Preserved social development but impaired executive function in a Shank3**- deficient rat model of Phelan-McDermid syndrome
(Full-text capture 2026-09-21; web artifacts lightly stripped; truncated.)
Running title: Late-emerging executive dysfunction in Shank3 rats
Anna C. Pearson MSc¹, Taylor M. Drazan BSc¹, Sean P. Bradley PhD², Audrey Thurm PhD³, Joseph D. Buxbaum PhD⁴, Jill L. Silverman PhD5,6, Yogita Chudasama PhD1,2
¹Section on Behavioral Neuroscience, ²Rodent Behavioral Core, ³Neurodevelopmental and Behavioral Phenotyping Service, National Institute of Mental Health, Bethesda, Maryland, USA
⁴Seaver Autism Center for Research and Treatment, Icahn School of Medicine at Mount Sinai, New York, NY USA.
⁵Department of Psychiatry and Behavioral Sciences, University of California, Davis, School of Medicine, Sacramento, CA, USA
6 MIND Institute, University of California, Davis, School of Medicine, Sacramento, CA, USA
Address for correspondence:
Yogita Chudasama, Ph.D.
National Institute of Mental Health
Building 35A, Room 2D-804
Bethesda, MD 20892
Tel: 301 594-4569
Email: yogita.chudasama@nih.gov bioRxiv preprint doi: https://doi.org/10.64898/2026.08.13.744651; this version posted August 18, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
ABSTRACT
Phelan-McDermid syndrome (PMS) is a genetic neurodevelopmental disorder caused by a microdeletion within chromosome 22q13.3 1-6, and accounts for ~1-3% of cases of autism spectrum disorders (ASD). Individuals with PMS typically present with neonatal hypotonia, severe speech delay, intellectual disability, motor impairments, and autism-related features, although additional manifestations such as epilepsy, sleep disturbances, and developmental regression are common. As in ASD, there is considerable heterogeneity in cognitive and behavioral impairments in PMS, making it difficult to determine which features arise directly from SHANK3/Shank3 deficiency and how deficits manifest across development. In the present study, we transferred the targeted Shank3 mutation, previously characterized on the Sprague Dawley background, onto the Long-Evans strain and performed a longitudinal behavioral assessment of Shank3-deficient rats from infancy to adulthood. The rats were evaluated for delays in early sensory and motor development, and ultrasonic vocalizations as neonates, social behavior, short term memory and gait as juveniles, and cognitive-executive dysfunction using a touchscreen-based visual discrimination and reversal learning task as adults. Despite profound reductions and/or complete loss of Shank3 protein expression, heterozygous and homozygous male and female rats showed normal physical and neurological reflexes across development, yet female Shank3 knockout pups showed a selective reduction in distress-associated ultrasonic vocalizations. As juveniles, subtle abnormalities emerged in short-term memory and limb coordination, while social preference for novelty and recognition was normal. Prominent behavioral abnormalities were observed in adults characterized by rapid and error-prone responding to visual stimuli during discrimination learning and reversal. These data suggest that rats with complete or partial Shank3 deficiency produce a selective behavioral profile in which high-order cognitive dysfunction is more pronounced than deficits in basic sensorimotor or select social behaviors. More broadly,
22\\mathfrak{q}13.3^{1-6}, $$ bioRxiv preprint doi: [https://doi.org/10.64898/2026.08.13.744651](https://doi.org/10.64898/2026.08.13.744651); this version posted August 18, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. these findings highlight executive dysfunction as a key consequence of the loss of _Shank3._ For the first time, we report the loss of _Shank3_ expression on a Long-Evans background strain as a valuable translational tool for investigating cognitive dysfunction and therapeutic windows in _Shank3_-associated disorders. * * * bioRxiv preprint doi: [https://doi.org/10.64898/2026.08.13.744651](https://doi.org/10.64898/2026.08.13.744651); this version posted August 18, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. #### INTRODUCTION _SHANK3_ encodes a postsynaptic scaffolding protein essential for glutamatergic synapse development and function1,2. A deletion or mutation of SHANK3 on chromosome 22q13.3 causes Phelan-McDermid Syndrome (PMS), a rare genetic neurodevelopmental disorder characterized by intellectual disability, language impairment, motor dysfunction and variable levels of autismrelated features3–5.Clinical manifestations of PMS exhibit substantial variability in cognitive and behavioral outcomes⁶. Increasing evidence indicates that deficits in executive function, particularly inattention and cognitive inflexibility, are a prominent feature of PMS and may be especially pronounced even in individuals with point mutations or small deletions⁷. These impairments are thought to contribute substantially to difficulties with learning and goal-directed behavior underscoring the importance of executive dysfunction as a translationally relevant phenotype. Several mouse models have advanced our understanding of the molecular and synaptic mechanisms of Shank3 loss2,8–10, but have been less successful in modeling the higher-order cognitive manifestations