reduction of its affinity for glycine (Halene et al., 2009; Labrie et al., 2008). The data in mice and recent data on facilitation of partner preference in the prairie vole by D-cycloserine implicate the NMDA receptor in regulation of sociability and support targeting this receptor as a pharmacotherapeutic strategy to improve sociability (Deutsch et al., 2011; Halene et al., 2009; Jacome et al., 2011a,b; Labrie et al., 2008; McAllister, 1994; Modi and Young, 2011). Because ASDs are neurodevelopmental disorders, whose recognition can occur within the first three years of life, and because early intervention may have disease-modifying effects that lead to improved functional outcomes, we wondered if D-cycloserine could improve deficits in sociability in younger 4-week old (i.e., one-week postweanling) Balb/c mice (Brodkin et al., 2004; Sankoorikal et al., 2006). Finally, because stereotypies observed spontaneously during social interaction can be a disabling symptom (Campbell et al., 1990; Goldman et al., 2008), we wanted to evaluate effects of D-cycloserine on this domain of psychopathology in 4-week old Balb/c and the outbred comparator 4week old Swiss Webster mice. The current experiments examined sociability and the effects of D-cycloserine (320 mg/kg, intraperitoneally) in 4week old Balb/c mice and the comparator strain of 4-week old Swiss Webster mice in a standard paradigm (Brodkin, 2007; Burket et al., 2010b; Crawley, 2004, 2007; Deutsch et al., 2011; Jacome et al., 2011a; Moy et al., 2007; Sankoorikal et al., 2006). Also, the 4-week old strains were compared to each other on several measures of spontaneously observed stereotypies during their 5-minute session of social interaction with 4-week old ICR social stimulus mice in a standard paradigm. The measurement of spontaneously observed stereotypies during free social interaction and assessment of drug effects on them is of interest because stereotypic behaviors in persons with ASDs often emerge during social interactions, interfering and/or competing with attention to salient social cues. Importantly, the data replicated the sociability deficit in 4week old Balb/c mice and showed that D-cycloserine improved several measures of impaired sociability in these younger Balb/c mice and 4-week old Swiss Webster mice (Deutsch et al., 2011; Sankoorikal et al., 2006). Also, similar to the 8-week old young adult mice, the 4-week old Swiss Webster comparator strain displayed more intense spontaneous stereotypic behaviors during social interaction than the 4-week old Balb/ c strain, which were attenuated by D-cycloserine. Because prosocial effects of the 320 mg/kg dose of D-cycloserine were observed in the test mice, we wondered about dose-dependent effects and whether sociability outcome measures differed in their sensitivity to prosocial effects of D-cycloserine; thus, we explored a range of D-cycloserine doses (i.e., 32.0, 56.0 100.0 and 180.0 mg/kg). A positive prosocial effect of D-cycloserine was observed at a dose as low as 32.0 mg/kg in Balb/c mice. Thus, although impaired sociability and spontaneous stereotypic behaviors observed during social interactions are dissociable dimensions of ASD psychopathology in Balb/c mice

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(Jacome et al., 2011c), D-cycloserine improves sociability and attenuates the severity of stereotypic behaviors; however, these effects may lack strain-selectivity.

2.

Results

The number of transitions between compartments is a measure of locomotor activity; analyses explored effects of strain and treatment condition on this measure of locomotor activity during session I. In session I, D-cycloserine and salinetreated Balb/c and comparator Swiss Webster mice were allowed to acclimate to the sociability apparatus for 5 min. A two-way ANOVA showed significant main effects for strain (i.e., Balb/c versus Swiss Webster; F[1,79] = 11.55, p < 0.01) and treatment condition (i.e., D-cycloserine versus saline; F[1,79] = 21.94, p < 0.0001) on a number of transitions between compartments (Fig. 1, panel A). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that salinetreated Balb/c mice (26.75 ± 2.67 [SEM]) made fewer transitions between compartments than saline-treated Swiss Webster mice (42.75 ± 2.77 [SEM]) (p < 0.01). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of Balb/c and Swiss Webster mice with D-cycloserine (320 mg/kg, ip) increased their number of transitions between compartments (47.05 ± 3.93 [SEM] and 53.80 ± 3.82 [SEM], respectively), relative to saline-treated Balb/c (p < 0.001) and Swiss Webster mice (p < 0.05), respectively. The number of transitions between compartments did not differ between Dcycloserine-treated Balb/c mice and saline-treated Swiss Webster mice (p > 0.05). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer transitions between compartments than saline-treated Swiss Webster mice (p < 0.01). Post-hoc comparisons with the Tukey– Kramer Multiple Comparison Test also showed that treatment of Balb/c mice with D-cycloserine (320 mg/kg, ip) increased their number of transitions between compartments, relative to saline-treated Balb/c mice (p < 0.001). Moreover, using the Tukey–Kramer Multiple Comparison Test, the number of transitions between compartments did not differ between D-cycloserine-treated Balb/c or Swiss Webster mice when compared to saline-treated Swiss Webster mice (p> 0.05). The number of transitions between compartments is a measure of locomotor activity; analyses explored effects of strain and treatment condition on this measure of locomotor activity during session II. When stimulus mice were enclosed, a two-way ANOVA showed significant main effects for strain (i.e., Balb/c versus Swiss Webster; F[1,79] = 25.30, p < 0.0001) and treatment condition (i.e., D-cycloserine versus saline; F[1,79] = 43.09, p < 0.0001) on number of transitions between compartments (Fig. 1, panel B). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Balb/c mice made fewer transitions between compartments when the stimulus mouse was enclosed (8.75± 2.99 [SEM]) than saline-treated Swiss Webster mice (22.15 ±2.14 [SEM]) (p < 0.01). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of Balb/c and Swiss Webster mice with D-cycloserine (320 mg/kg, ip) increased their number of transitions between compartments (26.75 ±3.03

