Sociability and motor functions in Shank1 mutant mice

Abstract

Autism is a neurodevelopmental disorder characterized by aberrant reciprocal social interactions, impaired communication, and repetitive behaviors. While the etiology remains unclear, strong evidence exists for a genetic component, and several synaptic genes have been implicated. SHANK genes encode a family of synaptic scaffolding proteins located postsynaptically on excitatory synapses. Mutations in SHANK genes have been detected in several autistic individuals. To understand the consequences of SHANK mutations relevant to the diagnostic and associated symptoms of autism, comprehensive behavioral phenotyping on a line of Shank1 mutant mice was conducted on multiple measures of social interactions, social olfaction, repetitive behaviors, anxiety-related behaviors, motor functions, and a series of control measures for physical abilities. Results from our comprehensive behavioral phenotyping battery indicated that adult Shank1 null mutant mice were similar to their wildtype and heterozygous littermates on standardized measures of general health, neurological reflexes and sensory skills. Motor functions were reduced in the null mutants on open field activity, rotarod, and wire hang, replicating and extending previous findings (Hung et al., 2008). A partial anxiety-like phenotype was detected in the null mutants in some components of the light ↔ dark task, as previously reported (Hung et al., 2008) but not in the elevated plus-maze. Juvenile reciprocal social interactions did not differ across genotypes. Interpretation of adult social approach was confounded by a lack of normal sociability in wildtype and heterozygous littermates. All genotypes were able to discriminate social odors on an olfactory habituation/dishabituation task. All genotypes displayed relatively high levels of repetitive self-grooming. Our findings support the interpretation that Shank1 null mice do not demonstrate autism-relevant social interaction deficits, but confirm and extend a role for Shank1 in motor functions.

Figure 1. Shank1 mice exhibit normal juvenile reciprocal social interaction behaviors

Juvenile social interaction between pairs consisting of a Shank 1 +/+, +/- or -/- mouse, age 19-22 days, with an unfamiliar sex and age-matched B6 control mouse. No significant differences were detected between the genotypes on the number of bouts of A) nose-to-nose sniff, B) anogenital sniff, C) body sniff, D) push and crawl, E) pushing past, and F) follow. N=9 per genotype. Data are shown as mean + standard error of the mean throughout Figures 1-10.

Figure 2. Sociability in Shank1 mice

Adult sociability, assayed in an automated photocell-equipped three-chambered arena, revealed unpredicted confounds. A) None of the Shank1 genotypes displayed normal sociability, defined as more time in the side chamber with the novel mouse than in the side chamber with the novel object. In contrast, control mice from the hybrid background B6/Jae, used to breed the Shank1 mutation, displayed normal sociability for time in the chamber containing the novel mouse versus time in the chamber containing the novel object. * p < 0.05. B) Shank1 -/- and B6/Jae displayed significantly more time spent sniffing the novel mouse than time spent sniffing the novel object. C) No genotype differences were seen in the number of entries into the either the left or right side chambers. D) No innate chamber side bias was present in any group during the 10 minute habituation phase before the start of the sociability test. N=15 +/+; N =8 +/-; N=17 -/-; N=12 B6/Jae.

Figure 3. No deficits in non-social and social olfactory capabilities in Shank1 mice

Olfactory habituation/dishabituation confirmed normal olfactory abilities in all three genotypes of Shank1 mice for non-social and social odors. All genotypes displayed significant habituation and dishabituation to non-social and social odors. The first presentation of a water-soaked swab elicited moderate sniffing that declined across the second and third exposure to water (habituation). The next presentation, a swab soaked in almond extract, elicited significantly more sniffing (dishabituation), which declined across the second and third presentation of the almond odor (habituation). Similarly, sniffing resumed at a high level to the next new odor, a swab soaked in banana flavoring (dishabituation), and declined across the three banana presentations (habituation). Shank1 +/+ mice sniffed the banana odor for a greater length of time compared to +/- and -/- littermates on the first trial. # p < 0.05. The next presentation, a swab soaked in almond extract, elicited significantly more sniffing (dishabituation), which declined across the second and third presentation of the almond odor (habituation). The next presentation, a social odor swab wiped across the bottom surface of a plastic cage that contained four unfamiliar mice of the same sex but a different strain, 129S1/SvImJ, elicited significantly more sniffing (dishabituation), which declined across the second and third presentation of this first social odor (habituation). The final presentation, a swab wiped across the bottom surface of a plastic cage that contained a second set of four unfamiliar mice of the same sex but a different strain, FVBS/Ant, elicited significantly more sniffing (dishabituation), which declined across the second and third presentation of the second social odor (habituation). N=15 +/+; N =18 +/-; N=14 -/-.

