c mouse model of autism spectrum disorders

Brain Research Bulletin 122 (2016) 29–34

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Characterization of gait and olfactory behaviors in the Balb/c mouse model of autism spectrum disorders Jessica A. Burket a , Chelsea M. Young b , Torrian L. Green a , Andrew D. Benson a , Stephen I. Deutsch a,∗ a b

Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, Norfolk, VA, United States Muhlenberg College, Allentown, PA, United States

a r t i c l e

i n f o

Article history: Received 11 January 2016 Received in revised form 17 February 2016 Accepted 19 February 2016 Available online 23 February 2016 Keywords: Balb/c Autism spectrum disorders Olfaction Cerebellum Gait

a b s t r a c t Abnormalities of gait and olfaction have been reported in persons with autism spectrum disorders (ASDs), which could reflect involvement of the cerebellum and nodes related to olfaction (e.g., olfactory bulb and ventral temporal olfactory cortex) in neural circuits subserving social, cognitive, and motor domains of psychopathology in these disorders. We hypothesized that the Balb/c mouse model of ASD would express “abnormalities” of gait and olfaction, relative to the Swiss Webster comparator strain. Contrary to expectation, Balb/c and Swiss Webster mice did not differ in terms of quantitative measurements of gait and mouse rotarod behavior, and Balb/c mice displayed a shorter latency to approach an unscented cotton swab, suggesting that there was no disturbance of its locomotor behavior. However, Balb/c mice showed significant inhibition of locomotor activity in the presence of floral scents, including novel and familiar floral scents, and a socially salient odor (i.e., concentrated mouse urine); the inhibitory effect on the locomotor behavior of the Balb/c mouse was especially pronounced with the salient social odor. Unlike the Swiss Webster strain, mouse urine lacks social salience for the Balb/c mouse strain, a model of ASD, which does not appear to be an artifact of diminished olfactory sensitivity or impaired locomotion. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Early anatomic imaging studies of the cerebellum in ASD suggested inconsistent abnormalities of the vermal lobules, including hypoplasia and hyperplasia, as well as changes in vermal volume that are selective for diagnostic subtypes (Bauman and Kemper, 2013; Ciesielski et al., 1997; Courchesne et al., 1994). Moreover, functional magnetic resonance imaging (fMRI) studies suggested that children with “high-functioning” ASD and age- and gendermatched controls differ in terms of connectivity between, and activation of, the supplementary motor area and anterior cerebellum (Bauman and Kemper, 2013). An early diffusion tensor imaging study is also consistent with impaired connectivity between the cerebellum and neocortex (Sivaswamy et al., 2010). Purkinje cell loss in the posterior inferior hemispheric regions of the cerebellum is a frequently noted histopathological abnormality in ASD (Bauman and Kemper, 2013; Ritvo et al., 1986). Pathology intrin-

∗ Corresponding author at: Anne Armistead Robinson Endowed Chair in Psychiatry, Professor and Chairman, Department of Psychiatry and Behavioral Sciences, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507, United States. E-mail address: [email protected] (S.I. Deutsch). http://dx.doi.org/10.1016/j.brainresbull.2016.02.017 0361-9230/© 2016 Elsevier Inc. All rights reserved.

sic to the cerebellum and its altered connectivity could be the basis of disturbances in “automating patterned motor behavior,” which could result in the abnormal acquisition of gesturing that is so important for effective social communication (Bauman and Kemper, 2013). In addition to connections with the cerebral cortex, direct connections exist between the fastigial nucleus of the cerebellum and amygdala, septal nuclei and hippocampus, and between the dentate nucleus of the cerebellum and dorsolateral prefrontal cortex, supporting roles for the cerebellum in affective behavior and higher cortical processes (Heath et al., 1978; Heath and Harper, 1974). Sniffing of objects is a stereotypic behavior that is frequently observed in young children with ASDs, especially those with comorbid intellectual disability (Cunningham and Schreibman, 2008). Whereas odor detection may not be impaired in persons with ASD, abnormal hedonic ratings of the pleasantness of food odors have been reported (Luisier et al., 2015). Moreover, reports of problems with odor identification in persons with ASD may be confounded by cognitive disturbance and reflect poorer language performance (Luisier et al., 2015). In any event, the possibility of an association between cerebellar dysfunction and stereotypic behaviors, including sniffing, which may be moderated by developmental age and

