Shank3 Exons 14–16 Deletion in Glutamatergic Neurons Leads to Social and Repetitive Behavioral Deficits Associated With Increased Cortical Layer 2/3 Neuronal Excitability

Shank3, an abundant excitatory postsynaptic scaffolding protein, has been associated with multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). However, how cell type-specific Shank3 deletion affects disease-related neuronal and brain functions remains largely unclear. Here, we investigated the impacts of Shank3 deletion in glutamatergic neurons on synaptic and behavioral phenotypes in mice and compared results with those previously obtained from mice with global Shank3 mutation and GABAergic neuron-specific Shank3 mutation. Neuronal excitability was abnormally increased in layer 2/3 pyramidal neurons in the medial prefrontal cortex (mPFC) in mice with a glutamatergic Shank3 deletion, similar to results obtained in mice with a global Shank3 deletion. In addition, excitatory synaptic transmission was abnormally increased in layer 2/3 neurons in mice with a global, but not a glutamatergic, Shank3 deletion, suggesting that Shank3 in glutamatergic neurons are important for the increased neuronal excitability, but not for the increased excitatory synaptic transmission. Neither excitatory nor inhibitory synaptic transmission was altered in the dorsal striatum of Shank3-deficient glutamatergic neurons, a finding that contrasts with the decreased excitatory synaptic transmission in global and Shank3-deficient GABAergic neurons. Behaviorally, glutamatergic Shank3-deficient mice displayed abnormally increased direct social interaction and repetitive self-grooming, similar to global and GABAergic Shank3-deficient mice. These results suggest that glutamatergic and GABAergic Shank3 deletions lead to distinct synaptic and neuronal changes in cortical layer 2/3 and dorsal striatal neurons, but cause similar social and repetitive behavioral abnormalities likely through distinct mechanisms.

We previously reported a GABAergic Shank3 (exons 14-16) deletion and described its impacts on synaptic and behavioral phenotypes in mice (Yoo et al., 2018); however, Shank3 is expressed in both glutamatergic and GABAergic neurons (Han et al., 2013;Yoo et al., 2018). In addition, although a glutamatergic Shank3 (exons 4-22) deletion has previously been generated using the NEX-Cre driver line (Bey et al., 2018), in our conditional knockout (cKO)-ready Shank3-mutant mice, exons 14-16 rather than exons 4-22 were targeted, affecting different Shank3 splice variants and leading to different phenotypes, based on the complex alternative splicing patterns in the Shank3 gene (Lim et al., 2001;Durand et al., 2007;Wang X. et al., 2011;Jiang and Ehlers, 2013;Wang et al., 2014a,b;Monteiro and Feng, 2017). We thus attempted to use Emx1-Cre that drives gene expression mainly in the cortex and hippocampus derived from the dorsal telencephalon (Gorski et al., 2002).
Different cortical layers such as layer 2/3 and layer 5 have been suggested to contribute to ASD. Some previous studies have characterized layer 5 cortical pyramidal neurons in the mPFC or somatosensory cortex in Shank3-mutant mice (Peixoto et al., 2016;Qin et al., 2019;Wang et al., 2019d). Layer 2/3 pyramidal neurons also receive diverse inputs from intracortical and subcortical afferents and provide excitatory inputs onto other cortical layers, including layer 5 (Gabbott et al., 2005;Hoover and Vertes, 2007;Xu and Sudhof, 2013;Lee et al., 2014;Virtanen et al., 2018), and have been implicated in cortical neuronal integration, cognitive functions, and brain disorders such as ASD, schizophrenia, and depression (Parikshak et al., 2013;Shrestha et al., 2015;Li et al., 2016;Page et al., 2018). Indeed, previous studies have highlighted the importance of layer 2/3 cortical neurons in ASD, reporting that superficial cortical layers in the human brain are enriched for genes that are coexpressed in ASD (Parikshak et al., 2013), that inhibitory synaptic transmission in layer 2/3 mPFC cortical neurons is reduced in neuroligin-2-mutant mice with cognitive and social dysfunctions (Liang et al., 2015), and that enhanced synapse remodeling in layer 2/3 pyramidal neurons may be a common pathology in two independent mouse models of ASD (Isshiki et al., 2014). A more recent study reported age-dependent changes in excitatory synaptic transmission and spine density in layer 2/3 mPFC pyramidal neurons . However, the role of layer 2/3 pyramidal neurons in ASD-related brain dysfunctions remains incompletely studied.

