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Comparative Study
. 2012 May 9;32(19):6525-41.
doi: 10.1523/JNEUROSCI.6107-11.2012.

Reduced excitatory neurotransmission and mild autism-relevant phenotypes in adolescent Shank3 null mutant mice

Mu Yang  1 , Ozlem Bozdagi, Maria Luisa Scattoni, Markus Wöhr, Florence I Roullet, Adam M Katz, Danielle N Abrams, David Kalikhman, Harrison Simon, Leuk Woldeyohannes, James Y Zhang, Mark J Harris, Roheeni Saxena, Jill L Silverman, Joseph D Buxbaum, Jacqueline N Crawley
Affiliations
Comparative Study

Reduced excitatory neurotransmission and mild autism-relevant phenotypes in adolescent Shank3 null mutant mice

Mu Yang et al. J Neurosci. .

Abstract

Mutations in the synaptic scaffolding protein gene SHANK3 are strongly implicated in autism and Phelan-McDermid 22q13 deletion syndrome. The precise location of the mutation within the Shank3 gene is key to its phenotypic outcomes. Here, we report the physiological and behavioral consequences of null and heterozygous mutations in the ankyrin repeat domain in Shank3 mice. Both homozygous and heterozygous mice showed reduced glutamatergic transmission and long-term potentiation in the hippocampus with more severe deficits detected in the homozygous mice. Three independent cohorts were evaluated for magnitude and replicability of behavioral endophenotypes relevant to autism and Phelan-McDermid syndrome. Mild social impairments were detected, primarily in juveniles during reciprocal interactions, while all genotypes displayed normal adult sociability on the three-chambered task. Impaired novel object recognition and rotarod performance were consistent across cohorts of null mutants. Repetitive self-grooming, reduced ultrasonic vocalizations, and deficits in reversal of water maze learning were detected only in some cohorts, emphasizing the importance of replication analyses. These results demonstrate the exquisite specificity of deletions in discrete domains within the Shank3 gene in determining severity of symptoms.

