GABA Neuronal Deletion of Shank3 Exons 14–16 in Mice Suppresses Striatal Excitatory Synaptic Input and Induces Social and Locomotor Abnormalities

Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14–16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14–16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.

Given that Shank3 is an important component of excitatory synapses (Boeckers et al., 1999;Lim et al., 1999;Naisbitt et al., 1999;Tu et al., 1999), and that the imbalance of excitation and inhibition (E/I) at synaptic and neuronal levels has been implicated in ASD (Yizhar et al., 2011;Nelson and Valakh, 2015;Lee E. et al., 2017), Shank3 dysfunctions may have significant influences on E/I imbalances associated with ASD. Importantly, however, because Shank3 is expressed in both excitatory and inhibitory neurons (Han et al., 2013), the consequences of Shank3 mutations in mixed neuronal populations are not easy to predict and should be assessed by direct cell type-specific Shank3 deletion in vivo for better understanding of related brain regions, cell types, and neural circuits. In further support of the importance of Shank3 expression in GABAergic neurons, Shank3 is highly expressed in the striatum (Peca et al., 2011), a brain region enriched with GABAergic neurons and known to be associated with various brain functions as well as neurological and psychiatric disorders (Balleine et al., 2007;Kreitzer and Malenka, 2008;Grueter et al., 2012;Báez-Mendoza and Schultz, 2013). In addition, GABAergic neurons in the striatum have dendritic spines where Shank3 may play important roles in the regulation of spinogenesis and axospinous synapse functions (Harris and Weinberg, 2012;O'Rourke et al., 2012).
To this end, we attempted a GABA neuron-specific deletion of Shank3 exons 14-16, which encodes the PDZ domain known to interact with many synaptic proteins, including GKAP/SAPAP (Kim and Sheng, 2004;Sheng and Kim, 2011), using the Viaat-Cre mouse line that drives Cre recombinase expression in widespread GABAergic neurons (Chao et al., 2010). The electrophysiological and behavioral phenotypes of these mice were compared with those from mice carrying a global Shank3 deletion (exons 14-16). We found that GABA neuron-specific Shank3 deletion induces a strong reduction in excitatory synaptic input onto dorsolateral striatal neurons and abnormal social and locomotor behaviors, while having moderate effects on repetitive and anxiety-like behaviors.

Animals
Mice carrying a deletion of exons 14-16 of the Shank3 gene flanked by LoxP sites were designed and generated by Biocytogen. The EGFP+ Neo cassette was eliminated by crossing these mice with protamine-Flp mic. EGFP+ Neo cassette-deleted Shank3 flox/+ mice were crossed with protamine-Cre mice, and the resulting mice were then crossed with wild-type (WT) mice to introduce the Shank3 ∆14-16 allele. Experimental Shank3 ∆14-16 global knockout mice were obtained by heterozygous mating (Shank3 ∆14-16/+ × Shank3 ∆14-16/+ ). To generate Shank3 ∆14-16 cell type-specific conditional knockout (cKO) mice in which Shank3 is knocked out in Viaat (vesicular inhibitory amino acid transporter)-expressing GABAergic neurons (Viaat-Cre;Shank3 fl/fl mice), homozygous Shank3 flox/flox female mice were crossed with double-heterozygous Viaat-Cre;Shank3 flox/+ male mice. The control group for the cKO mouse was Cre-negative Shank3 flox/flox littermates. Viaat-Cre, protamine-Flp and protamine-Cre mouse lines used in this study were maintained in a C57BL/6J genetic background for more than five generations, a breeding strategy that allowed us to compare all global and Viaat-Cre mouse line in the same pure C57BL/6J background. All 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 experimental procedures were approved by the Committee of Animal Research at KAIST (KA2016-30). All animals were fed ad libitum and housed under the 12 h light/dark cycle (light phase during 1:00 am to 1:00 pm). Polymerase chain reaction (PCR) genotyping of conventional knockout mice was performed using the following primers: for WT allele (276 bp): 5'-GGG TTC CTA TGA CAG CCT CA-3' and 5'-TTC TGC AGG ATA GCC ACC TT-3'; for deletion (del) allele (1,159 bp): 5'-GGG TTC CTA TGA CAG CCT CA-3' and 5'-AGC TCA GCC GTC ATG GAC-3'. Genotypes of Viaat-Cre;Shank3 fl/fl mice were determined by PCR using the following primers: for floxed (478 bp) or WT allele (276 bp): 5'-GGG TTC CTA TGA CAG CCT CA-3' and 5'-TTC TGC AGG ATA GCC ACC TT-3'; for Viaat-Cre allele (272 bp): 5'-GTG TTG CCG CGC CAT CTG C-3' and 5'-CAC CAT TGC CCC TGT TTC ACT ATC-3'. Only male mice were used for behavioral and electrophysiological experiments. Both male and female were used for biochemical experiments.