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Fig. 1 – Effect of D-cycloserine on locomotor activity. Bars represent means and ±SEM of the number of transitions made between compartments by 4-week-old male Balb/c and Swiss Webster mice during acclimation (panel A), in the presence of an enclosed 4 week-old male ICR stimulus mouse (panel B) and when test and stimulus mice were allowed to interact freely (panel C) 20 min after treatment with saline or D-cycloserine (320 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 using the Fisher's LSD Multiple Comparison Test. # p < 0.05, ##p < 0.01 and ###p < 0.001 compare number of transitions made between compartments within each group and between saline conditions using the Tukey–Kramer Multiple Comparison Test. n = 20 for all groups.

[SEM] and 43.50± 3.64 [SEM], respectively), relative to salinetreated Balb/c and Swiss Webster mice, respectively (p < 0.001). The number of transitions between compartments did not differ between D-cycloserine-treated Balb/c mice and saline-treated Swiss Webster mice (p > 0.05). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer transitions between compartments than saline-treated Swiss Webster mice (p < 0.05). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Balb/c and Swiss Webster mice with D-cycloserine (320 mg/kg, ip) increased their number of transitions between compartments, relative to saline-treated Balb/c and Swiss Webster mice, respectively (p<0.001). Moreover, using the Tukey–Kramer Multiple Comparison Test, the number of transitions between compartments did not differ between D-cycloserine-treated Balb/c mice and saline-treated Swiss Webster mice (p>0.05). The number of transitions between compartments is a measure of locomotor activity; analyses explored effects of strain and treatment condition on this measure of locomotor activity during session III. When test (i.e., Balb/c and Swiss Webster) and stimulus mice were allowed to interact freely, a two-way ANOVA showed significant main effects for strain (i.e., Balb/c versus Swiss Webster; F[1,79] = 32.98, p < 0.0001) and treatment condition (i.e., D-cycloserine versus saline; F [1,79] = 52.70; p < 0.0001) on number of transitions between compartments (Fig. 1, panel C). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Balb/c mice made fewer transitions between compartments when test and stimulus mice interacted freely (1.4 ± 1.20 [SEM]) than saline-treated Swiss Webster mice (33.30 ± 2.55 [SEM]) (p < 0.001). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of Balb/c and Swiss Webster mice with D-cycloserine

(320 mg/kg, ip) increased their number of transitions between compartments (39.70 ± 6.11 [SEM] and 56.25 ± 5.09 [SEM], respectively), relative to saline-treated Balb/c and Swiss Webster mice, respectively (p < 0.001). The number of transitions between compartments did not differ between D-cycloserinetreated Balb/c mice and saline-treated Swiss Webster mice (p > 0.05). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer transitions between compartments when test and stimulus mice interacted freely than saline-treated Swiss Webster mice (p < 0.001). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Balb/c or Swiss Webster mice with D-cycloserine (320 mg/kg, ip) increased their number of transitions between compartments, relative to salinetreated Balb/c (p<0.001) and Swiss Webster mice (p<0.01), respectively. Moreover, using the Tukey–Kramer Multiple Comparison Test, the number of transitions between compartments did not differ between D-cycloserine-treated Balb/c mice and salinetreated Swiss Webster mice (p>0.05). Effects of strain and treatment condition on discrete episodes of operationally-defined social approach during session III were analyzed. When test (i.e., Balb/c and Swiss Webster) and stimulus mice were allowed to interact freely, a twoway ANOVA showed significant main effects for treatment condition (i.e., D-cycloserine versus saline; F[1,79] = 43.60; p < 0.0001) and the interaction of strain and treatment condition (F[1,79] = 10.12, p < 0.01) on discrete episodes of social approach made by the test mice (Fig. 2). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of social approach when test and stimulus mice interacted freely (0.95 ± 0.66 [SEM]) than saline-treated Swiss Webster mice (7.00 ± 1.03 [SEM]) (p < 0.01). Post-hoc comparisons with the

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Fig. 2 – Effect of D-cycloserine on social approach in session III. Bars represent means and ±SEM of the number of discrete episodes of social approach made by 4-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 20 min after treatment with saline or D-cycloserine (320 mg/kg). *p<0.05, **p<0.01 and ***p<0.001 compare number of discrete episodes of social approach made by test mice during session III within and between groups. # p<0.05 and ###p<0.001 compare number of transitions made between compartments within each group and between saline conditions using the Tukey–Kramer Multiple Comparison Test. n=20 for all groups.