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Fig. 1. Effect of mouse strain on olfactory preferences. Scatter plots show the distribution of mice relative to time spent sniffing (exploring) the respective odor stimuli (N = 18 mice per group). A. Trial 1, time spent sniffing simultaneously presented water and floral scented cotton swabs over a 2-min period. B. Trial 2, time spent sniffing simultaneously presented familiar and novel floral scented cotton swabs over a 2-min period. C. Trial 3, time spent sniffing a novel floral scent versus a salient social odor (mouse urine) presented simultaneously over a 2-min period. ####p < 0.0001 comparison of time spent sniffing (exploring) the respective odor stimuli between Balb/c and Swiss Webster mice. *p < 0.05 and ****p < 0.0001 comparison of time spent sniffing (exploring) the respective odor stimuli within mouse strain.

IQ, exists (Radonovich et al., 2013). A recent study showed that the “sniff response,” which is the magnitude of sniffing modulated automatically in response to odor valence, such as pleasantness, was abnormal in 18 children with ASD (17 boys; mean age = 7 ± 2.3) compared to 18 typically developing (TD) controls (17 boys; mean age = 6.7 ± 2.1) (Rozenkrantz et al., 2015). Whereas the TD children adjusted their sniff response according to the odor valence (i.e., a larger response for a pleasant versus unpleasant odor), the sniff response of children with ASD did not differentiate between pleasant and unpleasant odors. The sniff response of the children with ASD correlated with the severity of social impairment (Rozenkrantz et al., 2015). Importantly, a normal sniff response is dependent on the integrity of connections between the ventral temporal olfactory cortex and cerebellum (Rozenkrantz et al., 2015). Because the Balb/c mouse has emerged as an important model of ASD and social odors have enormous salience for rodents, we wondered if Balb/c mice would display abnormalities of gait and odor discrimination, especially discrimination of salient social odors, relative to the Swiss Webster comparator strain (Benson et al., 2013; Burket et al., 2015; Deutsch et al., 2015, 2012, 2011).

2. Materials and methods 2.1. Animals Experimentally-naïve, 4-week old male, genetically-inbred Balb/c and outbred Swiss Webster (Harlan Laboratories, Frederick, MD, USA) test mice 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. 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.

2.2. Gait procedure A standard published procedure for the quantitative assessment of gait was utilized (Brooks and Dunnett, 2009; Carter et al., 1999; Glynn et al., 2005). To obtain footprint tracks, the soles of the front and hind paws were dipped in nontoxic red and brown paint, respectively. The mice were then allowed to walk down an enclosed runway lined with white paper, with three repetitions. The resultant footprints were analyzed by measuring the length of three strides for each front and hind leg and their front and hind base width from the middle portion of each run.