associated with PMS¹¹. This likely reflects, in part, the relatively limited cognitive repertoire of mice and the considerable variability in behavioral phenotypes across different genetic constructs and testing paradigms. The original _Shank3_-deficient rat model extended this work to a species with greater cognitive and social complexity, and has revealed impairments in attentional control and social communication12,13. However, characterization of this model has been exclusively restricted to the Sprague-Dawley strain. Compared with Sprague- Dawley rats, Long-Evans rats exhibit better visual function, greater exploratory behavior, and enhanced performance in cognitively demanding tasks, making them particularly well suited for studies of learning, attention, and executive function14,15. These differences are especially relevant for touchscreen-based assessments because Sprague-Dawley rats are albino and associated with altered retinal organization and reduced visual acuity20–23. Since PMS is a neurodevelopmental disorder and Shank3 expression peaks postnatally in cortico-striatal brain structures16–18, understanding when behavioral abnormalities first emerge is as important as identifying their adult manifestations. This issue is critical for translational research, as it is particularly relevant for higher-order cognitive functions which are commonly impaired in PMS but difficult to assess systematically in affected individuals because of intellectual disability, poor motor skills and language limitations19,20. To address this knowledge gap, we performed a longitudinal assessment of _Shank3_-deficient rats generated and maintained on a Long-Evans background strain, from infancy to adulthood to define the developmental emergence of social, emotional, motor, and cognitive abnormalities relevant to PMS. ##### METHODS #### Subjects Male and female _Shank3_ Sprague Dawley heterozygous rats were obtained as a gift from Dr. Joseph Buxbaum at Mount Sinai School of Medicine and rederived at the National Institute of Mental Health (NIMH) Transgenic Core facility on an outbred Long-Evans background. Full details of the generation of the _Shank3_ gene mutation are provided in Harony-Nicolas et al (2017)¹³. Heterozygous breeder rats were paired to generate the animals used in this study and backcrossed for at least seven generations ( **Fig. 1A).** We used male and female Long-Evans rats that were wildtype for Shank3+/+ (WT, n = 16), heterozygous for Shank3+/-and Shank3-/+ (HET, n = 34), and homozygous knockouts for Shank3-/-(KO, n = 18). These rats were tested as neonates and thereafter as juveniles and adults in a single cohort. * * * Behavioral testing was scheduled to minimize fatigue, repeated-testing effects, and potential carryover between assays while preserving the longitudinal design21,22. The order of testing was held constant across animals and was arranged to progress from short procedures to more socially or cognitive demanding assays as adults. A minimum of 24 hours separated successive tests. Ultrasonic vocalizations (USVs) were recorded on PD7 and PD14. Juvenile assessment proceeded in the following order: open field, gait analysis, grip strength, and novel object recognition (NOR). Social interaction testing was conducted at least 7 days after NOR, and touchscreen training began at least 2 weeks after the completion of social testing. All behavioral testing was conducted at approximately the same time of day to reduce variability associated with circadian influences on activity and performance. Upon weaning at postnatal day (PD) 21, rats were group-housed with mixed genotypes in a temperature-controlled room (23.3°C) with a 12:12h light:dark cycle according to ARRIVE guidelines²³. All behavioral testing occurred during the light phase. All experimental procedures were approved by the NIMH Institutional Animal Care and Use Committee, in accordance with the NIH guidelines for the use of animals. #### Genotyping and Western blotting for DNA, and protein expression On PD1-3, pups were tattooed with a non-toxic marker for identification. Tail clippings for genotyping took place at PD21. Thus, all neonatal testing (PD2-16) was conducted blind to the genotype and before tail clippings so as not to affect their neonatal milestones including vocalizations. Genotypes were confirmed with qPCR for RNA through TransnetYX Inc (Cordova, TN). In addition, to confirm the reduced expression of _Shank3_ in the _Shank3_-deficient rats, we performed a Western Blot analysis. Hemibrain lysates were prepared as outlined in Bozdagi et al. * * * (2010)², washed in a 4-12% Bis Tris 12-Well Gel (Invitrogen, #NPO322BO) and NuPAGE™ MES SDS Running Buffer (20X, Invitrogen, #NP0002), and transferred a polyvinylidene fluoride membrane. Anti-Shank 3 (1:1000, Cell Signaling Technologies, DK56R, 64555S Rabbit) and antibeta III tubulin (1:3000, Sigma, T9026 Mouse) antibodies were used for the detection of Shank 3 protein and beta III tubulin, respectively. SeeBlue™ Plus2 Pre-Stained Protein Standard was used as a marking ladder (Invitrogen, #LC5925). #### Behavioral testing #### Neonatal rats (postnatal day 2 – 16) _Neurodevelopmental reflexes._ To capture physical and neurological reflex delays and potential symptoms of hypotonia (low or weak muscle tone) in early life, we implemented a series of short behavioral tests adapted from Fox (1965)²⁴ to newborn rats from PD2 to PD1625. We focused on tests that assessed neurological reflexes which are involuntary and repetitive movements that demonstrate brain stem and spinal cord function ( **Fig 2A,** B). In brief, we examined: 1) body rotation and coordination (surface righting and negative geotaxis), 2) strength and vestibular imbalance (forelimb grasp and cliff aversion), and 3) sensory system maturation (rooting, auditory startle, ear twitch). A description of each test can be found in supplementary materials ( **Table S1).**PD16