Fisher's LSD Multiple-Comparison Test showed that treatment of Balb/c and Swiss Webster mice with D-cycloserine (320 mg/kg, ip) increased their discrete episodes of social approach (15.25±2.28 [SEM] and 12.00±1.36 [SEM], respectively), relative to saline-treated Balb/c (p<0.001) and saline-treated Swiss Webster (p<0.05) mice, respectively. Interestingly, D-cycloserinetreated Balb/c mice made a significantly greater number of discrete episodes of social approach than saline-treated Swiss Webster mice (p< 0.001). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of social approach when test and stimulus mice interacted freely than saline-treated Swiss Webster mice (p< 0.05). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Balb/c mice with D-cycloserine (320 mg/kg, ip) increased their discrete episodes of social approach, relative to saline-treated Balb/c mice (p < 0.001). Finally, post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test showed that D-cycloserine-treated Balb/c mice made a significantly greater number of discrete episodes of social approach than saline-treated Swiss Webster mice (p< 0.001). When test (i.e., Balb/c or Swiss Webster) and stimulus mice were allowed to interact freely, the mean number of discrete episodes of social avoidance made by all of the Swiss Webster mice treated with D-cycloserine (N = 20) was 0.00 (Fig. 3). Because of the absence of variance in this measure for this

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group (i.e., D-cycloserine-treated Swiss Webster mice), a twoway ANOVA could not be performed; thus, separate exploratory comparisons of mean values for number of discrete episodes of social avoidance between groups were conducted with Independent Samples t-tests (Fig. 3). Specifically, Independent Samples t-tests showed that D-cycloserine significantly reduced the number of discrete episodes of social avoidance made by Balb/c mice, relative to saline-treated Balb/c mice (t = 3.671; p < 0.001). Importantly, Independent Samples t-tests showed that saline-treated Balb/c mice made a significantly greater number of discrete episodes of social avoidance than saline-treated Swiss Webster mice (t = 4.947; p < 0.001). The number of discrete episodes of social avoidance made by D-cycloserine-treated Balb/c mice did not differ from the number of discrete episodes of social avoidance made by saline-treated Swiss Webster mice (t = 1.396; p > 0.05). 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. 4) 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. 5) are measures of the salience of the social stimulus mouse. Paired t-tests were used to determine effects of D-cycloserine on the salience of the enclosed social stimulus mouse for Balb/c and Swiss Webster mice; for both the saline and D-cycloserine treatment conditions, withinstrain 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

Fig. 3 – Effect of D-cycloserine on discrete episodes of social avoidance in session III. Bars represent means and ±SEM of the number of discrete episodes of social avoidance made by 4-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 20 min after treatment with saline or D-cycloserine (320 mg/kg). ***p < 0.001 compare number of discrete episodes of social avoidance made by test mice during session III within and between groups. n = 20 for all groups.

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Fig. 4 – Effect of D-cycloserine on time spent in social and nonsocial compartments in session II. Bars represent means and ±SEM of time spent (s) in the social and nonsocial compartments 20 min after i.p. treatment with saline or D-cycloserine (320 mg/kg) in Balb/c (panel A) and Swiss Webster (panel B) mice. *p < 0.05, **p < 0.01 and ****p < 0.0001 compare time spent in social and nonsocial compartments within groups. n = 20 for all groups.

compartment (Fig. 4, panel A) and time spent exploring (i.e., sniffing) the enclosed social stimulus mouse versus the empty inverted cup (Fig. 5, panel A) did not differ for the salinetreated Balb/c mice (p> 0.05); these data suggest that the social stimulus mouse lacked social salience for the 4-week old Balb/ c mice. However, as expected, saline-treated Swiss Webster mice spent significantly more time in the compartment containing the enclosed social stimulus mouse than the compartment containing the empty inverted cup (t = −2.761; p < 0.05; Fig. 4, panel B) and spent more time exploring (i.e., sniffing) the enclosed social stimulus mouse than the empty inverted cup (t = −2.761; p < 0.05; Fig. 5, panel B). Importantly, treatment of Balb/c mice with D-cycloserine resulted in their spending significantly more time in the compartment containing the

enclosed social stimulus mouse (t = −3.225; p < 0.01; Fig. 4, panel A) and exploring (i.e., sniffing) the enclosed social stimulus mouse (t=−6.393; p<0.0001; Fig. 5, panel A); these data suggest that D-cycloserine increased the salience of the social stimulus mouse for the Balb/c strain. Not surprisingly, D-cycloserinetreated Swiss Webster mice spent significantly more time in the compartment containing an enclosed social stimulus mouse (t = −9.255; p < 0.0001; Fig. 4, panel B) and significantly more time exploring (i.e., sniffing) the enclosed social stimulus mouse (t = −6.622; p < 0.0001; Fig. 5, panel B). Effects of strain and treatment condition on discrete episodes of operationally-defined anogenital sniffing during session III were analyzed. When test (i.e., Balb/c or Swiss Webster) and stimulus mice were allowed to interact freely,

Fig. 5 – Effect of D-cycloserine 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 20 min after treatment with saline or D-cycloserine (320 mg/kg) in Balb/c (panel A) and Swiss Webster (panel B) mice. *p < 0.05, ***p < 0.001 and ****p < 0.00001 compare time spent exploring (sniffing) social and nonsocial inverted cups within groups. n = 20 for all groups.