2.3. Olfaction Preference Testing began with a five minute acclimation period to a standard three-chamber apparatus, which 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). During acclimation, test mice were free to sniff/explore unscented cotton swabs saturated with water suspended in both the left and right compartments, providing measures of general exploratory behavior and locomotor activity. Thereafter, olfaction preference was measured during three 2-min trials. Specifically, cotton swabs saturated with water or a scented floral odor (Trial 1); a familiar or novel floral scent (Trial 2); or a novel floral scent paired with a salient social odor (Trial 3) were suspended in either the left or right compartments. The scent of each swab was randomly assigned to the left or right compartment within each trial and the presentation side was counterbalanced. Three minutes were taken between each trial to clean the apparatus of any residual odor and set-up for the following trial. Test mice were placed in the center of a three-compartment apparatus containing cotton swabs in opposing left or right compartments. Floral scents 1–3 were randomly selected permutations of floral odors chosen for their previously tested similarity in preference (i.e., jasmine, plumeria, and rose). The “social scent” was obtained by rubbing the cotton swab in week-old saturated bedding previously used by 5 male B6.129S2-Alox15 mice. All Sessions were conducted in dim lighting (< 3.5 lx) and videotaped using Sony HD Video Cameras (Sony Corp., Tokyo, Japan) in nightshot mode with infrared lighting for future viewing and data collection. Latency to approach a cotton swab and the time spent exploring (sniffing) the respectively scented swabs were recorded. 2.4. Rotarod test An automated apparatus was used to evaluate the ability of mice to maintain balance for up to 5 min on a rod rotating at a constant speed of 16 rpm (Biological Research Apparatus, Model 7600, Comerio Varese, Italy), as previously described (Deutsch et al., 2008, 1996). 2.5. Statistical analyses Within-strain comparisons of olfactory preferences were made with paired t-tests, whereas between-strain comparisons were made with independent t-tests (see Fig. 1). Analyses of initial latency to approach the cotton swabs (seconds) and measures

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Fig. 2. Effect of mouse strain on initial latency to approach scented and unscented cotton swabs. Bars represent means ±SEM of initial latency to approach the unscented (Acclimation) as well as floral (Trial 1), novel and familiar floral (Trial 2), and social scented (Trial 3) cotton swabs (N = 18 mice per group). Initial latency reflects the time (seconds) for a mouse to move from the middle compartment to either the left or right compartment in order to explore (sniff) cotton swabs saturated with either unscented (Acclimation), scented floral (Trials 1 and 2) or salient social (Trial 3) odors. Latency was recorded when the test mouse first approached the tip of the cotton swab. ##p < 0.01, ###p < 0.001 and ####p < 0.0001 comparison of latency to approach the cotton swab between Balb/c and Swiss Webster mice. ***p < 0.001 and ****p < 0.0001 within strain comparisons of Trials 1, 2 and 3 to Acclimation.

Table 1 Choice of First Approach to Cotton Swab. Acclimation Left None 7 5 Swiss Webster Balb/c 2 8 Total 9 13 Chi-Square (X2 = 3.756, df = 2; p > 0.05)

Right 6 8 14

Total 18 18 36

Trial 1: Water vs. Floral Scent Water None 0 9 Swiss Webster 7 5 Balb/c Total 7 14 Chi-Square (X2 = 8.743, df = 2; p = 0.013)

Floral Scent 9 6 15

Total 18 18 36

Trial 2: Familiar Scent vs. Novel Scent None Familiar Scent Swiss Webster 0 5 Balb/c 11 4 Total 11 9 Chi-Square (X2 = 17.36, df = 2; p = 0.0002)

Novel Scent 13 3 16

Total 18 18 36

Trial 3: Floral Scent vs. Social Scent None Floral Scent 0 5 Swiss Webster 15 1 Balb/c 15 6 Total Chi-Square (X2 = 25.73, df = 2; p < 0.0001)

Social Scent 13 2 15

Total 18 18 36

of gait (i.e., stride length and base width) were performed with ANOVA; when appropriate, post-hoc comparisons were made with the Tukey multiple comparison test (see Figs. 2 and 3). Significant differences in the proportion of mice first approaching the left or right cotton swab were analyzed with chi square (see Table 1). Fisher’s Exact Test was used to examine differences in the proportion of mice in each group able to maintain themselves on the rotating rod for 300 s. 3. Results The presence of a randomly-chosen floral scent (i.e., jasmine, plumeria, and rose) had no salience for the Balb/c and Swiss Webster mouse strains (Fig. 1A). However, unlike Balb/c mice, Swiss Webster mice showed preference for a novel floral olfactory stim-

