Synaptic and Molecular Mechanisms in 16p11.2 Copy Number Variations

& Advancement of Behavioral Assays for Sociability in Mice

By

Benjamin Rein

February 1, 2021

A thesis submitted to the faculty of the Graduate School of the University at Buffalo, The State University of New York in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Department of Physiology & Biophysics

Copyright by

Benjamin Rein

2021

All Rights Reserved

ii

Acknowledgments

I would like to express my most sincere appreciation and gratitude to all those who provided

support and guidance throughout my PhD, whether it was providing experimental direction, technical

training, troubleshooting tips, or conceptual chats. I thank you all for your contributions over these years.

To Dr. Zhen Yan, for always keeping your office door open and always having the right answer

to my questions, while trusting me to work independently. In times of uncertainty, I could always rely on

you to provide a solution and orient me in the right direction. Your wisdom and guidance have been a

great source of inspiration, and have been so instrumental in my development. You have shown me the

qualities that distinguish a great mentor, and I look forward to applying these principles in my future role

as a principal investigator. I thank you for everything you have taught me over these years.

To my committee members: Dr. Fraser Sim, Dr. Malcolm Slaughter, and Dr. Pablo Paez, for

providing incredibly valuable scientific advice and helping me think in new dimensions about my

research.

Thank you to Zijun Wang, Freddy Zhang, Jamal Williams, and Megan Conrow, for all of the time

and energy you’ve spent in our creative and conceptual discussions. Your open ears and valuable, creative

inputs always enabled the advancement of our projects. I have cherished the privilege of always having a

stimulating and enlightening conversation around the corner from my desk.

To Miss Chen, thank you so incredibly much for all that you do for the lab. Your hard work is so

very much appreciated. Thank you, thank you, thank you.

To Zijun Wang, Max Rapanelli, Luye Qin, Ping Zhong, Wei Wang, Qing Cao, and Tao Tan, for

taking the time to train me in the lab. None of this is possible without you, and I cannot thank you enough

for giving me your time and passing along your tips and tricks.

To Dr. Sim, for allowing me to drop in the office for spontaneous chats and always answering my

questions. Thank you for the guidance you’ve provided, the letters you’ve written, and the opportunities

you have created for me.

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To my wonderful, outrageously loving and supportive family. Thank you for always celebrating

me and touting my accomplishments. You are my greatest source of confidence and strength. You know

how much I love you all.

To Bella, for always being so much more than just “my rock.” Thank you for always being the

first audience to hear my presentations, for genuinely caring about my ideas even when they were foreign

to you, and for working so hard to understand my research material so we could talk about it. Thank you

for being the comfort I often needed.

This research was supported by Nancy Lurie Marks Family Foundation and National Institutes of

Health (R41-MH112237, R01-MH108842, R01-DA037618).

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Abstract

Copy number variations (CNVs; i.e. deletion or duplication) affecting the human chromosome

16p11.2 locus are among the strongest genetic risk factors for autism spectrum disorder (ASD), and

predispose for a range of other neurodevelopmental conditions including schizophrenia, epilepsy and

intellectual disability. Numerous case reports and populations studies have characterized the clinical

profiles of 16p11.2 deletions and duplications; however, the underlying molecular mechanisms remain

poorly understood due to insufficient basic science investigations. In Part I of this thesis, we characterize

the behavioral, synaptic, and cellular pathologies associated with 16p11.2 deletions and duplications in

mice and identify aberrant synaptic transmission in both models in the prefrontal cortex (PFC), a brain

region critical for sociability and higher-level cognitive functions. Specifically, 16p11.2 deletion mice

(16p11.2+/-) display impaired NMDA receptor-dependent glutamatergic transmission and regional

hypoactivity in PFC, while 16p11.2 duplication mice (16p11.2dp/+) exhibit GABAergic synaptic deficits

accompanied by neuronal hyperactivity in PFC. Notably, the divergent excitability profiles in PFC of

16p11.2+/- and 16p11.2dp/+ mice are the first opposing molecular phenotypes identified in 16p11.2 CNVs,

and offer an intriguing bidirectional explanation for the associated behavioral pathologies. Moreover,

restoring homeostatic excitability to PFC neurons in either model is sufficient to ameliorate the observed

social and cognitive deficits, implicating excitation-imbalance in PFC as a core mechanism. These

findings have increased the potential for therapeutic interventions by identifying a single molecular

endpoint in both CNVs which may be pharmacologically targetable for the alleviation of social and

cognitive symptoms.