Animals
Mice carrying a deletion of exons 14-16 of the Shank3 gene flanked by LoxP sites have been described (Yoo et al., 2018). Homozygous Shank3 14−16 cKO mice with a gene deletion restricted to dorsal telencephalic excitatory neurons (Emx1-Cre;Shank3 fl/fl mice) were produced by crossing homozygous Shank3 fl/fl female mice with double-heterozygous Emx1-Cre;Shank3 fl/+ mice. Cre-negative Shank3 fl/fl littermates, referred to as wild-type (WT) throughout the manuscript, were used as controls for Emx1-Cre;Shank3 fl/fl mice. The Emx1-Cre mouse line, purchased from the Jackson Laboratory (Jackson; #005628) and maintained in a C57BL/6J genetic background for more than five generations, was used for comparisons with all global and cKO mouse lines in the same pure C57BL/6J background. Mice were bred and maintained at the mouse facility of Korea Advanced Institute of Science and Technology (KAIST) according to Animal Research Requirements of KAIST, and all procedures were approved by the Committee of Animal Research at KAIST (KA2016-30). Animals were fed ad libitum and housed under a 12-h light/dark cycle (light phase from 1:00 am to 1:00 pm). Genotypes of Emx1-Cre;Shank3 fl/fl mice were determined by polymerase chain reaction (PCR) using the following primer pairs: floxed (478 bp) or WT allele (276 bp), 5 -GGG TTC CTA TGA CAG CCT CA-3 (forward) and 5 -TTC TGC AGG ATA GCC ACC TT-3 (reverse); Emx1-Cre (272 bp), 5 -GTG TTG CCG CGC CAT CTG C-3 (forward) and 5 -CAC CAT TGC CC TGT TTC ACT ATC-3 (reverse). Only male mice were used for behavioral and electrophysiological experiments, whereas both male and female were used for biochemical experiments.

Western Blot
Total brain lysates separated in electrophoresis and transferred to a nitrocellulose membrane were incubated with primary antibodies to Shank1 (#2100, guinea pig) (Ha et al., 2016), Shank2 (Synaptic Systems 162 202), Shank3 (#2036 guinea pig polyclonal antibodies raised against aa 1289-1318 of the mouse Shank3 protein)  and α-tubulin (Sigma T5168) at 4 • C overnight. Fluorescent secondary antibody signals were detected using Odyssey R Fc Dual Mode Imaging System.

Behavioral Assays
Male mice (2-8-mo-old) were used for all behavioral assays. Before behavioral experiments, mice were handled for 10 min per day for 3 days. All behavioral assays were initiated after a 30-min habituation in a dark booth. The behavioral tests for Emx1-Cre;Shank3 14−16 mice and Emx1-Cre mice were performed in the order indicated in Supplementary Table S1. The order of behavioral tests was designed to minimize stress to the animals.

Three-Chamber Test
Social approach and social novelty recognition were measured using the three-chambered social interaction test (Crawley, 2004;Nadler et al., 2004;Moy et al., 2009;Silverman et al., 2010) under illuminated (70-80 lux) conditions. The 3-chamber test apparatus is a white acrylic box (60 × 40 × 20 cm) divided into three chambers. Both left and right side chambers contained a cage in the upper or lower corner for an object or a stranger mouse. Experimental mice were isolated in a single cage for 3 days prior to the test, whereas unfamiliar stranger mice (129S1/SvlmJ strain) were group-housed (5-7 mice/cage). All stranger mice were age-matched males and were habituated to a corner cage during the previous day (30 min). The test consisted of three phases: empty-empty (habituation), stranger 1-object (S1-O), and stranger 1-stranger 2 (S1-S2). In the first phase (habituation), a test mouse was placed in the center area of the three-chambered apparatus, and allowed to freely explore the whole apparatus for 10 min. The mouse was then gently guided to the center chamber while an inanimate blue cylindrical object (O) and a WT stranger mouse (S1) were placed in the two corner cages. The positions of object (O) and stranger 1 (S1) were alternated between tests to prevent side preference. In the S1-O phase, the test mouse was allowed to explore the stranger mouse or the object freely for 10 min. Before the third phase (S1-S2), the subject mouse was again gently guided to the center chamber while the object was replaced with a new WT stranger mouse (S2). The subject mouse again was allowed to freely explore all three chambers and interact with both stranger mice for 10 min. The duration of sniffing, defined as positioning of the nose of the test mouse within 2.5 cm of a cage, was measured using EthoVision XT10 (Noldus) software.