ulus versus a familiar floral odor (p < 0.05; Fig. 1B). Although the time spent sniffing the novel versus familiar floral scent was statistically significantly greater in the Swiss Webster strain, the magnitude of the mean difference in seconds between sniffing the novel and familiar floral scent was small. When presented with a salient social odor, Balb/c mice spent significantly less time exploring (sniffing) the social odor than the Swiss Webster comparator strain (p < 0.0001); moreover, the Swiss Webster strain preferred the salient social odor to the novel floral scent (p < 0.0001; Fig. 1C). The presence of floral scents alone, and simultaneous presentations of novel and familiar floral scents and floral scents with a salient social odor significantly affected the initial approaches of both Balb/c and Swiss Webster mice from the middle chamber to the left or right chambers containing the suspended cotton swabs (Fig. 2). Specifically, a two-way ANOVA revealed significant main effects for strain (F1,136 = 45.63, p < 0.0001), trial (F3,136 = 4.822, p < 0.01) and their interaction (F3,136 = 25.23, p < 0.0001) on initial latency to approach the suspended cotton swabs; these data are consistent with the abilities of both mouse strains to detect the presence of the olfactory stimuli. Importantly, post-hoc comparisons showed that Balb/c mice were not impaired in exploratory or locomotor behavior, indicated by their shorter initial latency to approach the cotton swabs presented during acclimation than the Swiss Webster strain (p < 0.001). In Trial 1, the presence of a floral scent significantly reduced the initial latency to explore the cotton swabs in the Swiss Webster strain (p < 0.0001), whereas it had no effect on the exploratory behavior of the Balb/c strain. In Trial 2, when novel and familiar floral odors were presented simultaneously, the initial latency to sniff the cotton swabs was significantly increased in the Balb/c strain compared to its latency observed during acclimation (p < 0.001). In Trial 3, when the salient social odor was introduced simultaneously with a floral scent, there was a dramatic inhibition of the mean initial latency of the group of Balb/c mice to approach the suspended cotton swabs in the left or right chambers relative to acclimation (p < 0.0001). The latter data suggest that unlike the Swiss Webster comparator strain, the presence of a salient social odor inhibited the locomotor activity and exploratory behavior of the Balb/c strain. Interestingly, in Trial 1, whereas neither Swiss Webster nor Balb/c mice showed preference for the floral scent over water, the

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Fig. 3. Rotarod performance in Balb/c and Swiss Webster mice. Scatter plot showing the distribution of Balb/c (N = 7) and Swiss Webster (N = 5) mice able to maintain themselves on the rotating rod for up to 300 s.

presence of a floral scent inhibited exploratory behavior in the Balb/c mice (i.e., seven of 18 Balb/c mice displayed no exploratory behavior toward either the unscented or scented cotton swab) (␹2 = 8.743, p = 0.013; Table 1). In Trial 2, Swiss Webster mice preferred the novel floral (i.e., 13 out of 18 mice) over the familiar floral scent (i.e., 5 out of 18 mice); however, the simultaneous presentation of the novel and familiar floral scents significantly inhibited the exploratory/locomotor behavior of the Balb/c strain (i.e., 11 out of 18 Balb/c mice explored neither of these scents; ␹2 = 17.36, p = 0.0002; Table 1). Further, in Trial 3, the social odor significantly inhibited the exploratory behavior of Balb/c mice, as reflected in a significantly increased latency of initial approach to a scented cotton swab (p < 0.0001; Fig. 2) and 15 of 18 Balb/c mice displaying no exploratory behavior to either the floral scent or salient social odor (␹2 = 25.73, p < 0.0001; Table 1). In contrast, Swiss Webster mice showed preference for the social odor, as highlighted by both their significantly reduced initial latency to approach a cotton swab compared to both Balb/c mice and their performance during acclimation (p < 0.0001; Fig. 2), and 13 of 18 Swiss Webster mice choosing the socially scented cotton for their initial approach (␹2 = 25.73, p < 0.0001; Table 1). Fisher’s Exact Test revealed that the proportion of Balb/c (i.e., 4 out of 7) and Swiss Webster (i.e., 5 out of 5) mice able to maintain themselves on the rotating rod for 300 s did not differ (p = 0.205; Fig. 3). Moreover, one-way ANOVA revealed no significant differences between the Balb/c and Swiss Webster strains in terms of stride lengths and base widths. Thus, Balb/c mice showed no significant deviations in their motor coordination and gait compared to the Swiss Webster mouse strain (Fig. 4), indicating that the reduced locomotor activity of the Balb/c mouse strain in the presence of floral scents and the social odor was not due to an impaired ability to move (ataxia). 4. Discussion The cerebellum is an important node in the ‘motor control system,’ which appears to be disturbed in many persons with ASD. Compared to a group of 28 typically developing (TD) controls (ages 3.4–15.9 years; mean age = 8.31 ± 4.0[SD]), the mean postural sway area obtained during quiet standing was significantly greater in a group of 18 children and adolescents with ASD and nonverbal IQ scores greater than 70 (ages 3.9–15.7 years; mean age = 8.18 ± 3.4[SD]) (Radonovich et al., 2013). Moreover, the postural sway areas for about half of the ASD group were greater than 2 SD outside the range of the TD controls. Further, within the ASD group, there were significant positive correlations between pos-