Fluorescent in situ Hybridization (FISH)
In brief, frozen sections (14 µm thick) were cut coronally through the cortex and striatum formation. Sections were thaw-mounted onto Superfrost Plus Microscope Slides (Fisher Scientific #12-550-15). The sections were fixed in 4% formaldehyde for 10 min, dehydrated in increasing concentrations of ethanol for 5 min, and finally air-dried. Tissues were then pretreated for protease digestion for 10 min at room temperature. Probe hybridization and amplification were performed at 40 • C using HybEZ hybridization oven (Advanced Cell Diagnostics, Hayward, CA, USA). The probes used in this study were three synthetic oligonucleotides complementary to the nucleotide (nt) sequence 1488-2346 of Mm-Shank3, nt 62-3113 of Mm-Gad1-C3, nt 552-1506 of Mm-Gad2-C2, nt 464-1415 of Mm-Slc17a7/Vglut1-C2, and nt 1986-2998 of Mm-Slc17a6/Vglut2-C3 (Advanced Cell Diagnostics, Hayward, CA, USA). The labeled probes were conjugated to Alexa Fluor 488, Atto 550, and Atto 647. The sections were hybridized with the labeled probe mixture at 40 • C for 2 h per slide. Unbound hybridization probes were removed by washing the sections three times with 1× wash buffer at room temperature for 2 min. Following steps for signal amplification included incubations at 40 • C with Amplifier 1-FL  for 30 min, with Amplifier 2-FL for 15 min, with Amplifier  3-FL for 30 min and with Amplifier 4 Alt B-FL for 15 min. Each amplifier solution was removed by washing with 1× wash buffer at room temperature for 2 min. The slides were viewed, analyzed and photographed using TCS SP8 Dichroic/CS (Leica), and the ImageJ program (NIH) was used to analyze the images.

Western Blot
Total brain lysates separated in electrophoresis and transferred to a nitrocellulose membrane were incubated with primary antibodies to Shank3 (#2036 guinea pig polyclonal antibodies raised against aa 1289-1318 of the mouse Shank3 protein, 1:500; Lee et al., 2015) and α-tubulin (Sigma T5168; 1:1,000) at 4 • C overnight. Fluorescent secondary antibody signals were detected using Odyssey Fc Dual Mode Imaging System.

Rat Neuron Culture, Immunocytochemistry and Imaging
Primary hippocampal neuronal cultures were prepared from Sprague-Dawley rats at E18 as described previously (Goslin and Banker, 1991). Dissociated neurons were plated in coverslips coated with poly-L-lysine and laminin, and grown in neurobasal media supplemented with B27 (Invitrogen), 0.5 mM glutamax (Invitrogen) and 12.5 µM glutamate (plating media) in a 10% CO 2 incubator. After, this plating media and maintained media were replaced with feeding media (same as plating media only except for glutamate) every week. For immunocytochemistry, cultured neurons (at days in vitro or DIV 15) were fixed with 1% paraformaldehyde/1% sucrose (5 min) and methanol (5 min), permeabilized with 0.1% gelatin, 0.3% Triton X-100, 450 mM NaCl in phosphate buffered saline (PBS), and immunostained with primary antibodies against Shank3 (Santa Cruz H-160, 1:200) and GAD67 (Abcam ab26116, 1:200), and FITC-, and Alexa594-conjugated secondary antibodies (Jackson ImmunoResearch). The images were acquired using a confocal microscope (LSM780, Carl Zeiss) with a ×63 objective lens. The Z-stacked images were converted to maximal projection.

Behavioral Assays
Before behavioral experiments, all mice were handled for 10 min per day for 3 days. All behavioral assays were proceeded after 30 min habituation in a dark booth. All tested mice were 2-7 months male mice. The order of behavioral tests was designed in a way to minimize stress in animals. The behavioral tests for global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice were performed in the orders described in Supplementary  Table S1.