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a two-way ANOVA showed significant main effects for treatment condition (i.e., D-cycloserine versus saline; F[1,79] = 24.20; p < 0.0001) and the interaction of strain and treatment condition (F[1,79] = 4.91; p < 0.05) on number of discrete episodes of anogenital sniffing (Fig. 6). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that discrete episodes of anogenital sniffing when test and stimulus mice interacted freely did not differ between salinetreated Balb/c (0.70 ± 0.32 [SEM]) and saline-treated Swiss Webster mice (2.25 ± 0.45 [SEM]) (p > 0.05). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of Balb/c mice with D-cycloserine increased their discrete episodes of anogenital sniffing (5.85 ± 1.07 [SEM]), relative to saline-treated Balb/c mice (p< 0.001). Finally, D-cycloserinetreated Balb/c mice made a significantly greater number of discrete episodes of anogenital sniffing than saline-treated Swiss Webster mice (p< 0.001). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that discrete episodes of anogenital sniffing when test and stimulus mice interacted freely did not differ between salinetreated Balb/c and saline-treated Swiss Webster mice (p> 0.05). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Balb/c mice with D-cycloserine increased their discrete episodes of anogenital sniffing, relative to saline-treated Balb/c mice (p< 0.001). Finally, post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test showed that D-cycloserine-treated Balb/c mice made a significantly greater number of discrete episodes of anogenital sniffing than saline-treated Swiss Webster mice (p< 0.01).

Fig. 6 – Effect of D-cycloserine on discrete episodes of anogenital sniffing in session III. Bars represent means and ±SEM of the number of discrete episodes of anogenital sniffing made by 4-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 20 min after treatment with saline or D-cycloserine (320 mg/kg). ***p<0.001 compare number of discrete episodes of anogenital sniffing made by test mice during session III within and between groups. ##p<0.01 and ### p <0.001 compare number of transitions made between compartments within each group and between saline conditions using the Tukey–Kramer Multiple Comparison Test. n=20 for all groups.

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Effects of strain and treatment condition on discrete episodes of operationally-defined rearing during session III were analyzed. When test (i.e., Balb/c or Swiss Webster) and stimulus mice were allowed to interact freely for 5-min, a two-way ANOVA showed significant main effects for strain (i.e., Balb/c and Swiss Webster; F[1,79] = 14.02, p < 0.001), treatment condition (i.e., D-cycloserine and saline; F[1,79] = 10.46, p < 0.01) and their interaction (F[1,79] = 12.78, p < 0.001) on discrete episodes of rearing (Fig. 7, panel A). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of rearing when test and stimulus mice interacted freely (0.25 ± 0.10 [SEM]) than saline-treated Swiss Webster mice (4.55 ± 1.12 [SEM]) (p < 0.001). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of Swiss Webster mice with D-cycloserine reduced their number of discrete episodes of rearing (0.55 ± 0.31 [SEM]), relative to saline-treated Swiss Webster mice (p < 0.001). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of rearing when test and stimulus mice interacted freely than saline-treated Swiss Webster mice (p < 0.001). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Swiss Webster mice with D-cycloserine reduced their number of discrete episodes of rearing, relative to saline-treated Swiss Webster mice (p < 0.001). Effects of strain and treatment condition on discrete episodes of operationally-defined wall climbing during session III were analyzed. When test (i.e., Balb/c or Swiss Webster) and stimulus mice were allowed to interact freely for 5-min, a two-way ANOVA showed significant main effects for strain (i.e., Balb/c and Swiss Webster; F[1,79] = 48.80, p < 0.0001), treatment condition (i.e., D-cycloserine and saline; F[1,79] = 7.17, p < 0.01) and their interaction (F[1,79] = 16.72, p < 0.001) on discrete episodes of wall climbing (Fig. 7, panel B). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of wall climbing when test and stimulus mice interacted freely (2.05 ± 1.14 [SEM]) than saline-treated Swiss Webster mice (31.1 ± 4.12 [SEM]) (p < 0.001). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of Swiss Webster mice with D-cycloserine reduced their number of discrete episodes of wall climbing (13.35 ± 2.81 [SEM]), relative to saline-treated Swiss Webster mice (p < 0.001). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of wall climbing when test and stimulus mice interacted freely than saline-treated Swiss Webster mice (p < 0.001). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Swiss Webster mice with D-cycloserine reduced their number of discrete episodes of wall climbing, relative to saline-treated Swiss Webster mice (p < 0.001). Effects of strain and treatment condition on discrete episodes of operationally-defined grooming during session III were analyzed. When test (i.e., Balb/c or Swiss Webster) and stimulus mice were allowed to interact freely, a two-way ANOVA showed significant main effects for treatment