Mouse models are commonly used in ASD research, as mice are very social creatures. However,

the structure and significance of these social interactions are complex, making sociability a difficult

variable to assess experimentally. Several assays exist for evaluating sociability in mice, though these

tests fail to address certain facets of social behavior, and also suffer from inconsistent usage across

laboratories. A number of studies performed by different groups with identical ASD mouse models have

reported inconsistent findings about social behaviors, due to the use of different behavioral testing

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protocols. In Part II of this thesis, we sought to improve the existing methodology for assessing social

behavior in mice, both through the modification and optimization of existing protocols and the

development of entirely novel assays. We first describe a standardized protocol of the three-chamber

social preference test - a common method for evaluating sociability in ASD mouse models - which offers

more robust sensitivity to social deficits than a commonly used protocol in the literature. Secondly, we

propose a novel behavioral test for evaluating social motivation, which integrates components of the

social approach test and the elevated plus maze. Through these efforts, we hope to enable more accurate,

consistent and detailed behavioral investigations of mouse models across the ASD research field.

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Table of Contents

Acknowledgements…………………………………………………………….……... Page iii

Abstract …………………………………………………………………………......... Page v

List of figures and tables…………………………………………………………....... Page x

Part I

Chapter I: 16p11.2 Copy Number Variations and Neurodevelopmental Disorders Page 1

Abstract ………………………………………………………………………………... Page 2

The Link between 16p11.2 CNVs and Neurodevelopmental Disorders………………. Page 3

Analysis of Clinical Data from Humans with 16p11.2 CNVs ………………….……... Page 3

Prevalence of 16p11.2 CNVs …………………………………………..……... Page 3

Inheritance Patterns of 16p11.2 CNVs ………………………………………... Page 4

Neurodevelopmental Phenotypes Associated with 16p11.2 CNVs …………... Page 4

Insights from Preclinical Studies in 16p11.2 CNV Mouse Models……………..……... Page 7

Concluding Remarks ……………………………………....................................……... Page 12

Figures ……………………………………………………………………………...….. Page 14

Tables …………………………………………………………………………...……... Page 17

Chapter II: Chemogenetic Activation of Prefrontal Cortex Rescues Synaptic and Page 22

Behavioral Deficits in a Mouse Model of 16p11.2 Deletion Syndrome

Abstract…………………………………………………………………………..……... Page 23

Introduction ……………………………………………………………….……..……... Page 24

Results …………………………………………………………………………...……... Page 24

16p11.2+/- Mice Exhibit NMDA Receptor Hypofunction in PFC ……..…….... Page 24

Chemogenetic Activation of PFC Pyramidal Neurons in 16p11.2+/- mice…...... Page 26

Rescues NMDAR Hypofunction

Chemogenetic Activation of PFC Pyramidal Neurons in 16p11.2+/- mice .…..... Page 27

Ameliorates Cognitive and Social Deficits

Discussion ……………………………………………………………………….……... Page 28

Figures …………………………………………………………………………...……... Page 30

Chapter III: Reversal of Synaptic and Behavioral Deficits in a 16p11.2 Page 36

Duplication Mouse Model via Restoration of the GABA Synapse Regulator Npas4

Abstract………………………………………………………………………………..... Page 37

Introduction ……………………………………………………………….…….…….... Page 38

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Results …………………………………………………………………………..…….... Page 39

16p11.2dp/+ Mice Exhibit Social and Cognitive Behavioral Deficits …………... Page 39

Reminiscent of ASD and ID

GABAergic Synaptic Transmission is Impaired in 16p11.2dp/+ PFC …………... Page 41

Genome-wide Transcriptional Dysregulation in 16p11.2dp/+ PFC ……………... Page 42

Restoring Npas4 Expression in 16p11.2dp/+ mPFC Ameliorates ……………..... Page 43

Synaptic and Behavioral Deficits

Discussion …………………………………………………………………………….... Page 45

Figures ………………………………………………………………………………...... Page 48

Tables ………………………………………………………………………………...... Page 58

Part II

Chapter IV: A Standardized Social Preference Protocol for Measuring Social Page 65

Deficits in Mouse Models of Autism

Abstract……………………………………………………………………………...….. Page 66

Introduction ……………………………………………………………….………...….. Page 67

Comparison between the social preference test variants ………………...……. Page 67

Applications ………………………………..…………………………...……... Page 70

Limitations ………………………………..………………………….....……... Page 70

Experimental Design ………………………………..…………………………...……... Page 70

Materials ………………………………………………………………………………... Page 71

Procedure …………………………………………………………………..…..……..... Page 73

Expected Results ………………………………………………………………..…….... Page 78

Figures …………………………………………………………………………...……... Page 81

Chapter V: Diminished Social Interaction Incentive Contributes to Social Deficits Page 85

in Mouse Models of Autism Spectrum Disorder.