Direct Social Interaction Test
Direct social interaction tests were performed as described previously . All mice were isolated for 3 days prior to the day of the experiment. Each individual mouse was habituated to a gray box (30 × 30 × 30 cm; ∼25-30 lux) for two consecutive days (10 min/d). On day 3, pairs of mice of the same genotype (originally housed separately) were placed in the test box for 10 min. Time spent in nose-tonose interaction, following, and total interaction were measured manually in a blinded manner. Nose-to-nose interaction was defined as sniffing the head part of the other mouse. Following included regular following as well as nose-to-tail sniffing. Total interaction included nose-to-nose interaction, following, body contact, allo-grooming, and mounting.

Tube Test
The tube test assay was performed as described previously . Mice were group-housed (4 in a cage) for 2 weeks before behavioral experiments. We used transparent acryl tubes with 30-cm length and 3-cm inner diameter. During two-day training sessions, each mouse was trained to pass through the tube in either direction for eight times under illuminated (∼30 lux) conditions. As the mice hesitated to move, they were gently pushed by a plastic bar. After this, 3 days of test sessions were proceeded. Animals went through three more training trials before the test. For the test, two different mice were placed into the opposite ends of the test tube and carefully released to meet in the middle of the tube. The mouse that first retreated from the tube was marked as a "loser." Among six possible pairs between four cage-mates, two pairs were tested per day. Each mouse was ordered by its rank from 1 to 4.

Courtship Ultrasonic Vocalization
Adult subject male mice were isolated in their home cage for 3 days before the test, whereas age-matched intruder female mice were group-housed (6-7 mice/cage). We did not measure female cycles on the assumption that group housing might synchronize cycles. Basal ultrasonic vocalizations (USVs) of an isolated male mouse in its home cage under light conditions of ∼60 lux in a soundproof chamber were recorded for 5 min in the absence of a female intruder. Next, a randomly chosen stranger C57BL/6J female mouse was introduced into the cage, and female-induced courtship USVs were recorded for 5 min during free interaction between the male and female. Avisoft SASLab Pro software was used to automatically analyze the number of USV calls, latency to first call, and total duration of calls from recorded USV files. Signals were filtered from 1 to 100 kHz and digitized with a sampling frequency of 250 kHz, 16 bits per sample (Avisoft UltraSoundGate 116H). Spectrograms were generated using the following parameters: FFT length, 256; frame size, 100; window, FlatTop; overlap, 75%. These parameters yielded a frequency resolution of 977 Hz and a temporal resolution of 0.256 ms. Frequencies lower than 25 kHz were filtered out to reduce white background noise.

Repetitive Behavior and Self-Grooming Test
Each mouse was placed in a lighted (∼60-70 lux), fresh home cage with bedding and recorded for 20 min. Time spent in self-grooming and digging behavior, measured manually, was determined by analyzing the last 10 min. Self-grooming behavior was defined as stroking or scratching of the body or face, or licking body parts. Digging was defined as scattering bedding using the head and forelimbs. Self-grooming behavior was further analyzed by placing mice in an empty home cage without bedding and recording them for 20 min. Time spent in self-grooming behavior was counted manually in a blinded manner during the last 10 min.

Laboras Test
Each mouse was placed in a single cage and recorded for 96 consecutive hours from the start of the night cycle. The illumination condition during light-on periods was ∼60 lux. Basal activities (locomotion, climbing, rearing, grooming, eating, and drinking) were recorded and automatically analyzed using the Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS, Metris). Laboras results were not validated by our own manual analyses, given the availability of previous validation results ( Van de Weerd et al., 2001;Quinn et al., 2003Quinn et al., , 2006Dere et al., 2015). Mouse movements during the entire 4-day period were used for quantification of behaviors, except for repetitive behavior, for which analyses were restricted to movements during light-off periods, which yielded clearer results.