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Fig. 7 – Effect of D-cycloserine on stereotypic behaviors in session III. Bars represent means and ±SEM of the number of discrete episodes of rearing (panel A), wall climbing (panel B), and grooming (panel C) made by 4-week-old male Balb/c and Swiss Webster mice, 20 min after treatment with saline or D-cycloserine (320 mg/kg), when test and stimulus mice were allowed to interact freely. **p < 0.01 and ***p < 0.001 compare number of discrete episodes of stereotypic behavior (i.e., rearing [panel A], wall climbing [panel B] or grooming [panel C]) made by test mice during session III within and between groups. #p < 0.05, ## p < 0.01 and ###p < 0.001 compare number of transitions made between compartments within each group and between saline conditions using the Tukey–Kramer Multiple Comparison Test. n = 20 for all groups.

condition (i.e., D-cycloserine and saline; F[1,79]=6.85, p<0.05) and the interaction of strain and treatment condition (F[1,79] =5.99, p<0.05) on discrete episodes of grooming (Fig. 7, panel C). Posthoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of grooming when test and stimulus mice interacted freely (0.70±0.28 [SEM]) than saline-treated Swiss Webster mice (1.90±0.48 [SEM]) (p<0.01). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that treatment of

Swiss Webster mice with D-cycloserine reduced their number of discrete episodes of grooming (0.40±0.17 [SEM]), relative to saline-treated Swiss Webster mice (p<0.001). Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed that saline-treated Balb/c mice made fewer discrete episodes of grooming when test and stimulus mice interacted freely than saline-treated Swiss Webster mice (p <0.05). Post-hoc comparisons with the Tukey–Kramer Multiple Comparison Test also showed that treatment of Swiss Webster

Fig. 8 – Exploration of possible dose-dependent effects of D-cycloserine on social approach in session III. Bars represent means and ± SEM of the number of discrete episodes of social approach made by 4-week old male Balb/c (panel A) and Swiss Webster (panel B) mice towards a 4-week old ICR stimulus mouse when test and stimulus mice were allowed to interact freely 20 min after treatment with saline or D-cycloserine (32.0, 56.0, 100.0 or 180.0 mg/kg). *p < 0.05, **p < 0.01 represent post-hoc comparisons using Fisher's LSD Multiple Comparison Tests of the number of discrete episodes of social approach made by Swiss Webster mice treated with D-cycloserine versus Swiss Webster mice treated with saline during session III. Group sizes for all conditions were n = 16 with the exception of the saline (n = 14) and 56 mg/kg D-cycloserine-treated (n= 15) Balb/c mice.

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Fig. 9 – Exploration of possible dose-dependent effects of D-cycloserine on time spent in social and nonsocial compartments in session II. Bars represent means and ±SEM of time spent (s) in the social and nonsocial compartments 20 min after i.p. treatment with saline or D-cycloserine (32.0, 56.0, 100.0 or 180.0 mg/kg) in Balb/c (panel A) and Swiss Webster (panel B) mice. Paired t-tests were significant for all conditions in Swiss Webster mice; however, no significant differences were found in Balb/c mice (n= 16 for all groups). **p < 0.01, ***p < 0.001 compare time spent in social versus nonsocial compartments using paired t-tests.

mice with D-cycloserine reduced their number of discrete episodes of grooming, relative to saline-treated Swiss Webster mice (p<0.01). Possible dose-dependent effects of a range of D-cycloserine doses (i.e., 32.0, 56.0, 100.0 and 180.0 mg/kg) on discrete episodes of operationally-defined social approach in session III were explored separately in Balb/c and Swiss Webster mice (Fig. 8). A one-way ANOVA showed a significant main effect of treatment condition in the Swiss Webster strain (F[1,79] = 2.45, p = 0.05), whereas there was no significant main effect of treatment condition in the Balb/c strain (F[1,77] = 1.84, p > 0.05). Post-hoc comparisons with the Fisher's LSD Multiple-Comparison Test showed that saline-treated Swiss Webster mice made fewer discrete episodes of social approach when test and stimulus mice

interacted freely (8.13 ± 1.60[SEM]) than D-cycloserine-treated Swiss Webster mice at 100 mg/kg (11.69± 1.24[SEM]; p < 0.05) and 180 mg/kg (12.88 ± 0.99[SEM]; p < 0.01) doses. Post-hoc comparisons with the more conservative Tukey–Kramer Multiple Comparison Test showed no significant differences between groups. Possible dose-dependent effects of a range of D-cycloserine doses (i.e., 32.0, 56.0, 100.0 and 180.0 mg/kg) on time spent in social and nonsocial compartments (Fig. 9) and exploring (i.e., sniffing) an enclosed stimulus mouse versus an empty inverted cup (Fig. 10) in session II were explored separately for Balb/c and Swiss Webster mice using paired t-tests. Time spent in the social versus nonsocial compartment (Fig. 9, panel A) did not differ across the treatment groups for Balb/c

Fig. 10 – Exploration of possible dose-dependent effects of D-cycloserine 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 20 min after treatment with saline or D-cycloserine (32.0, 56.0, 100.0 or 180.0 mg/kg) in Balb/c (panel A) and Swiss Webster (panel B) mice. Paired t-tests were significant for all conditions with the exception of the Balb/c saline condition (n=16 for all groups). *p<0.05, **p<0.01, ***p<0.001 compare the time spent exploring/sniffing social and nonsocial inverted cups using paired t-tests.

