Methods were conducted as previously described (Bozdagi et al., 2010). Acute hippocampal slices were prepared from 4 to 6-week-old homozygous, heterozygous, and wildtype littermate mice. All experiments were conducted at 32°C on 2-3 slices per animal. Field EPSPs (fEPSPs) were recorded from stratum radiatum in CA1, evoked by stimulation of the Schaffer collaterals every 30 s with 100 μs pulses. LTP was induced with TBS (ten theta bursts of four pulses at 100 Hz, with an interval of 200 ms). LTD was induced by a low-frequency stimulation protocol (900 pulses at 1 Hz for 15 min, or by paired-pulse low-frequency stimulation (1 Hz for 15 min; 50 ms interstimulus interval).

Comprehensive behavioral phenotyping was conducted in male and female mice as previously described (Miyakawa et al., 2001; Paylor et al., 2001; Wrenn et al., 2004; Bailey et al., 2007; Chadman et al., 2008; Silverman et al., 2011). Paw tattoo identification markings were recorded after the behavioral test in all cases, to ensure that investigators were blind to genotype during all test sessions. For all tasks, all genotypes were tested on the same day in randomized order. Pups were tested for developmental milestones and separation-induced ultrasonic vocalizations between postnatal day 2 and postnatal day 14. Juveniles were tested for reciprocal social interactions between 21 and 25 days of age. Young adults were tested in the elevated plus-maze at 6 weeks of age. Adults were tested for sociability in our automated three-chambered social approach task between 10 and 16 weeks of age (WT-HET cohort and Cohort 1) or at 6-7 weeks of age (Cohort 2). One week after the social approach test, male subjects were tested for male-female reciprocal social interaction with concomitant ultrasonic vocalizations. One week later, male subjects were tested for social scent marking with concomitant recording of ultrasonic vocalizations. General health, neurological reflexes, vision, hearing, sensorimotor gating, nociception, open field activity, rotarod motor learning, grip strength, gait, light ↔ dark exploration test, repetitive self-grooming, and novel object recognition were conducted between 8 and 16 weeks of age, in that general order. Fear conditioning and Morris water maze were conducted at the end of this test battery, to avoid confounds of these two relatively stressful cognitive tasks on social and other behaviors. (Steiner et al., 2001; Wrenn et al., 2004; Bevins and Besheer, 2006; Bailey et al., 2007; Moy et al., 2007; Bainbridge et al., 2008; Chadman et al., 2008; McFarlane et al., 2008; Scattoni et al., 2008b; Stack et al., 2008; Yang et al., 2009; Bozdagi et al., 2010; Scattoni et al., 2011; Wohr et al., 2011; Yang et al., 2011a; Yang et al., 2011b).

Measures of general health and neurological reflexes were evaluated in adult mice as previously described (Steiner et al., 2001; Wrenn et al., 2004; Holmes et al., 2005; Bailey et al., 2007; Crawley et al., 2007; Chadman et al., 2008; Yang et al., 2009; Silverman et al., 2011). General health was assessed by fur condition, whisker condition, body weight, body temperature, body and limb tone, and three 15-minute observations of home cage behaviors at different phases of the circadian cycle. Neurological reflexes were assessed by forepaw reaching, righting reflex, trunk curl, whisker twitch, pinna response, eyeblink response and auditory startle. Behavioral reactivity was evaluated as responsiveness to petting, intensity of dowel biting, and level of sonic vocalization when handled. Empty cage behaviors were scored by placing the mouse into a clean, empty cage and noting wild running, stereotypies, and excessive exploration levels.

Measures of neuromuscular and motor functions were evaluated as previously described (Holmes et al., 2001; Wrenn et al., 2004; Bailey et al., 2007; Chadman et al., 2008; Yang et al., 2009; Silverman et al., 2011) Forelimb grip strength was tested with a Strength Meter (Columbus Instruments. Columbus, OH). A metal triangle grasping bar was fitted to a force transducer which connected to the Peak Amplifier. The grasping bar was positioned so that the bar was parallel to the horizontal plane. The mouse was held by the tail and carried toward the apparatus until it spontaneously reached out its forepaws to grab onto the bar. The mouse tail was then gently pulled back, with its body parallel to the horizontal plane. The force at the moment when the mouse lost its grip was recorded as the peak tension. The test was repeated 3 consecutive times for each mouse. The mean of all trials and the largest value from all trials were recorded as grip strength parameters. To measure wire hang, a mouse was placed on a wire cage lid. The cage lid was inverted and held approximately 50 cm above a cage lined with clean bedding, for a maximum of 60 s. Latency to fall was recorded for each mouse. General exploratory locomotion in a novel open field environment was tested in a VersaMax Animal Activity Monitoring System (Accuscan, Columbus, OH) for a 10 min session (Cohort 1) or a 30 min session (Cohort 2). Total distance traversed in the arena, horizontal activity detected by adjacent beam breaks in the lower photocell panels, vertical activity detected by beam breaks in the z upper photocell panels, and time spent in the center were automatically measured by software linked to the photocell detectors. Gait was evaluated using the footprint test. The subject’s forepaws and hind paws were respectively painted with green and red nontoxic paint (Crayola, PA). The mouse was then released onto a 50 cm-long, 20 cm-wide runway covered with a piece of white paper (Strathmore Artist Paper, WI), where the subject walked through a tunnel (10 cm × 10 cm × 30 cm) at the end of the runway. Forepaw width, hindpaw width, and stride length were measured to detect irregular gait or ataxia.

Social approach was assayed in our automated three-chambered apparatus (NIMH Research Services Branch, Bethesda, MD) using methods previously described (Crawley et al., 2007; Chadman et al., 2008; McFarlane et al., 2008; Moy et al., 2008; Yang et al., 2009; Silverman et al., 2010a; Silverman et al., 2010b; Silverman et al., 2011; Yang et al., 2011a; Yang et al., 2011c). The WT-HET cohort and the Cohort 1 were tested between 10 and 16 weeks of age. Cohort 2 was tested at 6-7 weeks of age. Novel target mice are 129/SvImJ mice between 8 and 16 weeks of age, of the same sex as the subjects. The apparatus was a rectangular, three-chambered box made of clear polycarbonate. Retractable doorways built into the two dividing walls controlled access to the side chambers. Number of entries and time spent in each chamber were automatically detected by photocells embedded in the doorways and tallied by the software. The test session began with a 10 min habituation session in the center chamber only, followed by a 10 min habituation to all three empty chambers. Lack of innate side preference was confirmed during the second 10 minute habituation. The subject was then briefly confined to the center chamber while the clean novel object (an inverted stainless steel wire pencil cup, Galaxy, Kitchen Plus, http://www.kitchen-plus.com) was placed in one of the side chambers. A novel mouse previously habituated to the enclosure, was placed in an identical wire cup located in the other side chamber. A disposable plastic drinking cup containing a lead weight was placed on the top of each inverted wire pencil cup to prevent the subject from climbing on top. The side containing the novel object and the novel mouse alternated between the left and right chambers across subjects. After both stimuli were positioned, the two side doors were simultaneously lifted and the subject was allowed access to all three chambers for 10 min. Time spent in each chamber and entries into each chamber were automatically tallied. Time spent sniffing the novel object and time spent sniffing the novel mouse during the 10 min test session were later scored from video recording, by an observer using two stopwatches. The apparatus was cleaned with 70% ethanol and water between subjects. 129Sv/ImJ was used as the target novel mouse because this strain is generally inactive, passive, and does not exhibit aggressive behaviors towards subject mice. Using a minimally active partner is a strategy that allows all approaches to be initiated by the subject mouse only. Up to four subject mice were tested in the same room at the same time, using a high-throughput multi-unit arrangement of the 4 test chambers.