Open-Field Test
Mice were placed in the center of an illuminated (90-100 lux) white acrylic box (40 × 40 × 40 cm), and their locomotion was recorded with a video camera for 1 h. The recorded video was analyzed using EthoVision XT10 software (Noldus). The center zone was defined as a 4 × 4-square area at the center of the entire 6 × 6-square region.

Elevated Plus-Maze
The maze consists of two open arms (30 × 6 cm, ∼180 lux) and two closed arms (30 × 6 cm, ∼20 lux) elevated 75 cm from the floor. Mice were introduced into the center of the apparatus with their head oriented toward the open arms and were allowed to freely explore the environment for 8 min. Amounts of time spent in open or closed arms and number of transitions were measured using EthoVision XT10 software (Noldus).

Light-Dark Test
The light-dark (LD) apparatus was divided into light (700 lux; 21 × 29 × 20 cm) and dark (∼5 lux; 21 × 13 × 20 cm) chambers separated by an entrance in the middle wall (5 × 8 cm). Mice were introduced into the light chamber with their head oriented toward the opposite side of the dark chamber and were allowed to freely explore the apparatus for 10 min. Amounts of time spent in light and dark chambers and number of transitions were analyzed using EthoVision XT10 software (Noldus).

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 5 software. Details of statistical analyses are presented in Supplementary Table S2. The normality of data distributions was determined using the D'Agostino and Pearson omnibus normality test, followed by Student's t-test (in the case of a normal distribution) and Mann-Whitney U test (in the case of a non-normal distribution). If samples were dependent on each other, a paired t-test (in the case of a normal distribution) or Wilcoxon signed rank test (in the case of a non-normal distribution) was used. Repeated-measures, two-way analysis of variance (ANOVA) with post hoc Bonferroni test (in the case of significant interactions) was used for time-varying analyses of open-field tests and Laboras tests. In cases where a Grubb's test showed that a single significant outlier ( * P < 0.05) caused data to be non-normally distributed, the outlier value was removed prior to analysis. A one-sample t-test was used for the analysis of Western blot data. P-values < 0.05 were considered statistically significant; individual P-values are indicated in figures as follows: * P < 0.05; * * P < 0.01; * * * P < 0.001; and ns, not significant.

RESULTS
Generation and Basic Characterization of Emx1-Cre;Shank3 14−16 Mice To analyze the effects of a Shank3 deletion restricted to glutamatergic neurons, we crossed Shank3 fl/fl mice (exons 14-16) with an Emx1-Cre mouse line, known to drive gene expression in glutamatergic neurons and glia with a dorsal telencephalic origin (Gorski et al., 2002).
The resulting Emx1-Cre;Shank3 14−16 mice, genotyped by PCR (Figure 1A), exhibited strong reductions in the levels of Shank3a and Shank3c/d variants in the hippocampus and cortex (Figures 1B,C), a finding in agreement with previous results on alternative splicing in Shank3 (Lim et al., 1999;Maunakea et al., 2010;Waga et al., 2014;Wang et al., 2014b). In contrast, Shank3 expression was largely unaffected in the thalamus, a brain region that is minimally affected by the Emx1 driver, and the striatum, which is mainly populated by GABAergic inhibitory neurons.
Levels of other members of the Shank family of proteins, namely Shank1 (Shank1a variant reported previously) (Lim et al., 1999;Naisbitt et al., 1999) and Shank2 (Shank2a and Shank2b reported previously) (Schmeisser et al., 2012;Won et al., 2012), were unaffected by Shank3 deletion in the tested brain regions (Figures 1D,E), indicative of the lack of compensatory changes.