_Vocalizations._ Isolation induced ultrasonic vocalizations (USVs) were recorded in pups on PD7 and PD14 **(Fig. 2C**) to capture developmental changes in vocal behavior across a period when isolation-induced USVs undergo marked changes in call rate, frequency, duration and acoustic structure during the first postnatal weeks26–28. At both timepoints, vocalizations were assessed under four sequential conditions to assess whether affective vocal output varied as a function of environmental and social context. This allowed us to compare vocalizations across a graded set of conditions ranging from minimal stimulation in isolation to increasing levels of familiarity and social relevance. The design allowed us to determine not only whether _Shank3_-deficiency altered the normal developmental trajectory of pup vocalizations, but also whether vocal responses were appropriately modulated by changing environmental and social context. Pups were separated from their mothers and placed individually on a heat pad in a Plexiglass recording chamber (L 41 cm × W 20 cm × H 20 cm). Pup vocalizations were recorded for three minutes in each condition. The recording conditions were run in the following order: 1) Novel, pup was alone in chamber with clean cage bedding; 2) Unfamiliar, pup was alone in chamber with cage bedding from an unfamiliar virgin female; 3) Familiar, pup was alone in chamber with cage bedding from its home cage; and 4) Social Interaction, the pup’s mother was placed in testing chamber behind a clear Plexigass wall with ventilation holes. In between conditions, the pup was placed on a heat pad in a padded Plexiglass holding chamber while the recording chamber was cleaned with 70% ethanol and allowed to air dry for three minutes before the next condition. Immediately after the final recording, pups were returned to their littermates. Litters were reintroduced to the mother after all recordings were complete. The entire procedure took 25 minutes. Vocalizations were acquired using an Avisoft CM16/CMPA condenser microphone and connected via an UltraSoundGate 416H USB audio device to a computer with Avisoft- RECORDER software (Avisoft Bioacoustics, Berlin, Germany). The recorded sounds were digitized at a sampling frequency of 250 kHz and analyzed with RavenPro (Cornell Lab of Ornithology; Ithaca, NY). Calls were identified by two band-limited entropy detectors, each with an entropy above background threshold of 1.5 dB. Calls were classified as 40kHz-like when captured by a detector band-limited from 30 kHz to 50 kHz. Calls were classified as 66kHz-like when captured between 55 kHz and 75 kHz. To avoid double-counting or harmonics, in cases where calls overlapped in both the 40 kHz and 66 kHz bands, only the putative vocalization with the highest entropy was counted. #### Juvenile rats (postnatal day 21 – 42) _Motor control and coordination._ To evaluate body movement, posture and gait, juvenile rats were placed in a clear Plexiglass square arena (L 40 cm × W 40 cm × H 29.8 cm) for five minutes in red light (i.e., darkness). Footprints were detected and recorded with a high-speed digital Basler Ace2 camera (Ahrensberg, Germany) located beneath the clear arena. Gait features were analyzed with FreeWalkScanTM2.0 tracking software (Clever Systems, Inc., Virginia, USA). Distance traveled was determined using TopScan (Clever Systems, Inc., Virginia, USA). _Muscular strength._ A Bioseb (BIO-GS4) grip strength monitor was used to assess muscular strength. The animal was allowed to grasp a metal grid while gently pulled backwards in a horizontal plane to determine the maximal peak force. The highest force exerted within three attempts was recorded. _Open field._ To assess locomotor activity, juvenile rats were placed in a square arena (L 44 cm× W 44 cm × H 40 cm) for thirty minutes. Animal movement was recorded with a digital camera positioned above the arena. Distance traveled and velocity were quantified using TopScan tracking software (Clever Systems, Inc., Virginia, USA). The arena was cleaned with 70% ethanol between trials to remove olfactory cues. _Novel object recognition._ We next assessed the rats’ ability to recognize and remember previously encountered objects as a measure of short-term recognition memory. Animals were placed in the same arena used for the open field containing two identical objects. The two objects were falcon tubes (H 12.0 cm × 1.7 cm diameter) secured with glue on a gray plaque and placed in opposite corners of the arena. Rats were allowed to freely explore the objects for five minutes during the acquisition phase. The objects were validated during the acquisition phase by