The male-female reciprocal social interaction test was conducted as previously described (Bozdagi et al., 2010; Scattoni et al., 2011). Results of the WT-HET group were published in a previous report (Bozdagi et al., 2010). Cohort 1 was tested between 10 and 16 weeks of age and Cohort 2 was tested at 9-10 weeks of age. All male subjects were sexually naïve at the time of testing. Each freely moving male subject was paired with a freely moving unfamiliar estrus C57BL/6J female for 5 min. 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). The WT-HET group and Cohort 1 were tested under red light illumination (10 lux). To explore whether more pronounced genotype differences would manifest if the reciprocal social interaction test was conducted under a brighter lighting condition, Cohort 2 was tested under white light illumination (250 lux). Videos from the male subjects were subsequently scored by two investigators uninformed of genotypes. Social sniffing (sum of nose-to-nose sniffing, nose-to-anogenital sniffing, and sniffing of other body regions) and exploratory activity were scored using Noldus Observer software (Noldus Information Technology, Leesburg, VA, USA) as previously described (Scattoni et al., 2011). Ultrasonic vocalization spectrograms were displayed using Avisoft software (Bozdagi et al., 2010). Ultrasonic calls were identified manually by two highly trained investigators blind to genotype. Summary statistics were calculated using Avisoft software. Interrater reliability was 95%.

One week after the male-female social interaction test, male subjects of the WT-HET group and Cohort 1 were tested for scent marking behaviors and ultrasonic vocalizations in the presence of female urinary pheromones, as previously described. (Roullet et al., 2011; Wohr et al., 2011). Briefly, the test was conducted in the open field arena and under red light illumination. To reduce background noise during ultrasonic vocalization recording, each open field was covered with a transparent Plexiglas lid equipped with ventilation holes. The floor of the open field was covered with a sheet of specialized drawing paper (Strathmore Drawing Paper Premium, recycled, microperforated, 400 series; Strathmore Artist Papers, Neenah, WI) which effectively absorbed drops of mouse urine. Each male subject was habituated to the paper-lined open field arena for 60 min, in the absence of female urinary odor. To reduce novelty-induced stress during habituation, a small amount of home cage bedding was put in the corner of each open field. At the end of the habituation, each subject was transferred to a clean temporary holding cage. Home cage bedding and fecal deposits were removed from each open field. Scent marks deposited on the paper during habituation were visualized under ultraviolet light using an ultraviolet lamp (Sleeklook Super 18” Black Light, eParty Unlimited, Chatsworth, CA). Visualized scent marks were outlined using a black pencil. 15 μl of fresh urine from a novel estrous B6 female was then pipetted onto the center of the paper. The subject mouse was placed back into the open field for 5 min. Scent marks deposited during the 5 min exposure to fresh female urinary pheromones were outlined with a blue colored pen. The open field was cleaned with 70% ethanol and water between subjects.

Juvenile reciprocal social interactions were tested on postnatal day 21, immediately before pups were weaned from the mother. The test was conducted in the Noldus PhenoTyper Observer 3000 chamber (25 cm × 25cm × 35 cm, Noldus, Leesburg, Virginia) as previously described (Yang et al., 2009; Yang et al., 2011b). The floor of the arena was covered with a 0.5 cm layer of clean bedding. Each subject mouse was singly housed in a clean cage for one hour before the test. After this brief isolation period, the freely moving subject mouse and a freely moving age- and sex-matched B6 partner mouse were simultaneously placed in the arena and their interactions were videotaped for 10 min. Social interactions were scored by a highly trained observer, using the Noldus Observer 5.0 software. Parameters of social behaviors included nose-to-nose sniff (sniffing the nose and snout region of the partner), front approach (moving towards the partner from a distance, in a head-on manner), follow (walking straight behind the partner, keeping pace with the one ahead), nose-to-anogenital sniff (sniffing the anogenital region of the partner), and push-crawl (pushing the head underneath the partner’s body or squeezing between the wall/floor and the partner, and crawling over or under the partner’s body are two similar behaviors which were combined as a single parameter), and avoidance to approach (moving sideways quickly or turning away from the partner when being approached). Besides social behaviors, non-social arena exploration (walking around the arena, rearing, or sniffing the wall) and bouts of self-grooming were scored as measures of exploratory activity and repetitive behavior, respectively. All behaviors were analyzed for frequency of occurrence, i.e. number of bouts.

Cohorts 1 and 2 were tested for assays of developmental milestones every other day from postnatal days 2 to 14, as previously described (Chadman et al., 2008; Scattoni et al., 2008a). Parameters of physical developmental milestones were body weight, body and tail lengths, fur development, eye opening, pinna detachment, and incisor eruption. Parameters of behavioral developmental milestones were righting reflex, negative geotaxis, cliff aversion, forepaw grasping reflex, auditory startle, level screen, screen climbing, and bar holding.