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Table 1 – Correlation matrix for dimensions of sociability and stereotypic behavior. Balb/c Sociability

Parameter Avoidance Approach by test Anogenital

Stereotypic behavior

Rearing Grooming Wall climbing

Sociability Avoidance r p r p r p r p r p r p

Stereotypic behavior

Approach by test 0.19 0.41

0.19 0.41 −0.12 0.62 −0.04 0.87 0.28 0.23 0.10 0.68

0.82 ***0.00 −0.19 0.42 −0.13 0.58 0.93 ***0.00

mice (p > 0.05) and time spent exploring (i.e., sniffing) the enclosed social stimulus mouse versus the empty inverted cup (Fig. 10, 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 the 4-week old Balb/c mice. Importantly, treatment of Balb/c mice with D-cycloserine resulted in their spending significantly more time exploring (i.e., sniffing) the enclosed social stimulus mouse across all treatment groups (32.0 mg/kg, t=−3.486, p<0.01; 56.0 mg/kg, t=−2.806, p<0.05; 100.0 mg/kg, t=−2.858, p<0.05; 180.0 mg/kg, t=−3.568, p<0.01) (Fig. 10, panel A); these data suggest that D-cycloserine increased the salience of the social stimulus mouse for the Balb/c strain. Not surprisingly, saline-treated and D-cycloserine-treated Swiss Webster mice spent significantly more time in the compartment containing an enclosed social stimulus mouse (32.0 mg/kg, t=−4.012, p<0.01; 56.0 mg/kg, t=−6.054, p<0.001; 100.0 mg/kg, t=−5.467, p<0.001; 180.0 mg/kg, t=−6.198, p <0.001) (Fig. 9, panel B) and significantly more time exploring (i.e., sniffing) the enclosed social stimulus mouse (32.0 mg/kg, t=−4.805, p<0.001; 56.0 mg/kg, t=−6.698, p<0.001; 100.0 mg/kg, t=−7.595, p<0.001; 180.0 mg/kg, t=−5.988, p<0.001) (Fig. 10, panel B). Interestingly, many within-strain measures of sociability and stereotypic behaviors obtained during the 5-minute session of free interaction between test and stimulus mice with the 320 mg/kg dose of D-cycloserine were not correlated significantly with each other; moreover, the

Anogenital − 0.12 0.62 0.82 ***0.00

− 0.29 0.21 − 0.20 0.40 0.73 ***0.00

Rearing −0.04 0.87 −0.19 0.42 −0.29 0.21

−0.05 0.84 −0.19 0.42

Grooming 0.28 0.23 −0.13 0.58 −0.20 0.40 −0.05 0.84

Wall climbing 0.10 0.68 0.93 ***0.00 0.73 ***0.00 −0.19 0.42 −0.10 0.69

−0.10 0.69

patterns of correlations differed between strains (see Tables 1 and 2 of correlation matrices).

3.

Discussion

Although ASDs are often recognized in toddlerhood, there is a growing effort to improve accuracy of diagnosis in infancy, even as early as age 6 months (Landa and Garrett-Mayer, 2006), because it is hoped that earlier diagnosis will stimulate development of earlier intervention with disease-modifying effects that could significantly improve functional outcomes. Thus, we were wondering if D-cycloserine could improve sociability in Balb/c mice younger than 8-weeks of age. D-Cycloserine improved the sociability deficits of 4-week old genetically inbred Balb/c mice, which were similar to those observed in older 8-week old Balb/c mice (Brodkin et al., 2004; Sankoorikal et al., 2006). Specifically, relative to 4-week old outbred Swiss Webster mice, 4-week old Balb/c mice showed diminished locomotor activity, based on number of transitions, in the presence of an enclosed and freely-behaving social stimulus mouse; made fewer discrete episodes of social approach during the 5-minute period of free interaction with the social stimulus mouse; displayed more social avoidance of the social stimulus mouse during their 5-minute period of free interaction; spent less time in the compartment containing the enclosed social

Table 2 – Correlation matrix for dimensions of sociability and stereotypic behavior. Swiss Webster Sociability