Increased Excitability in Global
Shank3 14−16 and Emx1-Cre;Shank3 14−16 mPFC Layer 2/3 Pyramidal Neurons Previous studies have associated Shank3 deletion with altered neuronal excitability in human and rodent neurons (Peixoto et al., 2016;Yi et al., 2016), suggesting the possibility of altered neuronal excitability in Shank3-deficient cortical neurons. To determine whether Shank3 deletion affects intrinsic excitability in layer 2/3 cortical pyramidal neurons in the mPFC, a brain region implicated in ASD, and whether glutamatergic neurons are involved, we measured and compared neuronal excitability in global Shank3 14−16 and Emx1-Cre;Shank3 14−16 pyramidal neurons in the prelimbic region of the mPFC.
Global Shank3 14−16 mice exhibited increased neuronal excitability in layer 2/3 pyramidal neurons, as shown by the current-spike curve and input resistance, two electrophysiolgical parameters that contribute to neuronal excitability in depolarizing and hyperpolarizing ranges of membrane potentials (Figures 2A-C). Emx1-Cre;Shank3 14−16 mice also showed similarly increased neuronal excitability in mPFC neurons (Figures 2D-F). These results suggest that glutamatergic neurons contribute to the increased neuronal excitability observed in global Shank3 14−16 mice.  (478 bp), and the primer set targeting general Cre generates a PCR band (272 bp) in Emx1-Cre;Shank3 14-16 mice, but not in WT mice. (B,C) Reduced levels of Shank3 protein variants in different brain regions of Emx1-Cre;Shank3 14-16 mice (12-13 weeks, male and female). Total brain lysates were analyzed by immunoblotting using a Shank3-specific antibody (#2036) (B). Neither the Shank3e isoform in the thalamus nor the Shank3c/d isoform in the striatum was quantified because of their low levels of expression in these regions. Th, thalamus; Str, striatum; Hpc, hippocampus; Ctx, cortex. cKO band signals normalized to α-tubulin are expressed relative to those from WT mice (C). Data are shown as mean ± SEM. n = 5 pairs (WT, cKO), * P < 0.05, * * P < 0.01, * * * P < 0.001, nd, not detectable, ns, not significant, and one sample t-test. (D,E) Normal levels of Shank1 and Shank2 protein variants in different brain regions of Emx1-Cre;Shank3 14-16 mice (12-13 weeks, male and female). Total brain lysates were analyzed by immunoblotting using a Shank1-specific antibody (#2100) and Shank2-specific antibody (162 202, SYSY) (D). cKO band signals normalized to α-tubulin are expressed relative to those from WT mice (E). Data are shown as mean ± SEM. n = 5 pairs (WT, cKO), ns, not significant, and one sample t-test.