Three-Chamber Test
Social approach was measured using the three-chambered test Nadler et al., 2004;Silverman et al., 2010). The apparatus is a white acrylic box (60 × 40 × 20 cm) divided into three chambers. The illumination condition was ∼10 lux for global Shank3 ∆14-16 mice and 70-80 lux for Viaat-Cre;Shank3 ∆14-16 mice. We used a dim light condition (∼10 lux) for global Shank3 ∆14-16 mice because a brighter light condition (∼70-80 lux) did not yield optimal results in WT mice. 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), stranger1-object (S1-O) and stranger1-stranger2 (S1-S2). In the first (habituation) phase, 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, termed stranger 1 (S1), were placed in the two corner cages. The positions of object (O) and 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 S1-S2 phase, the subject mouse was again gently guided to the center chamber while the object was replaced with a new WT stranger mouse, termed stranger 2 (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 from a cage, was measured using Ethovision XT10 (Noldus) software.

Direct Social Interaction Test
Each individual mouse spent 10 min in a gray box (30 × 30 × 30 cm; ∼25-30 lux) for two consecutive days for habituation. On day 3, pairs of mice of the same genotype (originally housed separately) were placed in the test box for 10 min. All mice were isolated for 3 days prior to the experimental day. Time spent in nose-to-nose 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 the behavior of a mouse following the other mouse as well as nose-to-tail sniffing. Total interaction included nose-tonose interaction, following, body contact, allo-grooming and mounting.

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 estrous cycles, assuming that group housing may synchronize the cycles. Basal ultrasonic vocalizations (USVs) of an isolated male mouse in its home cage under a light condition 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 males and females. 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 Hz to 100 kHz and digitized with a sampling frequency of 250 kHz, 16 bits per sample (Avisoft UltraSoundGate 116H). To generate spectrograms, the following parameters were used (FFT length: 256, frame size: 100, window: FlatTop, overlap: 75%), resulting in a frequency resolution of 977 Hz and a temporal resolution of 0.256 msec. Frequencies lower than 25 kHz were filtered out to reduce background white noises.

Repetitive Behavior and Self-Grooming Test
Each mouse was placed in a fresh home cage (∼60-70 lux) with bedding and recorded for 20 min. The last 10 min was analyzed manually to measure times spent in self-grooming and digging behavior. Self-grooming behavior was defined as stroking or scratching of its body or face, or licking its body parts. Digging was defined as the behavior of scattering bedding using its head and forelimbs. To further analyze self-grooming behavior, mice were placed in an empty home cage without bedding and were recorded for 20 min. Time spent in self-grooming behavior was counted manually during the last 10 min in a blind manner.

Laboras Test (Long-Term Monitoring)
Each mouse was placed in a single cage and recorded for 96 consecutive hours from the start of the night cycle. Illumination condition during light-on periods was ∼60 lux. Basal activities (locomotion, climbing, rearing, grooming, eating and drinking) were recorded and automatically analyzed by the Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS, Metris). Laboras results were not validated by 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 whole 4-day period were used for quantification, except for other behaviors, for which movements during light-off periods were used for more clear results.

Open-Field Test
Mice were put in the center of a white acrylic box (40 × 40 × 40 cm), and their locomotion was recorded with a video camera for 1 h. The illumination of the open field was 90-100 lux. The recorded video was analyzed using Ethovision XT10 software (Noldus). The center zone was defined as an area with 4 × 4 squares when the whole-field was 6 × 6 squares.

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

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

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 5 software. Details of statistical analyses and results are presented in Supplementary Table S2. The normality of the data distribution was determined using the D'Agostino and Pearson omnibus normality test, followed by Student's t-test (in the case of normal distribution) and Mann-Whitney U test (in the case of non-normal distribution). If, sample is dependent each other, paired t-test (in the case of normal distribution), and Wilcoxon signed rank test (in the case of non-normal distribution). Repeated-measures of two-way ANOVA and subsequent Bonferroni post hoc multiple comparison tests, performed only when there are significant interactions, were used for the time-varying analysis of open-field test and Laboras test. If a single value makes the data distribution as non-normal and is detected as significant outlier ( * P < 0.05) under the Grubb's test, we removed the data as outliers. One sample t-test was used for the analysis of western blot data. The statistical significance of values are indicated in the figure panels as follows: * P < 0.05, * * P < 0.01, * * * P < 0.001, nd, not detectable and ns, not significant.