The WT-HET cohort and Cohort 1 were tested for separation-induced ultrasonic vocalizations (USVs) on postnatal days 4, 6, 8 and 11, as previously described (Chadman et al., 2008; Scattoni et al., 2008a). 42 pups from 10 litters were tested in Bethesda. A separate cohort of 27 pups was tested at Mt. Sinai, on postnatal day 8. The pup was gently removed from the nest and placed in a glass bowl (10 cm × 8 cm × 8.5 cm) which was covered by a 0.5 cm layer of fresh bedding. The bowl was immediately placed in a sound-attenuating Styrofoam container, inside a sound-attenuating environmental chamber (ENV-018V; Med Associates, St. Albans, VT, USA). At the end of the 3 min recording session, each pup was weighed, its axillary temperature was taken (Microprobe digital thermometer with mouse probe, Stoelting Co., Wood Dale, IL, USA), and it was returned to the nest. Room temperature was maintained at 23 ± 1°C. Ultrasonic vocalizations were recorded by an Ultrasound Microphone (Avisoft UltraSoundGate condenser microphone capsule CM16, Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies of 10-180 kHz. The microphone was passed through a hole in the middle of the Styrofoam lid and affixed to it, leaving a distance of approximately 20 cm between the microphone and the mouse. Ultrasonic calls were recorded using the Avisoft Recorder (Version 3.4). Sampling rate was 250 kHz, format 16 bit. For acoustical analysis, recordings were transferred to Avisoft SASLab Pro (Version 5.0) and a fast Fourier transformation (FFT) was conducted. Spectrograms were generated with an FFT-length of 1024 points and a time window overlap of 50% (100% Frame, Hamming window). The spectrogram was produced at a frequency resolution of 488 Hz and a time resolution of 1 ms. A lower cut-off frequency of 15 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. Call detection was provided by an automatic threshold-based algorithm and a hold-time mechanism (hold-time: 0.01s). Three highly trained investigators, uninformed of the genotypes, manually scored total number of calls, average call duration, peak sound frequencies (frequencies with the highest sound pressure), and peak amplitude at the peak frequency (maximum of the spectrum). Concordance rate among the scorers was >95%. A separate cohort was tested at Mt. Sinai School of Medicine, on postnatal day 8 only, in a 10 minute session. Identical recording and scoring methods were used for experiments conducted at NIMH and Mt. Sinai School of Medicine.

Evaluation of responses to non-social and social odors was conducted as previously described (Wrenn et al., 2004; Chadman et al., 2008; Yang and Crawley, 2009). Each subject mouse was tested in a clean empty mouse cage containing a thin layer of fresh pinewood bedding. Odor-saturated cotton-tipped swabs (6 inches length, Solon Manufacturing Company, Solon, Maine) were used to deliver odor stimuli. To reduce novelty-induced exploratory activities, each subject was first habituated for 45 minutes in the empty testing cage containing one clean dry cotton swab. The test consisted of fifteen sequential 2 min trials: three presentations of plain tap water, three presentations of almond odor (prepared from almond extract, McCormick, Hunt Valley, MD; 1:100 dilution), three presentations of banana odor (prepared from imitation banana flavor, McCormick, Hunt Valley, MD; 1:100 dilution), three presentations of social odor from social cage 1, three presentations of social odor from social cage 2. Water, almond odor, and banana odor stimuli were prepared by dipping the cotton tip briefly into the solution. Social odor stimuli were prepared by wiping a swab in a zig-zag motion across a soiled cage of unfamiliar mice of the same sex. For each subject, one soiled cage of 129/SvImJ mice and one soiled cage of B6 mice were the sources of the two social odors. Time spent sniffing the swab was quantified with a stopwatch by an observer sitting 2 meters away from the testing cage. Sniffing was scored when the nose was within 1 cm of the cotton swab. The intertrial interval was approximately 1 minute.

Acoustic startle and prepulse inhibition of acoustic startle were measured using the SR-Laboratory System (San Diego Instruments, San Diego, CA) as described previously (Holmes et al., 2001; Wrenn et al., 2004; Chadman et al., 2008; Silverman et al., 2011). Test sessions began by placing the mouse in the Plexiglas holding cylinder for a 5-min acclimation period. For the next 8 min, mice were presented with each of six trial types across six discrete blocks of trials, for a total of 36 trials. The intertrial interval was 10–20 s. One trial type measured the response to no stimulus (baseline movement). The other five trial types measured startle responses to 40 ms sound bursts of 80, 90, 100, 110 or 120 dB. The six trial types were presented in pseudorandom order such that each trial type was presented once within a block of six trials. Startle amplitude was measured every 1 ms over a 65 ms period beginning at the onset of the startle stimulus. The maximum startle amplitude over this sampling period was taken as the dependent variable. Background noise level of 70 dB was maintained over the duration of the test session. For prepulse inhibition of acoustic startle, mice were presented with each of seven trial types across six discrete blocks of trials for a total of 42 trials, over 10.5 min. The intertrial interval was 10–20 s. One trial type measured the response to no stimulus (baseline movement) and another measured the startle response to a 40 ms 110 dB sound burst. The other five trial types were acoustic prepulse stimulus plus acoustic startle stimulus trials. The seven trial types were presented in pseudorandom order such that each trial type was presented once within a block of seven trials. Prepulse stimuli were 20 ms tones of 74, 78, 82, 86, and 92 dB intensity, presented 100 ms prior to the 110 dB startle stimulus. Startle amplitude was measured every 1 ms over a 65 ms period, beginning at the onset of the startle stimulus. The maximum startle amplitude over this sampling period was taken as the dependent variable. A background noise level of 70 dB was maintained over the duration of the test session.

Response to thermal stimulation of the feet and tail was measured as previously described (Wrenn et al., 2004; Chadman et al., 2008; Silverman et al., 2010a; Silverman et al., 2010c). For the hotplate test, the mouse was placed on the arena surface which was kept at a constant temperature of 55°C (IITC Life Science Inc., Woodland Hills, CA). Latency to first response, such as licking or shaking paws, was recorded. To prevent tissue damage, a cut-off latency of 30 seconds was applied. For the tail flick test, mice were gently restrained with the tail placed in the groove of the tail flick monitor (Columbus Instruments, Columbus, OH). An intense photobeam was directed at the tail. Latency for the mouse to move its tail out of the path of the beam was timed automatically by the apparatus. To prevent tissue damage, a cut-off latency of 10 seconds was applied.

Mice were scored for spontaneous grooming behaviors when placed individually in a clean, empty mouse cage without bedding, using methods previously described (Yang et al., 2007; McFarlane et al., 2008). Each mouse was given a 10 min habituation period in the empty cage and then rated for 10 min for cumulative time spent grooming all body regions. The test session was videotaped and scored later by two trained observers uninformed of the genotypes. The interrater reliability was > 95%.

