Shank3 wild-type and heterozygote breeding pairs were imported from Mount Sinai School of Medicine to the National Institute of Mental Health. Mice were maintained by breeding C57BL/6 wild-type mice with Shank3 heterozygotes and housed in a conventional temperature- and humidity-controlled vivarium. Littermates were housed by sex in mixed genotype groups of two to four per cage on a 12:12-h circadian cycle with lights on at 0600. Behavioral experiments were conducted between 1000 and 1600 in dedicated testing rooms.
Developmental milestones were tested across postnatal days 2-14, including measures of body weight, body length, tail length, pinnea detachment, eye opening, incisor eruption, fur development, righting reflex, negative geotaxis, cliff avoidance, grasping reflex, auditory startle, bar holding, level screen and vertical screen as previously described [43 (link),44 (link)]. In addition, the mice were evaluated in a standard, automated three-chambered social approach task as previously described [44 (link)].
Male-female social interactions were evaluated in a 5-min test session as previously described [43 (link),45 ], with the exception that subject males were group-housed and individually tested in clean cages with clean litter. Each of the 12 wild-type and 14 heterozygous male subject mice, ages 2.5-4 months, was paired with a different unfamiliar estrus C57BL/6J female. A digital closed-circuit television camera (Panasonic, Secaucus, NJ, USA) was positioned horizontally 30 cm from the cage. An ultrasonic microphone (Avisoft UltraSoundGate condenser microphone capsule CM15; Avisoft Bioacoustics, Berlin, Germany) was mounted 20 cm above the cage. Sampling frequency for the microphone was 250 kHz, and the resolution was 16 bits. The entire apparatus was contained in a sound-attenuating environmental chamber (ENV-018V; Med Associates, St. Albans, VT, USA) illuminated by a single 25-Watt red light. Videos from the male subjects were subsequently scored by an investigator uninformed of the subject's genotype on measures of nose-to-nose sniffing, nose-to-anogenital sniffing and sniffing of other body regions, using Noldus Observer software (Noldus Information Technology, Leesburg, VA, USA) as previously described. Ultrasonic vocalizations were played back and spectrograms were displayed using Avisoft software [43 (link),45 ]. Ultrasonic vocalizations were identified manually by two highly trained investigators blinded to genotype information, and summary statistics were calculated using the Avisoft package. Interrater reliability was 95%. Data were analyzed using an unpaired Student's t-test.
Olfactory habituation/dishabituation testing was conducted in male and female Shank3 wild-type and heterozygous mice ages 2.5-4 months using methods previously described [44 (link),46 (link),47 (link)]. Nonsocial and social odors were presented on a series of cotton swabs inserted into the home cage sequentially, each for 2 min, in the following order: water, water, water (distilled water); almond, almond, almond (1:100 dilution almond extract); banana, banana, banana (1:100 dilution artificial banana flavoring); social 1, social 1, social 1 (swiped from the bottom of a cage housing unfamiliar sex-matched B6 mice); and social 2, social 2, social 2 (swiped from the bottom of a second cage housing a different group of unfamiliar sex-matched 129/SvImJ mice). One-way repeated measures ANOVA was performed within each genotype for each set of habituation events and each dishabituation event, followed by a Tukey post hoc test.
Developmental milestones were tested across postnatal days 2-14, including measures of body weight, body length, tail length, pinnea detachment, eye opening, incisor eruption, fur development, righting reflex, negative geotaxis, cliff avoidance, grasping reflex, auditory startle, bar holding, level screen and vertical screen as previously described [43 (link),44 (link)]. In addition, the mice were evaluated in a standard, automated three-chambered social approach task as previously described [44 (link)].
Male-female social interactions were evaluated in a 5-min test session as previously described [43 (link),45 ], with the exception that subject males were group-housed and individually tested in clean cages with clean litter. Each of the 12 wild-type and 14 heterozygous male subject mice, ages 2.5-4 months, was paired with a different unfamiliar estrus C57BL/6J female. A digital closed-circuit television camera (Panasonic, Secaucus, NJ, USA) was positioned horizontally 30 cm from the cage. An ultrasonic microphone (Avisoft UltraSoundGate condenser microphone capsule CM15; Avisoft Bioacoustics, Berlin, Germany) was mounted 20 cm above the cage. Sampling frequency for the microphone was 250 kHz, and the resolution was 16 bits. The entire apparatus was contained in a sound-attenuating environmental chamber (ENV-018V; Med Associates, St. Albans, VT, USA) illuminated by a single 25-Watt red light. Videos from the male subjects were subsequently scored by an investigator uninformed of the subject's genotype on measures of nose-to-nose sniffing, nose-to-anogenital sniffing and sniffing of other body regions, using Noldus Observer software (Noldus Information Technology, Leesburg, VA, USA) as previously described. Ultrasonic vocalizations were played back and spectrograms were displayed using Avisoft software [43 (link),45 ]. Ultrasonic vocalizations were identified manually by two highly trained investigators blinded to genotype information, and summary statistics were calculated using the Avisoft package. Interrater reliability was 95%. Data were analyzed using an unpaired Student's t-test.
Olfactory habituation/dishabituation testing was conducted in male and female Shank3 wild-type and heterozygous mice ages 2.5-4 months using methods previously described [44 (link),46 (link),47 (link)]. Nonsocial and social odors were presented on a series of cotton swabs inserted into the home cage sequentially, each for 2 min, in the following order: water, water, water (distilled water); almond, almond, almond (1:100 dilution almond extract); banana, banana, banana (1:100 dilution artificial banana flavoring); social 1, social 1, social 1 (swiped from the bottom of a cage housing unfamiliar sex-matched B6 mice); and social 2, social 2, social 2 (swiped from the bottom of a second cage housing a different group of unfamiliar sex-matched 129/SvImJ mice). One-way repeated measures ANOVA was performed within each genotype for each set of habituation events and each dishabituation event, followed by a Tukey post hoc test.
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