Expression of Shank3 in Both Glutamatergic and GABAergic Neurons
To explore the contributions of Shank3 expression in excitatory and inhibitory neurons to synaptic functions and behaviors in mice, we first tested whether Shank3 is expressed in glutamatergic and GABAergic neurons using fluorescence in situ hybridization (FISH). Shank3 in situ signals were present in Vglut1-and Vglut2-positive glutamatergic neurons in brain regions including the medial prefrontal cortex (mPFC; Figures 1A,B), indicative of Shank3 expression in glutamatergic excitatory neurons. Shank3 signals were also present in Gad1-and Gad2-positive GABAergic neurons in brain regions including the mPFC and the dorsolateral region of the striatum (Figures 1C,D). These results suggest that Shank3 mRNA is expressed in both glutamatergic and GABAergic neurons. Shank3 mRNA signals outside of DAPI-labeled nuclei or neighboring cell body regions may represent dendritic (rather than somatic) Shank3 mRNA, as previously reported (Epstein et al., 2014).
To further characterize Shank3 expression in GABAergic neurons, we immunostained for Shank3 protein in GABAergic neurons in cultured rat hippocampal neurons. Shank3 signals were detected in dendrites of both GAD67 (encoded by Gad1)-positive GABAergic neurons and GAD67-negative cells ( Figure 1E). In addition, punctate Shank3 signals were observed at shaft excitatory synapses on dendrites of GAD67-positive GABAergic neurons ( Figure 1F). These results, together with the previously reported positive expression of EGFP-tagged Shank3 in GAD-6-positive GABAergic neurons (Han et al., 2013), suggest that Shank3 is expressed in both glutamatergic and GABAergic neurons.
Generation and Characterization of Global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 Mice To analyze the effects of cell type-specific Shank3 deletion, we first generated a new mouse line harboring a cassette containing exons 14-16 of the Shank3 gene flanked by flox sequences, and then crossed these mice with protamine-Flp and protamine-Cre mice to produce mice in which Shank3 exons 14-16 were globally and homozygously deleted (Shank3 ∆14-16 mice; Figure 2A). PCR confirmed the genotype of these mice (Figure 2B), and immunoblot analyses revealed that the two main splice variants of the Shank3 protein (Shank3a and Shank3c/d) were absent in several brain regions ( Figure 2C), a result expected based on previous studies on the alternative splicing of Shank3 (Lim et al., 1999;Maunakea et al., 2010;Waga et al., 2014;Wang et al., 2014).
We next generated mice carrying Shank3 ∆14-16 deletion restricted to GABAergic neurons by crossing Shank3 fl/fl mice with Viaat-Cre mouse lines, which drives gene expression globally in GABAergic neurons by the solute carrier family 32 (GABA vesicular transporter) member 1 (Slc32a1 or Viaat/vesicular inhibitory amino acid transporter) promoter (Chao et al., 2010;Kim et al., 2018). Viaat-Cre;Shank3 ∆14-16 mice, genotyped by PCR (Figure 2B), showed a strong reduction in Shank3a in the striatum (Figure 2D), a brain region enriched with GABAergic neurons. Notably, the hippocampus displayed a strong tendency for an increase in Shank3 expression, likely reflecting compensatory changes in the mutant pyramidal neurons caused by the Shank3 deletion in GABAergic neurons in the hippocampus or other brain regions.