Cohort 2 was tested for anxiety-like behaviors in the elevated-plus maze test and the light↔dark exploration test, using methods previously described (Crawley and Goodwin, 1980; Holmes et al., 2001; Bailey et al., 2007; Chadman et al., 2008; Yang et al., 2009). The elevated plus-maze consisted of two open arms (30cm × 5cm) and two closed arms (30 × 5 × 15 cm) extending from a central area (5 × 5 cm). Room illumination was approximately 30 lux. The test began by placing the subject mouse in the center, facing a closed arm. The mouse was allowed to freely explore the maze for 5 min. Time spent in the open arms and closed arms, and number of entries into the open arms and closed arms, were scored by an investigator, using Observer software (Noldus Information Technology, Leesburg, Virginia). The light ↔ dark exploration test was conducted in an automated chamber (NIMH Research Services Branch, Bethesda, MD). The test began by placing the mouse in the light compartment facing away from the partition. The animal was allowed to freely explore the apparatus for 10 min. Time spent in each compartment and number of transitions between the light (350 lux) and dark (3 lux) compartments were automatically recorded.

Spatial learning and reversal learning were assessed in the Morris water maze using procedures and equipment as previously described (Miyakawa et al., 2001; Wrenn et al., 2004; Bailey et al., 2007; Chadman et al., 2008). The apparatus was a circular pool (120 cm diameter) filled 45 cm deep with tap water rendered opaque with the addition of non-toxic white paint (Crayola, Easton, PA). Distal room cues were black and white cardboard patterns on the walls, approximately 1 meter from the circumference of the pool. Trials were videotaped and scored with WaterMaze video tracking software (Actimetrics, Inc., Wilmette, IL). Acquisition training consisted of 4 trials a day for approximately 5 days. Each training trial began by lowering the mouse into the water close to the pool edge, in a quadrant that was either right of, left of, or opposite to, the target quadrant containing the platform. The start location for each trial was alternated in a semi-random order for each mouse. The hidden platform remained in the same quadrant for all trials during acquisition training for a given mouse, but varied across subject mice. Mice were allowed a maximum of 60 s to reach the platform. A mouse that failed to reach the platform in 60 s was guided to the platform by the experimenter. Mice were left on the platform for 15 s before being removed. After each trial, the subject was placed in a cage lined with absorbent paper towels and allowed to rest under an infrared heating lamp for 60 s. Acquisition training continued for 5 days, or until the +/+ control group reached criterion. Three hours after the completion of training on day 5, the platform was removed and mice were tested in a 60 s probe trial, to confirm that their spatial learning was acquired by using distal environmental room cues. Parameters recorded during training days were latency to reach the platform, total distance traveled, swim speed, and thigmotaxis. Time spent in each quadrant and number of crossings over the trained platform location and over analogous locations in the other quadrants was used to analyze probe trial performance. Cohort 1 reversal training began 3 days after the completion of acquisition training. Cohort 2 reversal training began 35 days after the completion of acquisition training, to evaluate the robustness of the reversal phenotypes with different intervals after the initial training. In reversal training trials, the hidden platform was moved to the quadrant opposite to its location during acquisition training, for each mouse. Procedures for reversal training and probe trial were the same as in the initial acquisition phase.

Cohorts 1 and 2 were tested on standard delay fear conditioning as previously described (Paylor et al., 2001; Chadman et al., 2008). Training and conditioning tests took place in two identical chambers (Med Associates, E. Fairfield, VT) that were calibrated to deliver identical footshocks. Each chamber was 30 cm × 24 cm × 21 cm with a clear polycarbonate front wall, two stainless side walls, and a white opaque back wall. The bottom of the chamber consisted of a removable grid floor with a waste pan underneath. When placed in the chamber, the grid floor connected with a circuit board for delivery of scrambled electric shock. Each conditioning chamber was inside a sound-attenuating environmental chamber. A camera mounted on the front door of the environmental chamber recorded test sessions, which were later scored automatically, using VideoFreeze software (Med Associates, E. Fairfield, VT). For the training session, each chamber was illuminated with a white house light. An olfactory cue was added by dabbing a drop of imitation banana flavoring solution (1:100 dilution in water) on the metal tray beneath the grid floor. The mouse was placed in the test chamber and allowed to explore freely for 2 min. A pure tone (5kHz, 80 dB), which served as the conditioned stimulus was played for 30 s. During the last 2 s of the tone, a footshock (0.5 mA) was delivered as the unconditioned stimulus (US). Each mouse received three CS-US pairings, separated by 90 s intervals. After the last CS-US pairing, the mouse was left in the chamber for another 120 s, during which freezing behavior was scored by the VideoFreeze software (Med Associates, E. Fairfield, VT). The mouse was then returned to its home cage. Contextual conditioning was tested 24 h later in the same chamber, with the same illumination and olfactory cue present but without footshock. Each mouse was placed in the chamber for 5 min, in the absence of CS and US, during which freezing was scored. The mouse was then returned to its home cage. Cued conditioning was conducted 48 h after training. Contextual cues were altered by covering the grid floor with a smooth white plastic sheet, inserting a piece of black plastic sheet bent to form a vaulted ceiling, using near infrared light instead of white light, and dabbing vanilla instead of banana odor on the floor. The session consisted of a 3 min free exploration period followed by 3 min of the identical CS tone (5kHz, 80dB). Freezing was scored during both 3 min segments. The mouse was then returned to its home cage. The chamber was thoroughly cleaned of odors between sessions, using 70% ethanol and water.

The novel object recognition test was conducted with Cohort 2 mice in the open field arena, using methods previously described (Bevins and Besheer, 2006). The experiment consisted of two habituation sessions, a 10 min familiarization session, and a 5 min recognition test. On day 1, each subject was habituated to a clean empty open field arena for 30 minutes. Twenty four hours later, each subject was returned to the open field arena for a second habituation phase, this time for 10 min. The mouse was then removed from the open field and placed in a clean temporary holding cage for approximately 2 min. Two identical objects were placed in the arena. Each subject was returned to the open field in which it had been habituated, and allowed to explore freely for 10 min. After the familiarization session, subjects were returned to their holding cages, which were transferred from the testing room to a nearby holding area. The open field was cleaned with 70% ethanol and let dry. One clean familiar object and one clean novel object were placed in the arena, where the two identical objects had been located during in the familiarization phase. One hour after the familiarization session, each subject was returned to its open field for a 5 min recognition test, during which time it was allowed to freely explore the familiar object and the novel object. The familiarization session and the recognition test were videotaped and subsequently scored by two highly-trained investigators, uninformed of genotype, whose inter-rater reliability was ≥95%. Object investigation was defined as time spent sniffing the object when the nose was oriented toward the object and the nose-object distance was 2 cm or less. Recognition memory was defined as spending significantly more time sniffing the novel object than the familiar object. Total time spent sniffing both objects was used as a measure of general exploration. Time spent sniffing two identical objects during the familiarization phase confirmed the lack of an innate side bias.