showing no baseline preference for either object. Animals were then returned to their home cage for a retention interval of 60–90 minutes. During the test phase, rats were returned to the arena in which one familiar object was replaced with a novel object of different shape and texture (lego construction, H 12.8 cm × W 6.4 cm × L 3.84 cm) and allowed to explore the objects for five minutes while behavior was recorded with an overhead camera. Object exploration was defined as directing the nose toward the object within approximately 1 cm and/or touching the object with the nose or forepaws. The left/right location of the novel object was counterbalanced across animals to avoid object or side biases. The arena and objects were cleaned with 70% ethanol between trials to remove olfactory cues.12.8\ cm\times W\ 6.4\ cm\times L\ 3.84\ cm)
44.5\ cm\times W\ 106.7\ cm\times H\ 45.7\ cm)
contained the stimulus rat from day 2, which now served as a familiar rat. Again, the test rat was placed in the center chamber and allowed to explore both animals in each chamber. The stimulus rats were age-, strain-, and sex-matched to the test rat but were housed in a separate room in the vivarium to ensure novelty. The left/right side on which the stimulus rat was placed alternated between test subjects. Automated video tracking with TopScan (Clever Systems, Inc., Virginia, USA) was used to determine time spent in each zone. #### Adult rats (postnatal day 56 and above) Cognitive impairments associated with the _Shank3_-deficiency (WT, n=10; HET, n=11; KO, n=17) were assessed in rats during performance of a visual discrimination and reversal learning task conducted in automated operant touchscreen chambers. For this task, adult rats were maintained at 85% of their free-feeding weight and had access to water for at least one hour each day. The apparatus and online data collection were controlled by Animal Behavior Environment (ABET) II software (Lafayette Instruments, Indiana, USA). Each chamber was individually housed within a sound-attenuating cabinet, ventilated by low-level noise fans, and illuminated by a 3W house light mounted on the ceiling of the sound-attenuating cabinet. The ceiling and two sidewalls of the operant chamber were made of clear Plexiglas (one of which served as the door). One side of the operant chamber was fitted with a touch-sensitive monitor (9”W x 10”H). Images of white squares were presented at two of five discreet locations on the touchscreen ( **Fig.** 4). A black mask, approximately 1 cm from the surface of the display, served to restrict the rat’s access to the display except through response windows each measuring 2.06"L x 2.06"H. Opposite the(9W x10H)
2.06\mathrm{ L}\mathrm{ x}2.06\mathrm{~H}
\left(\mathrm{F}_{(2,2)}=0.135\right)
\left(\mathrm{F} {(7,2)}=\right. F{(2,2)}=3.317,\quad p=0.232
\mathrm{F}_{(7,2)}=42.936
\mathrm{F}_{(2,2)}=0.231
p=0.805
\mathrm{F}_{(2,65)}=0.98
F_{(2,65)}=0.82
\mathrm{F}(2,65)= 0.62;
#### _Shank3_ deficiency alters early life vocalizations in females ## Shank3 deficiency alters early life vocalizations in females Ultrasonic vocalizations (USV) are elicited when a pup is separated from the dam or littermates. These isolation-induced calls serve as a distress signal and widely used as developmental indicators of nervous system maturation and/or emotional reactivity30,31. Since USV production changes rapidly during early postnatal weeks, the ages of PD7 and PD14 were selected to capture distinct stages in the ontogeny of isolation-induced vocalizations. Rat pup USVs are robust during the first postnatal week and subsequently decline as pups approach weaning, with PD14 coinciding with major sensory and behavioral transitions, including eye opening26,32. Unlike mice, isolation-induced vocalizations in rats can persist beyond eye opening and decline progressively over subsequent development27,28. Vocalizations were recorded under four different contexts ranging from novel to familiar ( **Fig. 2C).** Across all genotypes (WT, n=16; HET, n=34; KO, n=18), total USV production declined overall for all conditions including the novel condition between PD7 and PD14 (all p<0.01; **Fig.** **2D; Table S2A,B),** consistent with the normal developmental reduction in isolation-induced vocalizations. An analysis of the call structure revealed that the proportion of negative affective calls (40kHz)33,34 remained high across all conditions and was not influenced by genotype at either age (PD7: F(2, 63) = 2.650, p = 0.078; PD14: F(2, 63) = 1.227, p = 0.300, **Fig. 2E; Table S2C,D).** While there was no effect of condition at PD7 (F(2.725, 163.5) = 1.875, p = 0.141), an effect did emerge at PD14 (F(2.672, 155.0) = 8.168, p < 0.0001, **Fig. 2E).** The most prominent genotype-dependent effects emerged in female _Shank3_ mutants at PD14. While no sex-dependent differences were detected at PD7 ( **Fig. 2F),** at PD14, _Shank3_ KO females exhibited a reduced proportion of ~40 kHz distress calls relative to KO males when tested in both the novel (Mean ± SEM: KO Males 0.841 ± 0.054, KO Females 0.500 ± 0.099, post hoc Tukey’s test, p = 0.025) and unfamiliar (Mean ± SEM: KO Males 0.946 ± 0.021, KO Females 0.580 ± 0.111, post hoc Tukey’s test, p = 0.012) isolation conditions ( **Fig. 2G).