Suppressed Excitatory Synaptic
Transmission in the Dorsolateral Striatum in Global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 Mice We first measured excitatory and inhibitory synaptic transmission in the dorsal striatum, a region with enriched with GABAergic neurons and implicated in the development of abnormal behaviors in Shank3-mutant mice (Peca et al., 2011;Peixoto et al., 2016). Both the frequency and amplitude of mEPSCs were substantially decreased in dorsolateral striatal neurons in global Shank3 ∆14-16 mice, with frequency exhibiting a larger decrease; in contrast, mIPSCs were normal (Figures 3A,B).
Similar changes were observed in dorsolateral striatal neurons in Viaat-Cre;Shank3 ∆14-16 mice: mEPSC frequency and amplitude were decreased, whereas mIPSCs were normal (Figures 3C,D). Collectively, these results suggest that global and GABAergic Shank3 deletions similarly suppress excitatory synaptic transmission in dorsolateral striatal neurons without affecting inhibitory synaptic transmission. To test the impact of global and GABA neuron-specific deletions of Shank3 exons 14-16 on behaviors in mice, we subjected Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice to a battery of behavioral tests. Shank3 ∆14-16 mice displayed normal social approach behavior in the three-chamber test, but increased social interaction in the direct social interaction test (Figures 4A,B). These mice also showed suppressed USVs upon encounter with a novel female stranger (courtship USVs; Figure 4C).
Viaat-Cre;Shank3 ∆14-16 mice showed normal social approach behavior in the three-chamber test but enhanced direct social interaction and suppressed courtship USVs (Figures 4D-F), similar to the behaviors observed in global Shank3 ∆14-16 mice. These results suggest that social interaction phenotypes induced by global Shank3 ∆14-16 deletion is largely recapitulated in mice with Shank3 exons 14-16 deletion restricted to GABAergic neurons.
Viaat-Cre;Shank3 ∆14-16 mice showed enhanced self-grooming and suppressed digging in home cages with bedding and enhanced rearing in Laboras cages (long-term monitoring), but normal self-grooming in a novel cage without bedding as well as in Laboras cages (Figures 5E-H); these results differed in some respects from those of the global Shank3 ∆14-16 mice.
Home-cage digging and Laboras-cage climbing were similarly reduced in global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice. These results suggest that the strong self-grooming induced by global Shank3 deletion is not fully recapitulated by the GABAergic Shank3 deletion, while the digging and climbing are similarly suppressed by both deletions.
Similar Novelty-Induced Hypoactivity in Global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 Mice In tests for locomotion, global Shank3 ∆14-16 mice displayed reduced locomotor activity in the open-field test, a novel environment ( Figure 6A). In Laboras cages (long-term monitoring), global Shank3 ∆14-16 mice showed strong hypoactivity during the first 2 h and modest hypoactivity measured over the first 6 h; during the last 72 h, a period after full habitation to the environment, Shank3 ∆14-16 mice exhibited normal locomotor activity ( Figure 6B). These results suggest that Shank3 ∆14-16 mice show hypoactivity in a novel, but not a familiar, environment.
Viaat-Cre;Shank3 ∆14-16 mice showed decreased locomotor activity in the open-field test and during the first 6 h in Laboras cages (long-term monitoring; Figures 6C,D), similar to the novelty-induced hypoactivity in global Shank3 ∆14-16 mice. These results suggest that global and GABAergic Shank3 deletion lead to similar novelty-induced hypoactivity in mice.

Partially Similar Anxiety-Like Behaviors in
Global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 Mice In tests for anxiety-related behaviors, global Shank3 ∆14-16 mice did not show anxiety-like behavior in the open-field test, as shown by the normal amount of time spent in the center region of the open-field arena ( Figure 7A). However, these mice were less anxious in the elevated plus-maze test, spending more time in the open arm (Figure 7B), and, conversely, more anxious in the light-dark apparatus, spending less time in the light chamber ( Figure 7C). Shank3 ∆14-16 mice also showed a reduced number of transitions between light and dark chambers in the light-dark apparatus, in line with the hypoactivity of the mice. These results suggest that global Shank3 ∆14-16 mice show differential anxiety-like behaviors.
Viaat-Cre;Shank3 ∆14-16 mice were more anxious in open-field and light-dark tests, spending less amount of time in the center region of the open-field arena ( Figure 7D) and in the light chamber of the light-dark apparatus ( Figure 7F).
However, these mice did not show anxiety-like behavior in the elevated plus-maze test, spending normal amount of time in the open arm ( Figure 7E).
These results suggest that global and GABAergic Shank3 deletions similarly induce anxiety-like behaviors in the light-dark test, whereas they have differential influences on other types of anxiety-like behaviors. Therefore, GABAergic Shank3 deletion seems to strongly contribute to the anxiety-like behavior of global Shank3 ∆14-16 mice in the light-dark test. These results also suggest that GABAergic Shank3 deletion does induce anxiety-like behavior in the open-field test, but this is masked by FIGURE 4 | Enhanced direct social interaction and suppressed social communication in global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice. (A) Normal social approach and social novelty recognition in Shank3 ∆14-16 mice (14-21 weeks) in the three-chamber test, as shown by time spent sniffing. S1, stranger; O, object; S2, novel stranger. Data are shown as mean ± SEM. n = 28 (WT) and 22 (KO), * * * P < 0.001, paired t-test. Details on the order of behavioral tests performed on Shank3 ∆14-16 mice and conditional Shank3 ∆14-16 mouse lines (see below) are described in Supplementary Table S1. (B) Enhanced social interaction in Shank3 ∆14-16 mice (14-20 weeks) in the direct social interaction test, as shown by nose-to-nose interaction, following and total interaction, the latter of which additionally includes allo-grooming and body contacts. Mean ± SEM. n = 20 (WT) and 16 (KO), * P < 0.05, * * P < 0.01, * * * P < 0.001, Student's t-test. global Shank3 deletion. In contrast, GABAergic Shank3 deletion seems to have minimal impacts on the anxiety-like behavior in the elevated plus-maze test, suggesting that non-GABAergic Shank3 deletions are more important for the anxiolytic-like behaivor of global Shank3 ∆14-16 mice in the elevated plusmaze.