Altered Excitatory and Inhibitory
Spontaneous Synaptic Transmissions in Global Shank3 14−16 , but Not Emx1-Cre;Shank3 14−16 , mPFC Layer 2/3 Pyramidal Neurons Given that neuronal excitability acts together with excitatory and inhibitory synaptic inputs to determine neuronal output function, we next measured excitatory and inhibitory synaptic transmission in Shank3-mutant mPFC neurons.
The frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs) was increased in the prelimbic region of the mPFC of global Shank3 14−16 layer 2/3 neurons compared with WT mice (Figure 3A). In contrast to mEPSCs, miniature inhibitory postsynaptic currents (mIPSCs) were not changed in global Shank3 14−16 mice ( Figure 3B).
We also measured excitatory and inhibitory synaptic transmission in the presence of network activity by excluding tetrodotoxin (a blocker of action potential firing) during slice recordings. The frequency and amplitude of spontaneous EPSCs (sEPSCs) were normal in global Shank3 14−16 layer 2/3 pyramidal neurons in the prelimbic region of the mPFC compared with those in WT neurons ( Figure 3E).
Notably, the frequency, but not amplitude, of spontaneous IPSCs (sIPSCs) was increased in global Shank3 14−16 layer 2/3 pyramidal neurons ( Figure 3F). In addition, both sEPSCs and sIPSCs were normal in Emx1-Cre;Shank3 14−16 mice (Figures 3G,H). These results collectively suggest that global Shank3 deletion leads to increases in mEPSC frequency and sIPSC frequency, whereas glutamatergic Shank3 deletion has no effects on any forms of spontaneous synaptic transmission in layer 2/3 mPFC pyramidal neurons.
Because our previous results revealed decreased excitatory synaptic transmission in dorsolateral striatal neurons in both global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice (Yoo et al., 2018), we next measured spontaneous excitatory and inhibitory synaptic transmission in dorsolateral striatal neurons.
However, there were no changes in the frequency or amplitude of mEPSCs in Emx1-Cre;Shank3 14−16 dorsolateral striatal neurons compared with WT neurons (Figure 4A). In addition, neither the frequency nor amplitude of mIPSCs was altered in Emx1-Cre;Shank3 14−16 dorsolateral striatal neurons ( Figure 4B). These results contrast with the strongly decreased mEPSC frequency and amplitude in dorsolateral striatal neurons in global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice (Yoo et al., 2018 In the three-chambered social interaction test, designed to measure social approach and social novelty recognition (Crawley, 2004;Moy et al., 2004;Silverman et al., 2010), Emx1-Cre;Shank3 14−16 mice showed social approach behaviors that are comparable to those of WT mice, as shown by time spent sniffing social and object targets ( Figure 5A). In addition, Emx1-Cre;Shank3 14−16 mice displayed normal social novelty recognition, as shown by time spent sniffing familiar and novel stranger mice.
Intriguingly, in experiments using genotype-and age-matched mouse pairs, Emx1-Cre;Shank3 14−16 mice showed enhanced social interaction in the direct social interaction test, as shown by time spent in nose-to-nose sniffing and total social interaction ( Figure 5B). These results indicate that Emx1-Cre;Shank3 14−16 mice display normal social approach and social novelty recognition, but abnormally enhanced direct social interaction, similar to the social behaviors of global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice in these tests (Yoo et al., 2018). These changes do not seem to involve altered social dominance, as supported by the lack of genotype difference in the Tube test ( Figure 5C). These results suggest that both glutamatergic and GABAergic neurons contribute  to the abnormally enhanced direct social interaction in global Shank3 14−16 mice. We next evaluated USVs, which are strongly associated with rodent behaviors and emotional states, including social communication (Knutson et al., 1998(Knutson et al., , 2002Portfors, 2007;Scattoni et al., 2009). Adult male Emx1-Cre;Shank3 14−16 mice encountering a novel female mouse emitted normal numbers of USVs compared with WT mice (Figure 5D). Notably, this result differs from the suppressed courtship USVs observed in global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice (Yoo et al., 2018), suggesting that GABAergic, but not glutamatergic, neurons strongly contribute to the USV deficits in global Shank3 14−16 mice.