tural sway area and measures of restricted, repetitive behaviors (Radonovich et al., 2013). There was also a suggestion that younger age and lower IQ scores influenced the larger postural sway areas observed in the ASD group. In any event, postural sway abnormalities could be associated with stereotypic sniffing behavior affect, and gait, and be referable to cerebellar abnormalities in at least some persons with ASD. Thus, it is interesting that based on rotarod performance and measures of stride length and base width, Balb/c mice, which have been shown to display impaired sociability in the standard three-chamber sociability apparatus, and the Swiss Webster comparator strain did not differ (Benson et al., 2013; Burket et al., 2015; Deutsch et al., 2015, 2012; Jacome et al., 2011). The concentration of darcin (MUP20), a major protein pheromone present in male urine, is significantly lower in male Balb/c mice than other comparator strains, such as the C57BL/6 (Roberts et al., 2010); darcin contributes to the attraction of female mice to male urinary scent marks. Recent work suggests that darcin stimulates hippocampal neurogenesis in female mice, an effect that could not be produced by urine obtained from male Balb/c mice (Hoffman et al., 2015). Clearly, we were studying the odor preferences of two strains of male mice, including exploratory interest in odors associated with concentrated male mouse urine. However, diminished production of darcin by male Balb/c mice is consistent with, and contributes to, the impaired sociability of this mouse model of ASD. In the current study, concentrated male mouse urine elicited little or no social interest in male Balb/c mice or, alternatively, elicited an actual aversive or disinterested response in this strain, compared to Swiss Webster mice. The data are consistent with prior replicated reports showing that Balb/c mice are less likely to explore conspecifics in the standard three-chamber apparatus than the Swiss Webster comparator strain, which could reflect the lack of salience of normal social cues, including urinary odors of conspecifics (Burket et al., 2015; Deutsch et al., 2015, 2012; Jacome et al., 2011). Pre- and postnatal exposure to valproic acid may produce neuronal and behavioral deficits relevant to ASDs, such as social impairment and alterations of motor functions and coordination (Furnari et al., 2014; Wellmann et al., 2014). Oxidative stress resulting from overproduction of reactive oxygen species (ROS) may mediate or contribute to the deficits resulting from exposure of offspring to valproic acid (Furnari et al., 2014). The fact that knockout mice lacking expression of ‘nuclear factor-erythroid 2 (NF-E2) related factor 2 (Nrf2), ’ which is a transcription factor that promotes expression of genes induced by ROS and involved in an antioxidant response, show greater sensitivity to valproic acid-induced behavioral deficits is consistent with a pathogenic mechanism of oxidative stress (Furnari et al., 2014). Interestingly, treatment of postnatal day 14 mice with valproic acid led to motor incoordination deficits assessed with rotarod performance, which are referable to cerebellar dysfunction; the deficits of motor coordination were more severe in the Nrf2 knockout mice than wild-type mice (Furnari et al., 2014). Importantly, the Balb/c mouse model of ASD showed no obvious deficits of gait or motor coordination, suggesting that cerebellar abnormalities may not contribute significantly to the neural circuitry responsible for the social deficits in this strain. Rat offspring exposed prenatally to valproic acid and tested between postnatal days 40 and 45 showed abnormalities of ultrasonic vocalizations, which are social communication signals in rodents, and social interactions (Wellmann et al., 2014). Interestingly, treatment with D-cycloserine, a partial glycineB agonist of the NMDA receptor, ameliorated the effects of prenatal exposure to valproic acid on ultrasonic vocalizations, normalized the increased “play fighting” in exposed male offspring, and increased social motivation in exposed female offspring (Wellmann et al., 2014). The data support a role for the NMDA receptor in the pathogenesis