DISCUSSION
In this study, we investigated the impacts of global and GABA neuron-specific deletion of Shank3 exons 14-16 on synaptic transmission and behaviors in mice. Global Shank3 ∆14-16 mice display decreased excitatory input onto dorsolateral striatal neurons and strong abnormalities in social, repetitive, locomotor and anxiety-like behaviors. The electrophysiological and behavioral (social and locomotor) phenotypes observed in global Shank3 ∆14-16 mice are strongly mimicked by Viaat-Cre;Shank3 ∆14-16 mice, although the repetitive and anxiety-like behavioral deficits in global Shank3 ∆14-16 mice are partially mimicked by Viaat-Cre;Shank3 ∆14-16 mice (summarized in Table 1). The result that both the frequency and amplitude of mEPSCs are reduced in global Shank3 ∆14-16 mice (Figure 3) further strengthens the notion that Shank3 is important for the development and function of excitatory synapses in the dorsal striatum. Similar decreases in the frequency and amplitude of mEPSCs in the dorsal striatum have been observed in the Shank3 mouse line lacking exons 13-16 (Shank3B −/− mice; Peca et al., 2011;Mei et al., 2016;Wang et al., 2017).
A more important finding from our study is that both mouse lines (global and Viaat-Cre) show similar decreases in the frequency and amplitude of mEPSCs in dorsolateral striatal neurons (Figure 3). This suggests that the suppressed excitatory input onto dorsolateral striatal neurons in these mouse lines are likely to be induced by the deletion of Shank3 in striatal GABAergic neurons in a cell autonomous manner.
dorsal striatum in addition to mPFC (Figure 1). In addition, the immunostaining result indicates that Shank3-positive punctate structures are observed on the dendrites of GAD67-positive GABAergic neurons in cultured hippocampal neurons. Given that Shank3 is an important component of the postsynaptic density at excitatory synapses (Sheng and Kim, 2000;Sheng and Sala, 2001;Boeckers et al., 2002;Sheng and Hoogenraad, 2007;Grabrucker et al., 2011;Sheng and Kim, 2011;Jiang and Ehlers, 2013;Sala et al., 2015;Monteiro and Feng, 2017;Mossa et al., 2017), the lack of Shank3 in dorsolateral striatal neurons may suppress normal development and maturation of the postsynaptic density, dendritic spines, and excitatory synapses. In addition, previous studies have reported a strong decrease in dendritic spine density in dorsal striatal neurons in Shank3B −/− mice (Peca et al., 2011), further suggesting that the decreased mEPSC frequency may be a consequence of postsynaptic changes. More recently, however, additional analyses of excitatory synaptic inputs onto D1 and D2 medium spiny neurons (MSNs) in the dorsal striatum of Shank3B −/− mice have revealed that D2 MSNs show reductions in both presynaptic release and spine density (Wang et al., 2017), suggesting that both pre-and postsynaptic factors may be involved. In addition, a previous study on Shank3B −/− mice showed that early abnormal excitability in pyramidal neurons in the somatosensory cortex driven by the limited inhibitory input from neighboring GABAergic neurons induces precocious development of excitatory synapses on dorsomedial striatal neurons that leads to a decrease in the mEPSC frequency at later stages (Peixoto et al., 2016). It is therefore possible that the decreased mEPSC frequency in dorsolateral striatal neurons in global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice may represent the consequences of the primary changes occurring in cortical GABAergic neurons.