** A similar differentiation between male and females was present in the familiar condition (Mean ± SEM: KO Males 0.889 ± 0.048, KO Females 0.641 ± 0.096) and during social interaction (Mean ± SEM: KO Males 0.927 ± 0.023, KO Females 0.659 ± 0.075) but the groups did not differ statistically (post hoc Tukey’s test for familiar, p = 0.236 and social interaction p = 0.147). Most call characteristics did not vary across genotypes. There was a modest increase in call entropy exhibited by the HET animals between PD7 and PD14 (post hoc Tukey’s test p = 0.012) whereas WT and KO animals showed no age-dependent change ( **Fig. S1A).** Call duration and peak frequency were unaffected by genotype or age **(Fig. S1B,C; Table S2E,J).** These data suggest that the loss of _Shank3_ selectively alters the expression of affective vocalizations in female pups during a critical developmental period, particularly in contexts associated with novelty or environmental uncertainty.\mathrm{(PD7:F_{(2,63)}=2.650,p=0.078;PD14:F_{(2,63)}=1.227,p=0.300,Fig}
\left(\mathrm{F}_{(2.725,163.5)}=1.875,\mathrm{p}=0.141\right)
\mathrm{F}_{(2.672,\ 155.0)}=8.168
(Mean\pm SEM:
0.841\pm0.054
0.500\pm0.099
p=0.025
\pm SEM:KO
0.946\pm0.021
p=0.012
0.580\pm0.111
0.889\pm0.048
(Mean\pm SEM)
0.641\pm0.096
(Mean\pm SEM)
0.927\pm0.023
0.659\pm0.075
p=0.236
p=0.147
p=0.012
#### Subtle memory deficit emerges before adulthood in _Shank3_ deficient rats * * * The novel object recognition test was conducted in juvenile animals (PD 21–28) as a shortterm memory assay and to reveal subtle cognitive impairments that might emerge before adulthood. The discrimination index provided a quantitative measure of how well the animals distinguished between the familiar and novel objects. There was high inter-individual variability in discrimination performance with data points widely dispersed even amongst the WT controls. The complete KO animals tended to have lower discrimination indices compared to HETs (F(2,63) = 2.124, p = 0.07; **Fig 3B),** suggesting a trend toward impaired recognition memory. Despite the wide range in individual scores, the _Shank3_-deficient rats tended to explore the novel object less. In fact, only two animals from this group showed memory for the novel object with high discrimination indices; the remaining 14 animals performed near or considerably below chance level. This effect was unrelated to general locomotor activity since their ambulatory movement in the open field was normal (F(10, 384) = 0.7244, p = 0.70; **Fig. 3C**) as was their speed (F(2, 64) = 2.648, p = 0.08; **Fig. 3D).** #### _Shank3_ deficient juveniles show altered limb coordination ## Shank3 deficient juveniles show altered limb coordination Developmental regression of motor skills is a key feature in patients with PMS35,36. We therefore examined motor function in animals lacking the _Shank3_ gene as juveniles since these animals showed normal motor coordination as neonates. First, we measured muscular strength but there was no detectable deficit; all animals regardless of genotype were able to exert the typical force for its size and age during a test of grip strength (Kruskal-Wallis H = 3.68, p > 0.05; **Fig.** **3E).** However, gait analysis revealed subtle coordination abnormalities. Ipsilateral paw overlap, which reflects the spatial and temporal coordination between forelimb and hindlimb movements on the same body side, were altered in a genotype- and side-specific manner. On the left side, juvenile rats both heterozygous for the _Shank3_ mutation (Mean ± SEM: 1.36 ± 56.43 mm) and the complete KO rats (Mean ± SEM: 56.91 ± 1.50 mm) exhibited reduced fore–hind paw distance relative to WT controls (Mean ± SEM: 63.39 ± 2.10 mm (P < 0.01 corrected with Tukey’s multiple correction), with no difference between mutant groups ( **Fig. 3F).** On the right side, only HET rats showed a significant reduction in paw overlap compared with WT (Mean ± SEM: HET, 56.26 ± 1.15 mm; WT, 62.43 ± 2.51 mm, p = 0.03), whereas KO animals were comparable to the other groups (Mean ± SEM: 57.65 ± 1.67 mm). These findings suggest that the mutation influences limb coordination, with the effect appearing more pronounced on one side.(\mathrm{Mean}\pm\mathrm{SEM}:1.36\pm56.43\mathrm{mm})
(Mean\pm SEM:56.91\pm1.50mm)
(\mathrm{Mean}\pm\mathrm{SEM}:63.39\pm2.10\mathrm{mm}(\mathrm{P}<0.01
(Mean\pm SEM:HET,56.26\pm
62.43\pm2.51
(Mean\pm SEM:57.65\pm1.67mm)
#### Social behavior is intact in juveniles with _Shank3_ mutation Social deficits have been reported in _Shank3_ deficient rodent models and are often considered a translationally relevant feature of Shank3-associated neurodevelopmental disorders2,8. We used the standard three-chamber assay to assess sociability and social novelty preference in juvenile rats, facilitating comparison with the extensive literature using this paradigm in mouse models of _Shank3_-deficiency. We found no evidence of impaired social behavior in _Shank3_ deficient animals. All genotypes displayed intact social preference by spending more time in the zone containing the unfamiliar conspecific (Stranger 1) than the zone containing the empty cup (P < 0.05; \*\*Fig. 3H, left panel).\*\* Consistent with this finding, the social preference index, reflecting time spent in close proximity to Stranger 1 relative to the empty cup, did not differ by genotype (\*\*Fig.