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Fig. 4. Characterization of gait abnormalities in Balb/c and Swiss Webster mice. Bars represent means ±SEM of (A) stride lengths and (B) base widths for Balb/c (N = 16) and Swiss Webster (N = 18) mice.

of the impaired sociability of the valproic acid model of ASD, similar to its hypothesized role in the Balb/c mouse strain (Benson et al., 2013; Burket et al., 2015; Deutsch et al., 2015, 2012). Also, the data extend support for the therapeutic targeting of the NMDA receptor to another rodent species and animal model of ASD (Benson et al., 2013; Burket et al., 2015; Deutsch et al., 2015, 2012; Wellmann et al., 2014). The Balb/c mouse model of ASD may have special heuristic value because it appears to better isolate the neural circuitry underlying impaired sociability without the confounding effects of nodes and circuits underlying repetitive stereotypic behaviors and gait abnormalities (Burket et al., 2015; Deutsch et al., 2015, 2012; Jacome et al., 2011). Specifically, relative to the BTBR T+ Itpr3tf/J (BTBR) and Swiss Webster mouse strains, Balb/c mice display less intense repetitive stereotypic rearing and grooming (Burket et al., 2015, 2014, 2013; Deutsch et al., 2015, 2012; Jacome et al., 2011; Silverman et al., 2012, 2010). Thus, the Balb/c mouse may be a preferred mouse strain to study the pathology of impaired sociability unencumbered by other symptom domains, and test targeted pharmacotherapeutic interventions to treat this core symptom domain of ASD. Funding The authors have nothing to disclose. Acknowledgement The authors acknowledge the support they received from the Families of Autistic Children of Tidewater (F.A.C.T.), and Office of the Dean of Eastern Virginia Medical School. References Bauman, M.L., Kemper, T.L., 2016. Chapter 3.4—The cerebellum in autism spectrum disorders. In: Hof, P. (Ed.), The Neuroscience of Autism Spectrum Disorders. Academic Press, San Diego, pp. 289–295. Benson, A.D., Burket, J.A., Deutsch, S.I., 2013. Balb/c mice treated with d-cycloserine arouse increased social interest in conspecifics. Brain Res. Bull. 99, 95–99. Brooks, S.P., Dunnett, S.B., 2009. Tests to assess motor phenotype in mice: a user’s guide. Nat. Rev. Neurosci. 10, 519–529. Burket, J.A., Benson, A.D., Tang, A.H., Deutsch, S.I., 2013. d-Cycloserine improves sociability in the BTBR T+ Itpr3tf/: J mouse model of autism spectrum disorders with altered Ras/Raf/ERK1/2 signaling. Brain Res. Bull. 96, 62–70. Burket, J.A., Benson, A.D., Tang, A.H., Deutsch, S.I., 2014. Rapamycin improves sociability in the BTBR T(+) Itpr3(tf)/: J mouse model of autism spectrum disorders. Brain Res. Bull. 100, 70–75. Burket, J.A., Benson, A.D., Green, T.L., Rook, J.M., Lindsley, C.W., Jeffrey Conn, P., Deutsch, S.I., 2015. Effects of VU0410120, a novel GlyT1 inhibitor, on measures

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