Behaviorally, global Shank3 ∆14-16 mice display altered social and repetitive behaviors, including suppressed courtship USVs and enhanced self-grooming (Figures 4, 5). These mice also show hypoactivity and altered anxiety-like behaviors (Figures 6, 7). Given that Shank3 ∆14-16 mice lack the PDZ domain-containing Shank3 variants, Shank3a and Shank3c/d, but retain Shank3e, our mouse line is likely to display behavioral phenotypes similar to those observed in the Shank3B -/mouse line, which globally lacks exons 13-16 encoding the PDZ domain (Peca et al., 2011). Indeed, Shank3 ∆14-16 and Shank3B −/− mice show largely similar behaviors, including suppressed courtship USVs, hypoactivity, and anxiety-like behavior (elevated zero maze and light-dark test), although Shank3B −/− mice additionally show suppressed social approach (Peca et al., 2011;Dhamne et al., 2017). Another Shank3-mutant mice similar to ours is the one lacking exon 13, encoding the PDZ domain (Shank3 E13 mice; Jaramillo et al., 2017). These mice show enhanced self-grooming and social interaction deficits, but normal locomotion and anxiety-related behavior; the partial similarity to our behavioral phenotypes is likely attributable to the different exon targeting strategy (insertion of a stop codon in front of exon 13) in Shank3 E13 mice.
Although the behavioral phenotypes of global Shank3 ∆14-16 mice are strong in multiple domains (social, repetitive, locomotor, and anxiety-like), the following points need to be further discussed. First, global Shank3 ∆14-16 mice show enhanced direct social interaction, which was unexpected and is at variance with the normal three-chamber social approach observed in these mice. Notably, a previous study on Shank3 ∆4-22 mice has reported a similar increase in direct social interaction where Shank3 ∆4-22 mice display frequently attempted but unsuccessful social interactions with a stranger C3H mouse, a different strain, that does not reciprocate and terminate the social interaction attempted by the subject mouse , suggesting that Shank3 ∆4-22 mice have normal social interest but struggle with persisting social failures. We could not test whether this is the case for our mice because we used genotypematched (WT-WT or KO-KO) mouse pairs where monitoring of non-reciprocated social interaction is difficult because of the same coat color and the confusion over retraction vs. rejection. However, our results suggest that social interest is normal in global Shank3 ∆14-16 mice, which is different from the significant social interaction deficits observed in many other Shank3-mutant mouse lines (Jiang and Ehlers, 2013;Monteiro and Feng, 2017). We propose that the difference in the specific Shank3 exons deleted in each mouse lines might explain the discrepancy. For instance, the exons deleted in our Shank3 mice (exons 14-16) are distinct from those deleted in Shank3B −/− mice (exon 13-16; Peca et al., 2011). In support of this possibility, a very small difference in the exons deleted in Shank2-mutant mice (i.e., exons 6 and 7 vs. exon 7) has been shown to cause strong differences in molecular, synaptic and behavioral phenotypes (Schmeisser et al., 2012;Won et al., 2012;Lim et al., 2017;Wegener et al., 2018).
Another notable result is that global Shank3 ∆14-16 mice display anxiolytic-like behavior in the elevated plus-maze whereas they show anxiety-like behavior in the light-dark apparatus and normal anxiety-like behavior in the center region of open-field arena. This could be due to the different anxiogenic components in these tests (Belzung and Griebel, 2001;Carola et al., 2002;Carobrez and Bertoglio, 2005), as exemplified by  Figure 6A. Data are shown as mean ± SEM. n = 23 (WT) and 22 (KO), ns, not significant, Student's t-test. (B) Anxiolytic-like behavior in Shank3 ∆14-16 mice (13-21 weeks) in the elevated plus-maze test. n = 22 (WT) and 20 (KO), * * P < 0.01, * * * P < 0.001, paired t-test (for the left panels), and Student's t-test (for the right panel). (C) Anxiety-like behavior in Shank3 ∆14-16 mice (14-21 weeks) in the light-dark test. Note that these mice are also hypoactive in this test, as shown by the number of transitions. n = 23 (WT) and 20 (KO), * P < 0.05, * * P < 0.01, Student's t-test. (D-F) Viaat-Cre;Shank3 ∆14-16 mice (8-9 weeks for D, 8-10 weeks for E, and 11-20 weeks for F) spend a reduced amount of time in the center region of the open-field area (D; locomotor activity results are described in Figure 6C the differential responses of nine different mouse strains to the elevated plus-maze and light-dark tests (Griebel et al., 2000). Notably, Shank3B -/mice (exons 13-16) also display differential anxiety-like behaviors in these assays, being partly similar to our results; normal anxiety-like behavior in elevated plus-maze, anxiety-like behavior in zero maze, light-dark apparatus and open-field center (Peca et al., 2011;Dhamne et al., 2017).