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Figures

Figure 1.
Figure 1.
Shank3 homozygous mice exibited impairment in synaptic transmission, induction, and maintenance of long-term potentiation. A, Input–output curve, representing the relationship between stimulus intensity and the size of the fEPSP slope, showed a significant impairment in Shank3 +/− and −/− in comparison with +/+. B, LTP induced by TBS in CA1 in acute hippocampal slices (N = 4–7/genotype) was significantly impaired in slices taken from the Shank3 +/− and −/− mice. C, D, Field recordings in CA1 in acute slices from Shank3 +/+ and +/− and −/− mice showed no differences between genotypes in NMDA receptor-dependent (C) or protein synthesis-dependent LTD (D). Error bars indicate SD.
Figure 2.
Figure 2.
Normal sociability in Shank3 mice tested in the automated three-chambered social approach task. Adult male Shank3 +/+, +/−, −/− and adult female Shank3 +/+, +/−, −/− all showed significant sociability, spending more time in the chamber containing the novel mouse than in the chamber containing the novel object (A, C). Similarly, all genotypes displayed significantly more time sniffing the novel mouse than the novel object (B, D). *p < 0.05, comparison between novel mouse and novel object. Error bars indicate SEM.
Figure 3.
Figure 3.
Adult reciprocal social interactions between male Shank3 and female B6 in freely moving dyads. In a 5 min test session, no significant genotype differences were found on measures of social interactions (scored as cumulative seconds spent by the male subject in sniffing the nose, anogenital, and other body regions of an unfamiliar adult estrus B6 female mouse). A, Cohort 1. C, Cohort 2. Number of ultrasonic vocalizations emitted during the social interaction test session in Cohort 1 (B) and Cohort 2 (D) show a nonsignificant trend toward +/− and −/− emitting lower levels USVs during social interactions than +/+ littermates. Error bars indicate SEM.
Figure 4.
Figure 4.
Adult scent marking and open-field activity in the presence of female urine. Behavioral and ultrasonic vocalization responses to female urinary pheromones in male Shank3 mice. No genotype differences were found in number of USVs emitted in the presence of female urine (A), number of scent marks left in the arena over a 60 min acclimation period, before the introduction of female urine (B), total number of scent marks left in the arena after the acclimation period and a 5 min exposure to a drop female urine (C). D, Total distance traveled during the 5 min test was lower in −/− males than in +/+ males. +/+, N = 13; +/−, N = 12; −/−, N = 17. *p < 0.05 versus +/+. Error bars indicate SEM.
Figure 5.
Figure 5.
Juvenile reciprocal social interaction behaviors in Shank3 mice tested between age day 21 and day 25. Each subject mouse was paired with an unfamiliar B6 partner of the same sex for a 10 min test in a Noldus Phenotyper arena. Each subject mouse was paired with an unfamiliar male B6 partner for a 10 min test in a Noldus Phenotyper arena. In the WT-HET cohort, no genotype differences were found in males on any measures (A–H). In females, +/− exhibited fewer bouts of anogenital sniffing (D), following (E), and push-crawl (F), compared with +/+ controls. In Cohort 1, significant genotype differences were found in males, with +/− exhibiting significantly fewer bouts of nose-to-nose sniffing (I), front approach (J), and push-crawl (N), compared with +/+ controls, and both +/− and −/− exhibiting fewer bouts of following (M) than +/+ controls. No significant genotype differences were found in females on any measures (I–P). In Cohort 2, significant genotype differences were found on several measures. Compared with +/+ controls, +/− exhibited fewer bouts of anogenital sniffing (T), whereas −/− exhibited fewer bouts of nose-to-nose sniffing (Q), push-crawl (T), and more incidences of avoidance to approach (S). Both +/− and −/− exhibited fewer bouts of arena exploration (W) than +/+. In females, no significant differences were found among genotypes on any measures (Q–X). *p < 0.05 compared with +/+. WT-HET cohort: male: +/+, N = 10; +/−, N = 14; female: +/+, N = 12; +/−, N = 16. Cohort 1: male: +/+, N = 15; +/−, N = 15; −/−, N = 14; female: +/+, N = 13; +/−, N = 14; −/−, N = 8. Cohort 2: male: +/+, N = 12; +/−, N = 12; −/−, N = 14; female: +/+, N = 9; +/−, N = 14; −/−, N = 10. Error bars indicate SEM.
Figure 6.
Figure 6.
Normal early developmental milestones and separation-induced pup ultrasonic vocalizations in Shank3 +/+, +/−, and −/−. Analysis of markers of developmental milestones revealed no genotype differences in Shank3 +/+, +/−, and −/− pups between postnatal days 2 and 14 on measures of body weight (A), body length (B), righting reflex (C), eye opening (D), pinna detachment (E), incisor eruption (F). Number of ultrasonic vocalizations emitted by pups separated from the nest did not differ significantly among genotypes, neither in the cohort tested on P8, at Mount Sinai (G), nor in the cohort tested between P4 and P11, at NIMH (H). No sex differences were found in any genotypes, therefore sexes were collapsed for the present analysis. Error bars indicate SEM.
Figure 7.
Figure 7.
Normal olfaction, sensory gating, startle reflex, and nociception in Shank3 mice. A, All three genotypes showed normal olfactory habituation and dishabituation responses to sequential presentations of water, two nonsocial odors, and two social odors. Habituation was significant for all genotypes on three consecutive trials of water presentations. Dishabituation was significant for all genotypes on water to almond. No significant genotype differences in sniff time were detected across trials. B, No genotype differences were found in amplitude of startle response to acoustic stimuli. C, No genotype differences were found in prepulse inhibition of acoustic startle. D, E, No genotype differences were found on latency to respond in the hot-plate test or tail flick test. No sex differences were detected in tests of sensory functions; therefore, sexes were collapsed for the present analysis. Error bars indicate SEM.
Figure 8.
Figure 8.
Increased repetitive self-grooming and normal anxiety-like behaviors in Shank3 mice. A, In Cohort 1, a nonsignificant trend was found for male +/− and −/− to exhibit higher levels of repetitive self-grooming, compared with +/+ controls. No significant differences of trends were found females. Male: +/+, N = 11; +/−, N = 15; −/−, N = 11; female: +/+, N = 9; +/−, N = 13; −/−, N = 9. B, In Cohort 2, male +/− and −/− exhibited significantly higher levels of repetitive self-grooming than +/+. No significant differences were found females. Male: +/+, N = 15; +/−, N = 12; −/−, N = 15; female: +/+, N = 8; +/−, N = 14; −/−, N = 14. *p < 0.05 versus +/+. C–H, No genotype differences were detected in the elevated plus-maze test, on measures of percentage open arm time (C, F), number of open arm entries (D, G), and total number of entries into open + closed arms (E, H). Males: +/+, N = 11; +/−, N = 13; −/−, N = 13; females: +/+, N = 9; +/−, N = 11; −/−, N = 14. I–N, No genotype differences were detected in the light ⇆ dark exploration test, on measures of number of transitions between compartments (I, L), time spent in the dark chamber (J, M), and latency to enter the dark chamber (K, N). Males: +/+, N = 11; +/−, N = 13; −/−, N = 13; females: +/+, N = 9; +/−, N = 11; −/−, N = 14. Error bars indicate SEM.
Figure 9.
Figure 9.
Learning and memory in Shank3 mice. Spatial learning in the Morris water maze test was normal in all three genotypes of male Shank3 mice. No genotype differences were found in either acquisition or reversal training. C, In the probe trial following acquisition training trials, all three genotypes exhibited selective quadrant search, spending more time in the trained quadrant than in two or more other quadrants. D, In the probe trial following reversal training trials, +/+ and +/− spent significantly more time in the trained quadrant than in other quadrants. −/− exhibited a nonsignificant trend for spending more time in the trained quadrant. E, Normal contextual and cued fear conditioning in Shank3 mice. No genotype differences were detected in freezing scores in the posttraining session on day 1. Contextual conditioning (day 2) and cued conditioning (day 3) did not differ significantly among genotypes. No significant sex differences were found; therefore, the sexes were collapsed for the present analysis. F, Impaired novel object recognition in Shank3 −/−. +/+ and +/− spent more time sniffing the novel object than the familiar object, whereas −/− exhibited low sniffing behavior toward both objects. Error bars indicate SEM. *p < 0.05 trained quadrant compared to at least two other quadrants.
Figure 10.
Figure 10.
Rotarod motor learning deficits in male Shank3 null mutant mice. Rotarod scores in Cohorts 1, 2, and 3 were evaluated for latency to either fall or to ride the rod around. Results collected using this criterion revealed significant genotype differences in all three cohorts of males. −/− males exhibited significantly shorter latencies to fall off the rotating rod or to ride the rod around, compared with +/+ males (A, C, E). In females, no significant genotypes differences were found in Cohorts 1 and 2 (B, D). In Cohort 3, −/− females exhibited significantly shorter latencies to fall or ride around compared with +/+ females (F). Cohort 3 was also scored with a more stringent criterion, latency to fall only. A nonsignificant trend was found for Cohort 3 −/− males to fall sooner than +/+ males (G). A similar nonsignificant trend was found in Cohort 3 females (H). *p < 0.05 compared with +/+ on each individual trial. Error bars indicate SEM.
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