Figure 4. Shank1 mice did not exhibit genotype differences in repetitive self-grooming

Cumulative time spent self-grooming was scored over a 10 minute session in a clean, empty mouse cage. Shank1 +/+, +/- and -/- mice did not differ on the amount of time spent self-grooming during a ten minute testing session. N=9 +/+; N =12 +/-; N=11 -/-; N=10 B6/Jae. The background strain, B6/Jae, scores are provided for illustrative purposes.

Figure 5. Mild anxiety-related behavior in Shank1 null mutant mice in the light ↔ dark test

Basal performance on an anxiety-related task, light ↔ dark exploration A) Shank1 null -/- mutants displayed reduced transitions between the light and dark compartments of the test apparatus compared to +/+ and +/- littermates. * p < 0.05. B) No significant differences were detected across Shank1 genotypes on time spent in the dark chamber. C) No significant differences were detected across Shank1 genotypes on latency to enter the dark portion of the apparatus. N=13 +/+; N =15 +/-; N=14 -/-; N=14 B6/Jae. Scores for the background strain, B6/Jae, are provided for illustrative purposes but were not included in the statistical analysis.

Figure 6. No genotype differences in anxiety-like behaviors on the elevated plus-maze

Elevated plus-maze data revealed no significant genotype differences in A) percentage of time spent on the open segments, B) entries into the open arm segments, C) total entries. N=13 +/+; N=15 +/-; N=14 -/-; N=14 B6/Jae. Score for the background strain, B6/Jae, are provided for illustrative purposes.

Figure 7. Shank1 null mutants display reduced exploratory locomotion in a novel open field

Total distance, center time, horizontal activity and vertical activity and were assayed in 5-minute time bins across a 30 minute session in a novel open field arena, for the three Shank1 genotypes and hybrid background mice, B6/Jae. A) Shank1 null mutants -/- traversed less total distance in a novel open field as compared to +/+ and +/- littermates. * p < 0.05. B) Shank1 null mutants -/- spent less time in the center of the open field as compared to +/+ littermates. * p < 0.05. C) Horizontal activity did not differ across genotypes and D) vertical activity did not differ across genotypes. N=14 +/+; N=14 +/-; N=14 -/-; N=10 B6/Jae. Scores for the hybrid background mice, B6/Jae, are provided for illustrative purposes.

Figure 8. Reduced motor coordination, balance and neuromuscular strength in Shank1 null mutant mice

Motor coordination, balance, and neuromuscular strength were assayed. Latency to fall from an accelerating rotarod was recorded with a 300 second maximum latency. Each subject was given 6 total trials over two days, 3 trials per day, with a 30–60 minute intertrial interval. Latency to fall from an inverted wire mouse cage lid was recorded with a 60 second maximum hang time. A) Shank1 null mutants -/- fell from the accelerating rotarod faster than +/+ and +/- littermates. * p < 0.05. N=16 +/+; N=23 +/-; N=21 -/-. B) Shank1 null mutants -/- fell from the inverted wire mouse cage lid faster than +/+ and +/- littermates. * p < 0.05. N=15 +/+; N=12 +/-; N=11 -/-; N=10 B6/Jae. Scores for the hybrid background mice, B6/Jae, are provided for illustrative purposes.

Figure 9. No genotype differences in acoustic startle or prepulse inhibition sensorimotor gating

A) No genotype differences were observed in the acoustic startle response at 6 dB levels. B) No genotype differences were observed in prepulse inhibition of acoustic startle at any prepulse level. N=9 +/+; N =9 +/-; N=13 -/-.

Figure 10. Normal pain sensitivity in Shank1 mice

Responsiveness to painful stimuli was measured using the hot plate and tail flick tasks. No genotype differences were observed for A) latency to jump, lick or vocalize in the hot plate task. N=10 +/+; N=8 +/-; N=10 -/-; N=10 B6/Jae. No genotype differences were observed for B) latency to flick the tail out of the path of an intense light beam. N=8 +/+; N=12 +/-; N=10 -/-; N=10 B6/Jae. Scores for the genetic background mice, B6/Jae, are provided for illustrative purposes.