Parameter Avoidance Approach by test Anogenital

Stereotypic behavior

Rearing Grooming Wall climbing

Sociability Avoidance r p r p r p r p r p r p

−0.44 *0.05 0.05 0.84 −0.28 0.23 0.32 0.17 −0.37 0.11

Approach by test − 0.44 *0.05

0.31 0.18 0.01 0.95 − 0.33 0.16 0.57 **0.01

Stereotypic behavior Anogenital 0.05 0.84 0.31 0.18

− 0.32 0.18 0.05 0.82 − 0.13 0.58

Rearing −0.28 0.23 0.01 0.95 −0.32 0.18

0.18 0.44 0.45 0.05

Grooming 0.32 0.17 −0.33 0.16 0.05 0.82 0.18 0.44

−0.24 0.30

Wall climbing − 0.37 0.11 0.57 **0.01 − 0.13 0.58 0.45 *0.05 0.24 0.30

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stimulus mouse and less time engaged in exploring (i.e., sniffing) the enclosed social stimulus mouse; and showed fewer episodes of anogenital sniffing, the latter is a quantitative measure of social exploration. Importantly, we selected the outbred Swiss Webster mouse as the comparator strain, as opposed to a more highly sociable strain, in order to lessen the likelihood of finding differences between strains (Jacome et al., 2011c). ASDs are chronic disorders and a major concern about pharmacotherapeutic agonist interventions is the potential loss of efficacy with sustained administration due to problems with agonist-induced desensitization. In this respect, a partial agonist may be preferred over a full agonist for chronic administration; however, a full agonist, such as D-serine, provided an additional proof of concept that therapeutic targeting of the glycineB site to improve sociability is viable. Also, demonstration of efficacy with both a full and partial agonist (i.e., D-serine and D-cycloserine, respectively) is consistent with the possibility that the major therapeutic action of D-cycloserine, a partial agonist, is mediated by the glycineB site (Deutsch et al., 2011; Jacome et al., 2011a,b; Long et al., 2006). The Balb/c mouse shows heightened sensitivity to behavioral effects of MK-801, a noncompetitive NMDA receptor antagonist; for example, relative to several inbred and outbred comparator strains, Balb/c mice are more sensitive to elicitation of irregular episodes of intense jumping, termed popping; antagonism of electrically precipitated seizures; and elicitation of circling behavior (Burket et al., 2010a; Deutsch et al., 1997a,b, 1998). These data suggest that there is altered endogenous tone of NMDA receptor-mediated neurotransmission in Balb/c mice, although their content of major immunoreactive NMDA receptor subunit polypeptides in hippocampus and cerebral cortex did not differ from an outbred Swiss mouse strain (Perera et al., 2008). Thus, we were stimulated to explore the effects of targeted NMDA receptor interventions in this sociability-impaired strain. Our results suggest that several discrete measures of sociability are also improved in the outbred comparator strain, raising questions about the strain-selectivity of this positive effect on sociability. However, in recent work, whereas D-cycloserine was shown to improve exploratory activity towards novel inanimate objects in both the Balb/c and Swiss Webster strains, social exploration was only enhanced in the Balb/c strain (Jacome et al., 2011b). Furthermore, in the study by Jacome et al. (2011c) and the current study, patterns of correlations among sociability measures differed between strains; thus, there is the possibility that between-strain differences may exist with respect to the sensitivity of different discrete measures of sociability to the effects of D-cycloserine. However, it is possible that improvement of at least some measures of sociability may be an epiphenomenon of a more direct effect on locomotor activity and exploration in general. Importantly, the data show that D-cycloserine can increase locomotor activity of both test strains, as reflected in transitions between compartments, even when no social stimulus mouse is present. Glutamate is the major excitatory neurotransmitter in mammalian brain and NMDA receptors are enriched within circuits and anatomic nodes (e.g., frontal cortex and hippocampus) that mediate various forms of learning, anxiety, reward-motivated behaviors, stress responsivity and seizure threshold, in addition to social behavior (Deutsch et al.,

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1997a; Rodgers et al., 2010, 2011; Thanos et al., 2011; Wlaz et al., 1994). Thus, not surprisingly, D-cycloserine has been shown to have a diverse inventory of behavioral actions in mice, including, but not limited to, anxiolytic effects in mice on their first exposure to the elevated plus maze (EPM) (Rodgers et al., 2010); post-training memory consolidation for preference of enclosed and avoidance of open arms of the EPM (Rodgers et al., 2011); facilitation of extinction to cued self-administration of cocaine (Thanos et al., 2011); and complex dose and time-dependent effects on electricallyprecipitated tonic hindlimb extension (Wlaz et al., 1994). Interestingly, although many behavioral effects of D-cycloserine are reported in mice at doses below 30 mg/kg, a pharmacokinetic analysis suggested that a low dose of D-cycloserine (i.e., 5 mg/kg) is cleared rapidly from plasma in mice (halflife less than 30 min) and its levels in frontal cortex were either undetectable or only trace amounts could be detected at 15 and 30 min after injection; 60 nmol/gram tissue was the limit of detection with the authors’ HPLC method (Wlaz et al., 1994). However, brain levels were 14% and 59% of plasma levels 15 and 60 min after injection, respectively, of a 320 mg/kg dose of D-cycloserine (Wlaz et al., 1994); 320 mg/kg was the dose used in most of our current experiments. Thus, it is likely that sufficient brain levels of D-cycloserine are achieved to mediate prosocial effects in Balb/c mice after injection of a 320 mg/kg dose. Of course, it may be that the failure to detect brain levels after injection of a 5 mg/kg dose of D-cycloserine in the study by Wlaz et al. (1994) reflected differences in route of injection (i.e., subcutaneously as opposed to intraperitoneally). As noted, because prosocial effects of the 320 mg/kg dose of D-cycloserine were observed, we examined the dose dependency of the prosocial effects of D-cycloserine in 4-week-old male Balb/c and comparator Swiss Webster mice, examining 0.0, 32.0, 56.0, 100.0 and 180.0 mg/kg, intraperitoneal doses. Interestingly, none of these doses of D-cycloserine increased the number of discrete episodes of social approach made by Balb/ c mice toward the social stimulus mouse during 5 min of free social interaction in session III, nor did any of them increase the amount of time (sec) Balb/c mice spent in the compartment containing the enclosed social stimulus mouse. However, each of these doses increased the amount of time (sec) Balb/c mice spent exploring/sniffing the enclosed social stimulus mouse in session II; thus, this latter measure appears to be very sensitive to a prosocial effect of D-cycloserine in Balb/c mice. Importantly, the dose–response data support the selection of the 320 mg/kg dose in this current and previous investigations (Deutsch et al., 2011; Jacome et al., 2011b); specifically, the 320 mg/kg dose of D-cycloserine showed significant prosocial effects in 4 and 8-week-old male Balb/c mice on a variety of dependent sociability measures. Finally, the data suggest that D-cycloserine improves measures of both sociability and stereotypic behaviors observed spontaneously during social interaction, irrespective of strain. This is very important in a medication strategy for ASDs because it would clearly be undesirable to attenuate the severity of stereotypic behaviors, while worsening measures of sociability. In summary, the data lend additional support to medication development strategies that target the NMDA receptor for the treatment of persons with ASDs. Importantly, D-cycloserine was reported to attenuate the severity of social withdrawal in