\*\* \*\*3H, right panel).\*\* Likewise, when stranger 1 (now familiar) was paired with a novel conspecific (stranger 2), animals in all groups preferentially investigated the novel animal and neither investigation time in the novel versus familiar zones nor the corresponding discrimination index differed by genotype (P > 0.05; **Fig. 3I).** In addition, latencies to first investigate the familiar or stranger rat were comparable across groups **(Fig. S2).** Thus, there was no evidence of this type of impaired social approach and recognition behaviors in juvenile _Shank3_-deficient rats, using this specific assay, as previously reported12,13. #### Side-specific perseveration in adult _Shank3_ deficient rats ## Side-specific perseveration in adult Shank3 deficient rats When rats reached adulthood (PD > 60), we assessed cognitive performance in an operant touchscreen discrimination task requiring them to learn and update stimulus-reward associations. Animals were first required to learn a visual discrimination, transferred the learned rule to a novel stimulus pair, adapted their responses following reversal of the reward contingencies, and finally performed the discrimination when the stimuli when presented in variable spatial locations ( **Fig.** **4A, B).** Genotype had a major influence on task performance (F(2, 138) = 7.368, P < 0.001, \*\*Fig.\*\* \*\*4C).\*\* \*Shank3\* KO rats were impaired during the acquisition phase committing more errors to criterion than both HET and WT groups (Mean ± SEM: WT 166.4 ± 34.19; HET 153.7 ± 20.26; KO 257.6 ± 36.51). Despite this learning difficulty, the KO rats successfully transferred the rule to a novel stimulus pair performing comparably to WT and HET groups (p > 0.05). However, when the stimulus-reward contingencies were reversed, KO rats again were impaired, committing many errors relative to controls (Mean ± SEM: WT 317.2 ± 39.91, HET 368.2 ± 37.22, KO 472.4 ± 39.52). Once the reversed rule was learnt, all animals performed similarly during the variablelocation phase (Mean ± SEM: WT 83.80 ± 17.32, HET 88.00 ± 20.83, KO 86.47 ± 19.10) indicating intact stimulus discrimination independent of stimulus position.(\mathrm{PD}\geq60)
\left(\mathrm{F}_{(2,138)}=7.368\right. (Mean\pm SEM;
166.4\pm34.19;
153.7\pm20.26;
\mathrm{K0}~257.6\pm36.51
(Mean\pm SEM:WT317.2\pm39.91
368.2\pm37.22
\pm39.52)
(Mean\pm SEM:WT83.80\pm17.32
88.00\pm20.83
86.47\pm19.10)
\mathrm{F}_{(2,;35)}=4.585
\mathrm{F}_{(2,;35)}=1.747
\mathrm{F}_{(2,;34)}=2.871
F_{(2,105)}=7.951,;p= \mathrm{F}_{(2,103)}=18.50
\mathrm{F}_{(2,;35)}=5.922
genotype ( **all P>0.05; Fig. G, Fig. S3E, F).** #### DISCUSSION The present study provides the first longitudinal behavioral assessment of _Shank3_-deficiency in rats maintained on a Long-Evans background spanning neonatal, juvenile, and adult stages of development. The Long-Evans background offers important advantages for modeling higherorder cognitive features of PMS, including intact visual function and robust performance on cognitively demanding tasks, making it particularly well suited for touchscreen-based assessment of learning and executive function. Despite profound reduction or absence of Shank3 protein expression, mutant animals exhibited largely intact sensory and motor development, preserved social interactions, and modest abnormalities during the juvenile period. In contrast, the most prominent behavioral deficit emerged in adulthood during touchscreen-based cognitive testing, where _Shank3_-deficient rats displayed impairments in discrimination and reversal learning that were driven by repeated responding to previously selected locations. These findings establish the Long-Evans _Shank3_-deficient rat as a valuable model of PMS and reveal a developmentally evolving behavioral profile in which deficits in higher-order cognitive control are more pronounced than deficits in basic sensorimotor or specific social behaviors. We observed several aspects of behavior that were unexpectedly preserved. Neither HET nor KO mutants exhibited delays in the acquisition of neonatal reflexes, sensory responsiveness, or gross motor development. Likewise, juvenile mutants showed a robust preference for a social stimulus over the nonsocial alternative with no evidence of impaired social recognition, findings that contrast with reports of social impairments of this type in some Shank3 mouse models2,8,37. In rats, however, the absence of a preference for the novel conspecific should not necessarily be interpreted as impaired social recognition. Social novelty preference requires not only discrimination between familiar and novel individuals, but also a motivational bias toward investigating the novel animal. Rats are highly social and strongly motivated to engage in direct social contact, but because the three-chamber paradigm restricts direct physical interaction, continued investigation of the familiar conspecific may reflect persistent social motivation rather than a failure to recognize the familiar animal. Thus, the equivalent investigation of familiar and novel animals may reflect limitations in the expression of social recognition within the threechamber paradigm rather than an inability to discriminate social familiarity. These speciesspecific