Modestly Enhanced Repetitive Self-Grooming, but Normal Digging, in Emx1-Cre;Shank3 14−16 Mice We next evaluated repetitive behaviors, a core component of ASD-related behavior, in Emx1-Cre;Shank3 14−16 mice. Emx1-Cre;Shank3 14−16 mice displayed enhanced self-grooming in a new home cage with bedding but normal self-grooming in a new home cage without bedding (Figures 5E,F), suggesting that the presence of bedding is required for repetitive behavior in addition to a new cage or environment. This result shows similarities to the strong self-grooming behaviors in global Shank3 14−16 mice observed in all three environments (new home cage with bedding, new home cage without bedding, and Laboras cages), but is more comparable to the mildly enhanced self-grooming in Viaat-Cre;Shank3 14−16 mice, observed only in a new home cage with bedding (Yoo et al., 2018). Measurements of digging, another method for quantifying repetitive behavior, showed no changes in Emx1-Cre;Shank3 14−16 mice compared with WT mice, even in the presence of bedding. This contrasts with the decreased digging observed in both global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice (Yoo et al., 2018).
Emx1-Cre;Shank3 14−16 mice subjected to the Laboras test, designed to measure mouse behaviors for a long period of time (i.e., four consecutive days) in a light/dark-cycling environment with bedding (Quinn et al., 2003(Quinn et al., , 2006, showed normal levels of self-grooming (Figures 5G,H). The results of these tests, in which mice were fully habituated, especially on days 2-4, suggest that Emx1-Cre;Shank3 14−16 mice show enhanced selfgrooming only in a particular environment (i.e., novel home cage with bedding). Other behaviors of Emx1-Cre;Shank3 14−16 mice, including climbing, rearing, drinking and eating, were unchanged in Laboras cages ( Figure 5H). Because hyperactivity and anxiety are observed in ASD, PMS and schizophrenia, we also evaluated locomotor activities of Emx1-Cre;Shank3 14−16 mice. In the open-field test, representing a novel environment, Emx1-Cre;Shank3 14−16 mice showed normal levels of locomotor activity, as shown by distance moved during 60 min ( Figure 6A). In Laboras cages, representing a familiar environment, Emx1-Cre;Shank3 14−16 mice showed normal levels of locomotor activities during the last 72 h ( Figure 6B). Locomotion in Laboras cages was also unchanged during the first 6 h, similar to the results of the open-field test. These results suggest that glutamatergic Shank3 deletion does not affect locomotor activity, in contrast to the reported hypoactivity of both global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice (Yoo et al., 2018).
In anxiety-related behavioral tests, Emx1-Cre;Shank3 14−16 mice spent a normal amount of time in the center region of the open-field arena ( Figure 6A), but spent an increased amount of time in the open arm of the elevated plus-maze (EPM) (Figure 6C), and a normal amount of time in the light chamber of the LD apparatus ( Figure 6D) (Yoo et al., 2018). In addition, the normal light-chamber time in the LD test in Emx1-Cre;Shank3 14−16 mice differs from the reduced light-chamber time (anxiety-like behavior) in global Shank3 14−16 and Viaat-Cre;Shank3 14−16 mice (Yoo et al., 2018) (summarized in Table 1). Therefore, the two contrasting anxiety-like behaviors in global Shank3 14−16 mice-anxiolytic-like behavior in the EPM and anxiety-like behavior in the LD apparatus-seem to more strongly involve glutamatergic and GABAergic neurons, respectively.
Control Emx1-Cre Mice Show Normal Locomotor Activity, Anxiety-Like Behavior, Direct Social Interaction, and Repetitive Behavior It is conceivable that control mice harboring Emx1-Cre alone might show behavioral abnormalities. To test this, we analyzed the behaviors of Emx1-Cre mice. These mice showed normal behaviors in Laboras cages, including locomotion, climbing, and rearing (Supplementary Figure S1A). In addition, Emx1-Cre mice showed normal levels of locomotor activity in the open-field test and time spent in the center region of the open-field arena (Supplementary Figure S1B). These mice also showed normal levels of time spent in the open arm of the EPM (Supplementary Figure S1C), direct social interaction (Supplementary Figure S1D), and self-grooming and digging in home cages with bedding (Supplementary Figure S1E). These results suggest that control Emx1-Cre mice show normal locomotion, repetitive behavior, and anxiety-related behaviors.
Then how might a Shank3 deletion lead to an increase in mEPSC frequency in global Shank3 14−16 layer 2/3 pyramidal neurons? Increased mEPSC frequency may involve increased excitatory synapse number or increased excitatory synaptic transmission through mechanisms, including increased neuronal excitability of presynaptic neurons and increased efficiency of presynaptic release. Therefore, one possibility is that the increased neuronal excitability in global Shank3 14−16 mPFC neurons increases the output function of these neurons and activates the intra-cortical network between layer 2/3 neurons, promoting the development of excitatory synapses in target layer 2/3 cortical neurons. Intriguingly, a previous study has shown that Shank3 could be detected in axonal compartments and nerve terminals and negative regulates presynaptic NMDARs (Halbedl et al., 2016), suggesting that the loss of presynaptic Shank3 might contribute to the increased mEPSC frequency. Alternatively, the increased neuronal excitability induced by loss of the interaction between Shank3 and HCN channels (Yi et al., 2016) may promote excitatory synaptic transmission and excitatory synapse development in a cell-autonomous manner; however, this is an unlikely possibility, as noted above.