Motor learning was assessed using a mouse accelerating rotarod (Ugo Basile, Collegeville, PA). Mice were placed on the rotating drum that accelerated from 4 to 40 revolutions per minute over 5 minutes. Cohort 1 was tested for three trials a day, for two consecutive days. Cohorts 2 and 3 were tested for two trials a day, for three consecutive days. The intertrial interval was 1 hour for all three cohorts. Rotarod scores were scored for latency to either fall or ride the rod around for all three cohorts. Cohort 3 was also scored for latency to fall, a more stringent criterion.

Adult Shank3 mice were evaluated for general health and neurological reflexes between 10 and 16 weeks of age. Similar results were obtained in three cohorts. Representative data from Cohort 1 are shown in Table 1. Mice from Cohort 2 were used for one parameter, grip strength. The three genotypes scored similarly on measures of body weight, neurological reflexes, motor functions including open field activity, wire hang and gait, and responsivity to handling. No balding patches were observed in mice evaluated during this age range. Observations of home cage behaviors revealed no abnormalities in general activity, group huddling, and nesting. No excessive aggressive behaviors were observed in adult males. Nursing females showed normal maternal behaviors. Average litter size was 7.2 pups (range of 3 to 11). Bald patches were occasionally observed in +/−, especially in mice older than 10 months. Forelimb grip strength did not differ significantly across genotypes in males (F_{2, 32} = 0.065, NS) (Table 1) or females (F_{2, 27}= 2.53, NS, data not shown). Footprint analysis revealed no significant genotype differences in gait, as measured by forepaw width (F_2,24=0.73, NS), hindpaw width (F_2,24=0.05, NS), and stride length (F_2,24=2.54, NS). In Cohort 1, open field exploratory locomotion did not differ significantly across genotypes on measures of total distance traveled (F_2,40=1.64, NS), vertical activity (F_2,40=0.01, NS), and center time (F_2,40=0.90, NS). A significant genotype effect was found in horizontal activity (F_2,40=4.13, p<.05), with −/− showing lower levels of horizontal activity than +/+ (p<.05) (Table 1). In females (data not shown), no significant genotype differences were found on total distance traveled (F_2,27=1.96, NS), horizontal activity (F_2,27=1.46, NS), vertical activity (F_2,27=0.96, NS), and center time (F_2,27=0.94, NS). In Cohort 2 (data not shown), open field exploratory locomotion in a 30 min session was somewhat lower in mutants as compared to wildtype littermates on some parameters. In Cohort 2 males, small but significant genotype differences were found in total distance traveled (F_2,33=3.57, p<.05) and horizontal activity (F_2,33=5.63, p<.05). Post hoc comparisons revealed that −/− exhibited lower levels of horizontal activity than +/+ controls (p<.05). No significant genotype differences were found in vertical activity (F_2,33=1.96, NS) and center time (F_2,33=2.99, NS). In Cohort 2 females, significant genotype differences were found in total distance traveled (F_2,34=3.58, p<.05), horizontal activity (F_2,34=6.90, p<01), and vertical activity (F_2,34=6.20, p<.01). Post hoc comparisons revealed that +/− females exhibited higher levels of horizontal activity (p<.05) and vertical activity (p<.01) as compared to +/+. Center time did not differ significantly across genotypes (F_2,34=1.47, NS). While reasons for the discrepancy in open field results between cohorts remain unknown, noise from construction of a nearby building occurred sporadically during some of the days when open field testing was conducted in Cohort 2.

Normal sociability was found in all three cohorts of Shank3 mice of all genotypes. Figure 2 displays the significant sociability detected in adult Shank3 mice of Cohort 1. In both sexes, significantly more time was spent in the chamber containing the novel mouse than in the chamber containing the novel object, and more time was spent sniffing the novel mouse than the novel object. Chamber time: (A) male +/+, F_1,17=42.88, p<.001; male +/−, F_1,20=6.00, p<.01; male −/−, F_1,20=5.76, p<.05; (C) female +/+, F_1,9=10.32, p<.01; female +/−, F_1,16=33.00, p<.001; female −/−, F_1,10=36.01, p<.001. Sniff time: (B) male +/+, F_1,17=33.56, p<.001; male +/−, F_1,19=73.45, p<.001; male −/−, F_1,20=52.65, p<.001; (D) female +/+, F_1,9=106.53, p<.001; female +/−, F_1,16=83.86, p<.001; female −/−, F_1,10=74.55, p<.001. Similarly, all three genotypes of Cohort 2 displayed normal sociability (data not shown). Chamber time: male +/+, F_1,10=82.00, p<.001; male +/−, F_1,11=22.11, p<.001; male −/−, F_1,10=26.38, p<.001; female +/+, F_1,9=7.60, p<.05; female +/−, F_1,10=30.00 p<.001; female −/−, F_1,15=14.33, p<.01. Sniff time: male +/+, F_1,10 =80.00, p<.001; male +/−, F_1,11=60.22, p<.001; male −/−, F_1,10=55.180.00, p<.001; female +/+, F_1,9=8.60, p<.05; female +/−, F_1,10=30.44 p<.001; female −/−, F_1,15=48.7, p<.001. Similar sociability was seen in the previously published WT-HET group (data not shown): Chamber time: male +/+, F_1,13=8.50, p<.01; male +/−, F_1,11=44.72, p<.001; female +/+, F_1,7=8.13, p<.05; female +/−, F_1,12=31.32, p<.001. Sniff time: male +/+, F_1,13=63.00, p<.001; male +/−, F_1,11=45.82, p<.001; female +/+, F_1,7=8.97, p<.05; female +/−, F_1,12=50.25, p<.001.

Figure 3 shows duration of total social sniffing and number of ultrasonic vocalizations in male Shank3 subjects paired with unfamiliar estrous B6 females in a 5 min social interaction test. In Cohort 1, minor trends were seen for male +/− and −/− to show less total social sniffing (Figure 3A, F_2,41=1.55, p=.23, NS) and fewer ultrasonic vocalizations (Figure 3B, F_2,41=1.50, p=.24, NS) than +/+ controls. In Cohort 2, no trends or significant genotype differences were seen for total social sniffing (Figure 3C, F_2,41=0.11, NS) or ultrasonic vocalizations (Figure 3D, F_2,41=0.47, NS).