considerations may be particularly important in Long-Evans rats and suggest that the social novelty phase of the three-chamber assay should be interpreted cautiously when used as a measure of social recognition in this strain. We note that previous studies of _Shank3_-deficient rats reported intact short-term social recognition despite impairments in more demanding forms of social memory12,13,38. Our KO rats showed a tendency toward impaired novel object recognition early in their juvenile period, suggesting that deficits in short-term memory may emerge before detectable impairments in social behavior. Although differences in genetic background and testing procedures may contribute to variation across studies, the preservation of aspects of sociability observed here mirrors the clinical heterogeneity seen in individuals with PMS, where social functioning varies substantially despite at least some shared genetic etiology³⁹. Unlike rodent behavioral tasks that are designed to capture broad social deficits observed in other ASDassociated conditions, differences in social deficits associated with _Shank3_ disruption may reflect specific attention and memory alterations that impact social functioning⁴⁰ * * * Although early sensory and motor development was largely preserved, several more subtle alterations emerged during early development. One subtle change was in affective vocal communication during infancy. Isolation-induced ultrasonic vocalizations are widely used as an index of emotional reactivity and changes in pup–dam communication are often interpreted as reflecting alterations in the neural circuits regulating affective state12,30,31. Consistent with normal development, overall call production declined substantially between PD7 and PD14 across all genotypes. However, by PD14, only female _Shank3_ KO pups emitted a lower proportion of ~40 kHz distress-associated calls, particularly when exposed to novel or unfamiliar environments. This effect was not present at PD7 and occurred in the absence of changes in total call output or call structure suggesting that _Shank3_ deficiency selectively influenced the affective quality rather than the overall production of vocalizations. The female-specific nature of the effect suggests that the behavioral consequences of _Shank3_ deficiency may, under some conditions, be modulated by sexdependent developmental processes. However, sex differences are not a consistent clinical feature of PMS. Developmental behavioral studies in multiparous species like rats are inherently susceptible to litter-related variability, and subtle sex-specific effects require substantially larger sample sizes for robust confirmation. Nevertheless, the restriction of this effect to homozygous knockout animals suggests a more severe loss of _Shank3_ function may be necessary for this alteration in affective vocal signaling to emerge. A modest increase in call entropy was observed in HET animals during development. Entropy is commonly interpreted as a measure of acoustic complexity28, suggesting that distress calls became slightly less stereotyped with age in this group. However, the effect was restricted to heterozygous animals and was not accompanied by changes in call duration or frequency, indicating that it is unlikely to account for the more prominent female-specific alterations in affective vocal signaling observed in KO pups. Although robust sex differences in social functioning have not been consistently reported in individuals with PMS, both clinical and preclinical studies suggest that _Shank3_-related phenotypes may be differentially modulated in males and females12,41. Importantly, while the functional significance of the altered isolation induced distress-call profile remains unclear, these vocalizations have a communicative function in pup–dam interactions, as playback of pup isolation calls elicits maternal approach and retrieval behavior¹². Thus, the selective reduction in distress associated calls may reflect a subtle alteration in early affective communication between pup and dam. Our findings further suggest that this behavior may be influenced by biological sex, and emerge early in life, even when overt behavioral impairments are not apparent. _Shank3_ deficiency did not disrupt the initial development of basic motor function, but more complex aspects of motor skill involving gait become affected when the rats were juvenile. This temporal dissociation is noteworthy given that motor regression and deterioration of motor skills are well-recognized features of PMS35,36,42. Although the present data do not demonstrate true regression, as motor performance was not tracked longitudinally within individual animals, the appearance of coordination deficits after a period of apparently normal development in infancy is consistent with the notion that _Shank3_ deficiency may interfere with the maturation or maintenance of motor circuits rather than their initial formation. Such an interpretation is compatible with the established role of _Shank3_ in synaptic stabilization and plasticity⁴³ and suggests that motor abnormalities may emerge as neural circuits become increasingly refined during postnatal development. Future studies would need to determine whether these early coordination deficits remain stable