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an open-label, dose escalation study of 10 children with autistic disorder diagnosed according to DSM-IV criteria (Posey et al., 2004).

4.

Experimental procedures

4.1.

Subjects

Experimentally-naïve, 4-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 4week old male ICR mice, housed 4 per cage. Housing conditions were adopted from prior literature (Sankoorikal et al., 2006). Mice were individually weighed prior to drug administration and up to 20 mice were tested in each condition. 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. 4.2.

Drugs

D-cycloserine

(Sigma-Aldrich Co.; St. Louis, MO) was dissolved in 0.9% saline and prepared each day of the experiment. D-cycloserine was injected intraperitoneally in a volume of 0.01 ml/g of body weight 20 min prior to testing sociability. 4.3.

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, as required by Eastern Virginia Medical School's Institutional Animal Care and Use Committee. 4.4.

Sociability procedure

The laboratory adopted an established mouse behavioral procedure for the quantitative assessment of sociability (Brodkin, 2007; Burket et al., 2010b; Crawley, 2004, 2007; Deutsch et al., 2011; Jacome et al., 2011a,c; Moy et al., 2007; Sankoorikal et al., 2006). 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-minute 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-minute 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 are 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 is measured in the second 5-minute session. The following measures of sociability, stereotypic behaviors and locomotor activity are reliably obtained in the third 5-minute session of free interaction between test and stimulus mice and analyzed in this report: discrete episodes of social approach; discrete episodes of social avoidance; discrete episodes of anogenital sniffing; discrete episodes of rearing; discrete episodes of wall climbing; discrete episodes of self-grooming; and number of transitions between compartments (Jacome et al., 2011c; Silverman et al., 2010). Social approach is defined as a discrete episode of initiation of sniffing the social stimulus mouse by the test mouse within at least a 2-cm vicinity of each other. Social avoidance is a nonsocial response of the test mouse defined as a discrete episode of freezing, withdrawing, or turning its head away while within a 2-cm vicinity of the socially-salient stimulus mouse. Anogenital sniffing is a social behavior displayed by the test mouse defined as a discrete episode of sniffing the anogenital area of the stimulus mouse within a two-cm vicinity. 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 number of discrete episodes the test mouse is engaged in licking and rubbing of fur with forelimbs during the 5-minute period of free social interaction (i.e., third 5-minute session). A transition between compartments is defined as the number of times all four extremities cross between compartments, measured in all three sessions, which can indicate changes in locomotor activity. 4.5.

Statistics

A two-way ANOVA was used to examine effects of strain (Balb/c vs. Swiss Webster), treatment condition (i.e., fixed dose of 320 mg/kg of D-cycloserine vs. saline), and their interaction on the number of transitions between compartments; discrete episodes of social approach; discrete episodes of social avoidance; discrete episodes of anogenital sniffing; discrete episodes of rearing; discrete episodes of wall climbing; and discrete episodes of self-grooming. Importantly, because positive effects of the fixed 320 mg/kg dose of D-cycloserine on sociability were observed, possible dose dependent effects of D-cycloserine on measures of sociability, exploring a range of doses (i.e., 32.0, 56.0, 100.0 and 180.0 mg/kg), were studied separately in Balb/c and Swiss Webster mice using a oneway ANOVA. When ANOVA was significant, exploratory Fisher's LSD Multiple Comparison Tests were applied in post-hoc comparisons, where appropriate. Thereafter, when Fisher's

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LSD tests were significant, more conservative Tukey–Kramer Multiple Comparison Tests were also conducted. When a two-way ANOVA could not be performed (see Fig. 3) because of an absence of variance in one of the groups, exploratory multiple comparisons of mean values between groups were performed with Independent Samples t-tests. Paired t-tests were used to determine effects of D-cycloserine on the salience of the enclosed social stimulus mouse for Balb/c and Swiss Webster mice; specifically, for both the saline and D-cycloserine 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 inverted cup containing the social stimulus mouse.

Acknowledgment The authors acknowledge support received from the Office of the Dean of Eastern Virginia Medical School.

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