Figure 4 shows scent marking behaviors and ultrasonic vocalizations in Cohort 1 males in response to 15 μl of female urine deposited in the center of an open field arena. During the 5 min urine exposure, no significant genotypes differences were found in (A) number of ultrasonic vocalizations (F_2,36=1.04, NS) and (C) number of scent marks deposited in the open field (F_2,36=0.81, NS). A significant genotype effect was found in (D) total distance traveled (F_2,36=4.02, p<.05). Post hoc analysis revealed that total distance travelled was shorter in −/− as compared to +/+ during urine exposure (p<.05) During the 60 min habituation period before the introduction of the female urine, no significant genotype differences were found in (B) number of scent marks deposited in the clean open field (F_2,36=2.51, NS). In the WT-HET Cohort (data not shown), male +/− emitted significantly fewer ultrasonic vocalizations than +/+, in the presence of female urinary pheromone (F_1,24=4.37, p<.05). No significant genotypes differences were found in the number of scent marks deposited in the open field during the 5 min urine exposure (F_1,19=2.40, NS). The effect of genotype was not significant for total distance traveled in the open field during female urine exposure (F_1,19=0.13, NS). During the 60 min of habituation period in the empty open field, Shank3 +/− males deposited fewer scent marks than +/+ controls (F_1,19=4.58, p<.05). These results indicate a partial reduction in responses to social olfactory cues, whose significance was inconsistent across cohorts.

Figure 5 shows results of reciprocal social interactions in three cohorts of Shank3 juveniles. In the WT-HET Cohort, deficits were found on some but not all parameters. Shank3 +/− juveniles paired with B6 partners were lower on some parameters as compared to +/+. Female +/− showed significantly fewer bouts of (D) nose-to-anogenital sniffing (F_1,26=4.78, p<.05), (E) following (F_1,26=8.49, p<.01), and (F) push-crawls (F_1,26=4.49, p<.05), as compared to +/+ controls. No significant differences were found in (A) nose-to-nose sniff (F_1,26=0.27, NS), (B) front approach (F_1,26=0.07, NS), (G) arena exploration (F_1,26=1.04, NS), and (H) bouts of self-grooming (F_1,26=1.86, NS). Males showed no significant genotype differences on nose-to-nose sniff (F_1,26=0.74, NS), front approach (F_1,26=0.65, NS), avoidance when approached (F_1,26=2.95, NS), nose-to-anogenitial sniff (F_1,26=0.00, NS), following (F_1,26=1.06, NS), push-crawl (F_1,26=0.80, NS), arena exploration (F_1,26=1.60, NS), and bouts of self-grooming (F_1,26=0.40, NS). In Cohort 1, significant genotype differences were found in males, on (I) nose-to-nose sniff (F_2,46=3.50, p<.05), (J) front approach (F_2,46=11.30, p<.01), (M) following (F_2,46=12.76, p<.01), and (N) push-crawl (F_2,46=3.67, p<.05). Post hoc comparisons with the Scheffe test indicated that the significant differences were between +/− and +/+ on each of these parameters (p<.05), and between −/− and +/+ on following (p<.01). Trends for genotype differences were also seen in (K) avoidance when approached (F_{2, 46}=2.95, NS) and (L) nose-to-anogenitial sniff (F_2,46=1.88, NS). No significant genotype differences were found in the non-social parameters: (O) arena exploration (F_2,46=0.21, NS) and (P) bouts of self-grooming (F_2,46=0.06, NS). In females, no significant genotype differences were found on any parameters, including nose-to-nose sniff (F_2,32=1.45, NS), front approach (F_2,32=2.92, NS), avoidance when approached (F_2,32=1.54, NS), anogenital sniff (F_2,32=0.11, NS), following (F_2,32=1.11, NS), push-crawl (F_2,32=1.28, NS), arena exploration (F_2,32=0.47, NS), and bouts of self-grooming (F_2,32=0.52, NS). In Cohort 2, significant genotype differences were found in males, on (Q) nose-to-nose sniff (F_2,35=3.76, p<.05), (S) avoidance when approached (F_2,35=6.82, p<.01), (V) push-crawl (F_2,35=4.93, p<.01), and (W) arena exploration (F_2,35=8.72, p<.01). As compared to +/+ males, −/− males exhibited significantly fewer bouts of nose-to-nose sniffs (p<.05), more avoidances (p<.01), and fewer push-crawls (p<.05). +/− and −/− exhibited less arena exploration than +/+ controls (p<.01 for each comparison). Genotype differences were not significant for (R) front approach (F_2,35=.52, NS), (T) nose-to-anogenitial sniff (F_2,35=1.24., NS), (U) following (F_2,35=0.25, NS),) and (X) bouts of self-grooming (F_2,35=0.27, NS). Females showed minimal genotype differences in juvenile social interactions. No significant genotype effects were found in bouts of nose-to-nose sniff (F_2,30=0.73, NS), front approach (F_2,30=2.62, NS), avoidance to approach (F_2,30=0.82, NS), follow (F_2,30=1.61, NS), push-crawl (F_2,30=2.26, NS), arena exploration (F_2,30=0.38, NS), and self-grooming (F_2,30=0.10, NS). A significant genotype effect was found in nose-to-anogenital sniff (F_2,30=3.40, p<.05). However, post hoc comparisons did not reveal significant differences among the three genotypes on this measure. Taken together, these results show a profile of reduced juvenile reciprocal social interactions in Shank3 null and heterozygous mutant males on some parameters. In contrast, females displayed comparatively normal juvenile reciprocal social interactions.

Figure 6 shows normal early physical development and separation-induced ultrasonic vocalizations in Shank3 pups of Cohort 1. No significant genotypes differences were detected on measures of early developmental milestones, including (A) body weight (F_2,55=0.34, NS), (B) body length (F_2,55=0.73, NS), (C) righting reflex (F_2,55=0.36, NS), (D) pinna detachment (F_2,55=0.21, NS), (E) incisor eruption (F_2,55=0.76, NS), and (E) eye opening (F_2,22=0.74, NS). Similar results were found in the WT-HET Cohort (data not shown). No significant genotypes differences were detected on measures of early developmental milestones, including body weight (F_1,22=1.08, NS), body length (F_1,22=0.003, NS), righting reflex(F_1,22=52, NS), pinna detachment (F_1,22=0.18, NS), incisor eruption (F_1,22=1.29, NS), and eye opening (F_1,22=1.88, NS). Separation-induced ultrasonic vocalizations did not differ among genotypes in three cohorts of Shank3 pups. In +/+ and +/− pups tested at Mt. Sinai on age day 8, no significant genotypes differences were detected on total number of ultrasonic calls over a 10 minute test session (t₂₅=1.587, NS), nor over the first 3 minutes of the 10 min session (Figure 4G, t₂₅=0.051, NS). Similar results were found in pups of the WT-HET Cohort tested at NIMH, on postnatal days 4, 6, 8, and 11, for 3 minutes on each day (data not shown). Repeated Measures ANOVAs revealed no significant genotype differences in total number of ultrasonic calls (F_1,25=1.81, NS), average call duration (F_1,25=0.54, NS), or average peak call frequency (F_1,25=1.14, NS). A significant genotype difference was found in average peak call amplitude (F_1,25=5.87, p<.05), with calls of +/− pups being at higher amplitudes as compared to those of +/+ littermate controls in the WT-HET Cohort (p<.05). Ultrasonic vocalizations during juvenile interactions did not differ between genotypes in the WT-HET Cohort, data not shown. Similarly, no significant genotype differences were found on total number of calls in Cohort 1 (Figure 4H, F_2,39=0.69, NS). No genotype differences were found on average call duration (F_2,39=0.24, NS) and peak call amplitude (F_2,39=1.33, NS) (data not shown). A significant main effect of genotype was detected for average peak call frequency (F_2,39=4.65, p<.05, data not shown). Post hoc analysis with the Newman-Keuls test indicated that peak call frequency was lower in +/− pups as compared to −/− pups (p<.05). Neither +/− nor −/− pups differed significantly from +/+ pups on this measure (data not shown).