or progress with age, to closely model the regressive motor features reported in some individuals with Phelan-McDermid syndrome. * * * The clearest behavioral abnormality in the KO rats was a deficit in learning and executive function observed in adulthood during touchscreen discrimination learning. _Shank3_-deficient rats required more trials to acquire and reverse stimulus–reward contingencies yet successfully transferred learned rules to novel stimuli and performed normally when stimulus locations varied. These deficits were not attributable to impaired stimulus discrimination, generalized side bias, or reduced sensitivity to negative feedback. Instead, the _Shank3_ deleted animals repeatedly responded to previously selected response locations suggesting a difficulty disengaging from prior actions. A similar pattern of perseverative responding has been reported in _Shank3_-deficient mice in the Barnes Maze, where animals repeatedly returned to previously searched or previously reinforced escape locations despite changing task demands, supporting a conserved association between Shank3 deficiency and impaired behavioral flexibility across species44. Intriguingly, the rats responded unusually fast during the acquisition phase; response latencies normalized during reversal and reward collection latencies remained intact throughout testing. Thus, during initial learning, _Shank3_-deficient rats were not slower or less engaged than controls but instead responded rapidly while making more errors. This pattern is consistent with an altered speed–accuracy tradeoff in which choices are executed hastily rather than engaging in deliberate stimulus evaluation. Together, these findings suggest that _Shank3_ deficiency disrupts executive control over response selection, rather than learning capacity itself. This distinction is clinically important, as it suggests that _SHANK3_ disruption may impair the ability to regulate and adapt behavior rather than producing a generalized cognitive deficit. Such a profile is consistent with dysfunction within corticostriatal networks that regulate action selection, cognitive flexibility, and inhibitory control45,46, systems that are known to be sensitive to Shank3 disruption9,47,48. * * * In conclusion, we report that _Shank3_ deficiency produces a selective and developmentally evolving behavioral profile in which higher-order cognitive dysfunction is more pronounced than deficits in basic sensorimotor or social behaviors. While several aspects of the phenotype were preserved, the evidence suggests that _Shank3_ loss influences the maturation of neural circuits involved in affective communication, motor coordination, and short-term memory before the emergence of more pronounced cognitive deficits in adulthood. More broadly, the results mirror the marked heterogeneity observed in PMS and support the view that learning and executive dysfunction may represent a particularly sensitive consequence of _Shank3_ disruption. By defining the developmental emergence of behavioral abnormalities, this work establishes the Shank3- deficient Long-Evans rat as a valuable translational model for investigating the neural mechanisms underlying cognitive dysfunction in _Shank3_-associated neurodevelopmental disorders and for identifying developmental windows that may be most amenable to therapeutic intervention. #### ACKNOWLEDGEMENTS This research was supported by the Intramural Research Program of the National Institute of Mental Health (ZIA MH002951 and ZIC MH002952 to Y.C.), part of the National Institutes of Health (NIH). The contributions of the NIH author(s) are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Schematic illustrations of rats and behavioral assays were created with both Biorender.com and Adobe Illustrator v30.7; all other figure elements were generated by the authors. We thank Dr. Jim Pickle for rederiving the rats on a Long-Evans background and Mrs. Anamika Singh for training us in Western Blotting. We also thank the NIMH Rodent Behavioral * * * Core staff for their help and guidance with behavioral testing infrastructure, testing protocols and training. We are indebted to the NIMH Veterinary Medicine and Resources Branch (VMRB) and NINDS Animal Health and Care Section (AHCS) for their services in animal health, hygiene and husbandry. ACP is currently a graduate student at Georgetown University, Washington DC, USA, and AT is now an Associate Professor at Boston Children’s Hospital in the Department of Psychiatry, Harvard Medical School, Boston MA, USA. #### CONFLICT OF INTEREST The authors have no biomedical financial interests or potential conflict of interests to report. #### REFERENCES 1 Ross MM, Aizenman E. 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Shank3 immunoreactivity was detected at approximately 180 kDa, and tubulin was used as a loading control. **(C)** Quantification of Shank3 protein expression normalized to tubulin. Shank3 expression was reduced in HET animals and absent in KO animals relative to WT controls. Data points represent individual animals. Bars indicate mean ± SEM. \*\* p < 0.01; \* p < 0.05. **(D)** Experimental timeline to show longitudinal behavioral assessment of Shank3-deficient rats from infancy to adulthood.p<0.01;*p<0.05.
180
45
\mathrm{P}=0.07