Our measurements of sEPSCs and sIPSCs provide additional insight into the role of network activity in the context of a Shank3 deletion. Specifically, global Shank3 14−16 layer 2/3 pyramidal  Table summarizes increases or decreases in various electrophysiological and behavioral phenotypes in a given mouse line relative to WT/control mice, but is not intended to compare phenotypic severities across different mouse lines. * P < 0.05, * * P < 0.01, and * * * P < 0.001; NS, no significant change; NM, not measured; up and down arrows, increases and decreases, respectively. Areas shaded in orange represent similar phenotypes.
neurons showed normalized sEPSC frequency and increased sIPSC frequency (Figures 3E,F), findings that contrast with the increased mEPSC frequency and normal mIPSC frequency in the same neurons (Figures 3A,B). These sEPSC/sIPSC phenotypes likely represent compensatory changes that serve to suppress the increased mEPSC frequency as well as the increased neuronal excitability in these neurons and thus normalize the neuronal output. Indeed, the fact that sEPSCs in global Shank3 14−16 layer 2/3 pyramidal neurons are normalized suggests that these compensatory changes could actually normalize the neuronal output, at least in the slice preparation, which likely represents baseline conditions. However, the consequence of these compensatory effects seems to be abnormally increased inhibitory synaptic transmission onto pyramidal neurons that still maintain their increased neuronal excitability, as measured in the presence of network activity. Therefore, although neuronal output was apparently normalized in layer 2/3 neurons, the balance between excitatory and inhibitory synaptic transmission, and neuronal activity, might be disrupted. In keeping with this, an imbalance in excitation/inhibition ratio has been implicated in ASD (Rubenstein and Merzenich, 2003;Yizhar et al., 2011;Lee et al., 2015;Nelson and Valakh, 2015;Lee E. et al., 2017;Selimbeyoglu et al., 2017). A disruption in excitation/inhibition balance may also alter network properties such as brain rhythms.
Notably, a recent study on Shank3 4−22 mice carrying deletions in exons 4-22 (not exons 14-16, as in the current study) reported behavioral phenotypes that are surprisingly similar to those observed in our global Shank3 14−16 mice (Yoo et al., 2018), including normal social approach, enhanced direct social interaction, suppressed courtship USVs, enhanced selfgrooming, open-field hypoactivity, and anxiolytic-like behavior (EPM) . In addition, a more recent related study investigated the impacts of a Shank3 (exons 4-22) deletion restricted to Nex-positive glutamatergic neurons in the cortex, hippocampus, and amygdala (Nex-Cre;Shank3 4−22 mice) (Bey et al., 2018). Intriguingly, Nex-Cre;Shank3 4−22 mice recapitulated many behavioral phenotypes of global Shank3 4−22 mice, including normal social approach and enhanced self-grooming, similar to the results from global Shank3 14−16 and Emx1-Cre;Shank3 14−16 mice reported here. In addition, these Nex-Cre;Shank3 4−22 mice did not recapitulate the suppressed courtship USV or hypoactivity phenotypes of global Shank3 4−22 mice. Again, this is similar to the results from our mice (global and Emx1), which together with our demonstration that Viaat-Cre;Shank3 14−16 mice display suppressed courtship USVs suggests (Yoo et al., 2018) that GABAergic neurons may be important for the courtship USV phenotype in Shank3-deficient mice.
However, Nex-Cre;Shank3 4−22 mice not only failed to recapitulate the hypoactivity of global Shank3 4−22 mice, they actually showed increased locomotor activity in open-field tests (Bey et al., 2018), results in contrast with the normal locomotor activity behavior in our Emx1-Cre;Shank3 14−16 mice. Whether these differences involve differentially altered striatal synaptic transmission remains unclear because the previous study on Nex-Cre;Shank3 4−22 mice analyzed synaptic transmission only in the hippocampus (Bey et al., 2018). However, these discrepancies could be attributable to differences in the specific exons of Shank3 deleted or specific characteristics of Nex-Cre versus Emx1-Cre mice (Guo et al., 2000;Gorski et al., 2002;Goebbels et al., 2006).
In conclusion, our results suggest that glutamatergic Shank3 (exons 14-16) deletion increases neuronal excitability in layer 2/3 mPFC cortical neurons, but has no effect on synaptic transmission in dorsal striatal neurons. It also induces social and repetitive behavioral deficits, similar to the effects of global and GABAergic Shank3 deletions.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The animal study was reviewed and approved by the Committee of Animal Research at Korea Advanced Institute of Science and Technology (KAIST) (KA2016-30).