Figure 7 shows normal olfaction, sensory gating, startle response, and pain sensitivity in Shank3 mice. Figure 7A shows intact olfactory abilities in Shank3 mice of all three genotypes on the olfactory habituation/dishabituation task. Habituation, indicated by decreased time spent in sniffing the sequence of three same odors, and dishabituation, indicated by increased time sniffing the different odor, was normal for social and nonsocial odor cues in all three genotypes. Habituation to water, main effect: +/+ F_2,10= 68.743, p < .001; +/− F_2,11= 22.300, p <.001; −/− F_2,12 = 41.843, p <.001; dishabituation water to almond: + F_1,10= 91.827, p <.001; +/− F_1,11 = 26.547, p < .001; −/− F_1,12= 56.314, p <.001. Habituation to almond, +/+ F_2,10= 19.97, p <.001; +/− F_2,11= 115.215, p <.001; −/− F_2,12= 42.54, p <.001; dishabituation almond to banana: +/+ F_1,10= 22.518, p <.001; +/− F_1,11= 96.201, p <.001; −/− F_1,12 = 44.692, p <.001. Habituation to banana, +/+ F_2,10= 23.047, p <.001; +/− F_2,11= 54.941, p <.001; −/− F_2,12= 41.524, p <.001; dishabituation banana to social odor 1: +/+ F_1,10= 39.336, p <.001; +/− F_1,11= 82.609, p <.001; −/− F_1,12=25.816, p <.001. Habituation to social odor 1, +/+ F_2,10= 27.468, p <.001; +/− F_2,11= 34.912, p<.001; −/− F_2,12 = 4.754, p<.05. Dishabituation social odor 1 to social odor 2: +/+ F_1,10= 32.797, p<.001; +/− F_1,11= 26.230, p<.001; −/− F_1,12= 20.636, p<.001. Habituation to social odor 2, +/+ F_2,10= 29.474, p<.001; +/− F_2,11= 14.307, p<.001; −/− F_2,12=26.636, p<.001. No significant genotype differences were detected across trials. No sex differences were detected and sexes were collapsed for statistical analysis. Figure 7B shows normal acoustic startle responses at 5 different decibel levels across genotypes (F_2,62=0.45, NS). Figure 7C show normal sensorimotor gating in prepulse inhibition of acoustic startle at all prepulse levels across genotypes (F_2,65=0.05, NS). Figure 7D and E show pain sensitivity in Shank3 mice. No significant genotype differences were detected on hotplate (F_2,63=0.70, NS) and tail flick (F_2,61=1.18, NS). No sex differences were found in the hotplate test and the tail flick test.

Figure 8A shows repetitive self-grooming in Cohort 1. A trend was seen in males for +/− and −/− to show higher levels of self-grooming than +/+ in a 10 min test conducted in an empty cage (F_2,34=2.02, p=.15, NS). In females, self-grooming was similar across genotypes (F_2,28=0.18, NS). In Cohort 2 (Figure 8B), male Shank3 mutants displayed high self-grooming (F_2,39=13.56, p<.001), with +/− (p<.05) and −/− (p<.01) exhibiting significantly higher levels of self-grooming than +/+. No significant genotype differences were found in females (F_2,33=1.25, NS).

No genotype differences were detected on measures of anxiety-related behaviors in Shank3 mice. On the elevated plus-maze (Figure 8C-H), no significant genotype differences were found in either males or females on percent time spent in the open arms (males: F_2,34=0.73, NS; female: F_2,31=2.06, NS), open arm entries (males: F_2,34=2.12, NS; female: F_2,31=2.99, NS), or total arm entries (males: F_2,34=0.33, NS; female: F_2,31=1.97, NS). In the light ↔ dark exploration test (Figure 8I-N), no significant genotype differences were found in either males or females on number of transitions (males: F_2,22=0.91, NS; female: F_2,29=1.62, NS), time spent in the dark compartment (males: F_2,22=0.58, NS; female: F_2,29=0.88, NS), or latency to enter the dark compartment (males: F_2,22=1.41, NS; female: F_2,29=1.54, NS).