Our results indicate that GABAergic neurons contribute to some of the abnormal behaviors observed in global Shank3 ∆14-16 mice. Specifically, the enhanced direct social interaction, suppressed courtship USVs, and novelty-induced hypoactivity observed in global Shank3 ∆14-16 mice were also observed in Viaat-Cre;Shank3 ∆14-16 mice. In contrast, the strong self-grooming behavior observed in global Shank3 ∆14-16 mice were only partially mimicked by Viaat-Cre;Shank3 ∆14-16 mice. In anxiety-like behaviors, only the light-dark test results were similar in global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice. Therefore, GABA neuronal Shank3 deletion seems to be more important for social and locomotor behaviors than repetitive and anxiety-like behaviors.
A recent study reported the effects of a deletion of Shank3 exons 4-22 restricted to Nex-positive glutamatergic neurons in the cortex, hippocampus and amygdala (Nex-Shank3 cKO mice) and Dlx5/6-positive GABAergic neurons in the striatum (Dlx5/6-Shank3 cKO mice; Bey et al., 2018). Neither Nex-Shank3 nor Dlx5/6-Shank3 cKO mice exhibit social approach deficits, results similar to the normal social approach behavior reported by the same group using mice with a global Shank3 ∆4-22 mice . This phenotype is also similar to the normal social approach behavior observed in our global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice.
The suppressed courtship USV and hypoactivity phenotypes observed in global Shank3 ∆4-22 mice  were not recapitulated in either Nex-Shank3 or Dlx5/6-Shank3 cKO mice (Bey et al., 2018). These results are different from our findings that both global Shank3 ∆14-16 and Viaat-Cre;Shank3 ∆14-16 mice show suppressed courtship USV and hypoactivity. Furthermore, the enhanced self-grooming observed in global Shank3 ∆4-22 mice  was observed in Nex-Shank3 cKO mice, but not in Dlx5/6-Shank3 cKO mice (Bey et al., 2018). These results are slightly different from our finding that the enhanced self-grooming in global Shank3 ∆14-16 mice was partially recapitulated in Viaat-Cre;Shank3 ∆14-16 mice. These results indicate that two different global Shank3 deletions (exons 14-16 and 4-22) in mice lead to remarkably similar behavioral phenotypes in mice in social, repetitive, locomotor and anxiety-like behavioral domains, but that these similarities are minimized by two different cKOs restricted to GABAergic neurons (Dlx5/6 and Viaat). These discrepancies could be attributable to differences in the specific exons of Shank3 deleted and/or specific characteristics of Dlx5/6-Cre vs. Viaat-Cre mice (Oh et al., 2005;Goebbels et al., 2006;Chao et al., 2010). For instance, Dlx5/6-Cre primarily targets GABAergic neurons in the striatum (Monory et al., 2006), whereas Viaat-Cre targets the majority of GABAergic neurons in the brain (Chao et al., 2010). In addition, it could be subtle differences in mouse housing conditions or experimental details.
In conclusion, our results suggest that the deletion of Shank3 exons 14-16 restricted to GABAergic neurons in mice induces phenotypes that are similar to those induced by global Shank3 deletion. These include strongly suppressed excitatory synaptic onto dorsolateral striatal neurons and strongly altered social and locomotor behaviors but modestly altered repetitive and anxiety-like behaviors.

FUNDING
This study was supported by the Institute for Basic Science (IBS-R002-D1 to EK).