Figure 9 shows performance of Cohort 2 males in the Morris water maze spatial learning task. During the acquisition training, all three genotypes showed similar learning curves over 5 days. The main effect of day was significant for latency to reach the platform within each of the three genotypes (Figure 9A, +/+, F_4,28=12.69, p<.001; +/−, F_4,36=30.95, p<.001; −/−, F_4,28=12.18, p<.001). Latency to reach the hidden platform was not significant across genotypes (F_2,23= 1.70, NS). Swim speed was similar across genotypes, with no differences in the main effect of day (data not shown, +/+, F_4,28=1.30, NS; +/−, F_4,36=1.22, NS; −/−, F_4,28=2.13, NS) and in the genotype effect (F_2,23=0.49, NS). In the probe trial conducted 3 hours after the final training trial, all genotypes spent significantly more time in the training quadrant as compared to the other three quadrants (Figure 9C, +/+, F_3,21=12.87, p<.001; +/−, F_3,30=11.70, p<.001; −/−, F_3,18=6.17, p<.01). Selective quadrant search was also measured by platform crossings. +/− and −/− made significantly more crossings over the previous platform location than equivalent locations in at least two other quadrants. +/+ showed a trend for selective quadrant search (data not shown, +/+, F_3,21=2.01, p=.14, +/−, F_3,30=9.72, p<.001; F_3,18=5.10, p<.05). During reversal training, which started 35 days after the completion of acquisition trials, all three genotypes acquired the task within 4 days. In all three genotypes, the main effect of day was significant for latency to reach the new hidden platform (Figure 9B, F_3,21=9.75, p<.001; +/− F_3,24=13.85, p<.001; −/−, F_3,18=4.10, p<.05). The genotype effect for latency to reach the hidden platform was not significant (F_2,21=0.49, NS). The main effect of day was not significant for swim speed in all three genotypes (data not shown, +/+, F_3,21=0.37, NS; +/−, F_3,24=1.07, NS_; −/−, F_3,18=1.32, NS). In the probe trial conducted 3 hours after the last reversal training trial, +/+ and +/− showed significant selective quadrant search, spending more time in the new training quadrant than time in at least two other quadrants, whereas the −/− group did not (Figure 9D, +/+, F_3,21=10.64, p<.01; +/− F_3,24=6.06, p<.01; −/−, F_3,18=2.29, NS). Similarly, +/+ and +/− made more crossings over the new platform location than equivalent locations in at least two other quadrants, whereas the −/− did not (data not shown, +/+, F_3,21=3.07, p<.05; +/− F_3,24=4.86, p<.01; −/−, F_3,18=0.58, NS). Similar results were found in Cohort 1 male mice (data not shown). All three genotypes displayed similar learning curves across the 5 days of acquisition. The main effect of day was significant for latency to reach the platform within each of the three genotypes (+/+, F_4,48=24.50, p<.001; +/−, F_4,52=25.23, p<.001; −/−, F_4,52=34.23, p<.001). A significant genotype effect was found for latency to reach the hidden platform (F_2,38=5.197, p<.01). Scheffe post hoc tests indicated a significant difference between +/− and −/− (p<.01), but neither +/− nor −/− was significantly different from +/+ controls. Similarly, the main effect of day was significant for swim speed in all three genotypes (+/+, F_4,48=3.31, p<.05; +/−, F_4,52=2.89, p<.05; −/−, F_4,52=4.87, p<.01), and the genotype factor was significant for swim speed (F_2,38=3.62, p<.05). Scheffe post hoc tests revealed that −/− swam at a significantly slower speed than +/+ controls (p<.01), along with a trend for slower swim speed in +/− as compared to +/+ (p=0.057, NS). In the probe trial conducted 3 hours after the final training trial, all genotypes displayed selective quadrant search, spending significantly more time in the training quadrant as compared to the other three quadrants (+/+, F_3,36=13.37, p<.001; +/−, F_3,39=20.84, p<.001; −/−, F_3,39=4.56, p<.05). +/+ and +/− made significantly more crossings over the previous platform location than equivalent locations in the other three quadrants. −/− showed a nonsignificant trend for more crossings over the previous platform location (+/+, F_3,36=13.08, p<.001; +/−, F_3,39=27.63, p<.001; F_3,39=2.60, p=.066). During reversal training, which started 3 days after the completion of acquisition trials, all three genotypes acquired the task within 4 days. In all three genotypes, the main effect of day was significant for latency to reach the new hidden platform (+/+, F_3,21=21.22,p<.001; +/− F_3,24=9.57, p<.001; −/−, F_3,24=11.31, p<.001) and for swim speed (+/+, F_3,21=6.56, p<.01; +/− F_3,24=5.11, p<.01; −/−, F_3,24=2.95, p<.05). In the probe trial conducted 3 hours after the last reversal training trial, all three genotypes showed significant selective quadrant search, spending more time in the new training quadrant than the other three quadrants (+/+, F_3,21=5.87, p<.01; +/− F_3,24=6.39, p<.01; −/−, F_3,24=9.76, p<.01) and making more crossings over the new platform location than equivalent locations in the other quadrants (+/+, F_3,21=19.67, p<.01; +/− F_3,24=3.97, p<.05; −/−, F_3,24=7.79, p<.001). Thus, it appears that Shank3 mutant mice acquired the hidden platform location using distal spatial cues, while showing deficits in the reversal probe trial, an impairment that was inconsistently across cohorts.

As shown in Figure 9, no genotype differences were found on contextual and cued fear conditioning in Cohort 2 mice. Since no sex differences were found on any measures, data were analyzed with sexes combined. In the training session, freezing behavior before presentations of cue-shock pairings was minimal and did not differ across genotypes (F_2,29=0.64, NS, data not shown). No significant genotype differences in post-shock freezing were found on the training day (F_2,29=1.97, NS). In the contextual conditioning test, no significant genotype differences were found on freezing (F_2,29=1.54, NS). In the cued conditioning test, no significant genotype differences were found in freezing behavior, either before cue presentation (F_2,29=0.49, NS) or after cue presentation (F_2,29=0.25, NS). Cohort 1 similarly displayed no genotype differences in fear conditioning. In the training session, minimal levels of freezing behavior were seen before the presentations of cue-shock pairings (data not shown). A significant genotype effect was found during the training session in post-shock freezing (F_2,79=6.65, p<.01), with +/− and −/− displaying higher levels of freezing as compared to +/+ controls (p<.05 for each comparison). In the contextual conditioning test session, no significant genotype differences were found on freezing (F_2,79= 2.29, NS). In the cued conditioning test session, no significant genotype differences were found in freezing behavior, either before cue presentation (F_2,79=1.07, NS) or after cue presentation (F_2,79= 1.42, NS). Therefore, on this test of emotional memory, Shank3 mutants displayed normal performance.

Cohort 2 null mutants displayed an apparent deficit in novel object recognition, as shown in Figure 9F. Preference for the novel object over the familiar object was found in male +/+ (F_1,9=21.24, p<.01) and +/− (F_1,10=7.55, p<.05), but not in male −/− (F_1,17=3.91, NS). Similar results were found in females (data not shown). Preference for the novel object over the familiar object was found in female +/+ (F_1,7=36.85, p<.001) and +/− (F_1,14=6.33, p<.05), but not in female −/− (F_1,13=.16, NS). Males showed a significant genotype effect on total sniff time (F_2,44=7.18, p<.01), with −/− lower than +/+ (p<.001) and +/− (p<.001). Females showed a non-significant trend (F_2,40=2.85, p=0.08) for less total sniff time in −/−. Consistent results were found in Cohort 3 (data not shown). Preference for the novel object over the familiar object was found in +/+ (F_1,9=17.93, p<.01) and +/− (F_1,9=25.00, p<.01), but not in −/− (F_1,8=2.12, NS). As in Cohort 2, −/− of Cohort 3 exhibited lower total sniff time as compared to +/+ controls (F_2,26=8.31, p<.01), with −/− lower than +/+ (p<.01) and +/− (p<.05). Lower overall exploration complicates the interpretation of apparent cognitive deficits in novel object recognition.