Abstract………………………………………………………………………….…….... Page 86

Introduction ………………………………………………………………,…….…….... Page 87

Results …………….………………………………..…………………………...……... Page 88

Shank3 and BTBR Mouse Models of ASD Exhibit Diminished Social .……... Page 88

Interaction Incentive in a Modified Elevated Plus Maze

Diminished Social Interaction Incentive in ASD Mouse Models is not ….…… Page 90

Revealed Under Less Aversive Barrier Conditions

viii

Discussion …….……………………………………………..………………….……... Page 91

Figures …………………………………………………………………………...……... Page 95

Chapter VI: Discussion and Conclusions …………..………………………………… Page 102

Chapter VII: Materials and Methods …………………….…………………...……... Page 105

Animals……………………………………………………………..…………...……... Page 105

Behavioral Techniques…………………………………………………...……...……... Page 106

Electrophysiological Recordings…………….………………………………………... Page 110

Viral Vectors and Animal Surgery………………..…………………………………... Page 111

Biochemical Techniques……………………..………………………………………... Page 111

References ……………………..………………………………………........................ Page 114

ix

List of Figures and Tables

Chapter 1: 16p11.2 Copy Number Variations and Neurodevelopmental Disorders

Figure 1. Common phenotypes in carriers of 16p11.2 CNVs.

Figure 2. Summary of main preclinical findings from mouse models of 16p11.2 deletion and duplication

Figure 3. Schematic showing molecular and cellular mechanisms of 16p11.2 genes and interacting

partners in nucleus, soma and synapses.

Table 1. Prevalence of 16p11.2 CNVs in the general population and within groups of clinical cohorts

with various neurodevelopmental disorders.

Table 2. Penetrance of neurodevelopmental disorders and physical abnormalities among 16p11.2 deletion

and 16p11.2 duplication carriers.

Table 3. Behavioral pehnotypes present in various mouse models carrying 16p11.2 deletion or

duplication.

Chapter II: Chemogenetic Activation of Prefrontal Cortex Rescues Synaptic and Behavioral Deficits in a

Mouse Model of 16p11.2 Deletion Syndrome

Figure 1. 16p11.2+/- mice exhibit diminished NMDAR-mediated synaptic responses in PFC.

Figure 2. AMPAR-mediated synaptic transmission is unchanged in PFC of 16p11.2+/- mice.

Figure 3. The total and synaptic levels of glutamate receptor subunits are unchanged in PC of 16p11.2+/-

mice.

Figure 4. Chemogenetic activation of PFC restores neuronal excitability in 16p11.2+/- mice.

Figure 5. Chemogenetic activation of PFC restores NMDAR function and elevates NR2B

phosphorylation in 16p11.2+/- mice.

Figure 6. Chemogenetic activation of PFC ameliorates cognitive and social deficits in 16p11.2+/- mice.

Chapter III: Reversal of Synaptic and Behavioral Deficits in a 16p11.2 Duplication Mouse Model via

Restoration of the GABA Synapse Regulator Npas4

Figure 1. 16p11.2dp/+ mice exhibit social deficits, repetitive behaviors, and cognitive impairment

reminiscent of ASD and ID symptoms.

Figure 2. 16p11.2dp/+ mPFC pyramidal neurons exhibit GABAergic synaptic deficits and elevated

excitability.

Figure 3. RNA-sequencing identifies numerous upregulated genes in PFC of 16p11.2dp/+ mice.

Figure 4. RNA-sequencing identifies downregulated genes from diverse classes in 16p11.2dp/+ PFC,

including the GABA-synapse regulator Npas4.

Figure 5. Restoring Npas4 expression in PFC ameliorates the social and cognitive deficits and restores

GABAergic synaptic transmission in 16p11.2dp/+ mice.

Table S1. Information regarding human postmortem tissue.

x

Table S2. qPCR primers.

Table S3. Detailed statistical information for all experiments described.

Table S4. Significantly upregulated genes in 16p11.2 duplication PFC identified via RNA-seq.

Table S5. Significantly downregulated genes in 16p11.2 duplication PFC identified via RNA-seq.

Chapter IV: A Standardized Social Preference Protocol for Measuring Social Deficits in Mouse Models

of Autism

Figure 1. Social behavioral data obtained from several transgenic mouse models using the 3-phase S-NS

protocol.

Figure 2. Social behavioral data obtained from several ASD mouse models using the 2-phase S-E

protocol.

Chapter V: Diminished Social Interaction Incentive Contributes to Social Deficits in Mouse Models of

Autism Spectrum Disorder.

Figure 1. Genotype confirmation for Shank3 mice.

Figure 2. Graphic illustrating the three elevated plus maze (EPM) protocols used in the current study.

Figure 3. Shank3 mice exhibit diminished social interaction incentive in a modified elevated plus maze

(EPM) protocol.

Figure 4. BTBR mice exhibit diminished social interaction incentive in a modified elevated plus maze

(EPM) protocol.

Figure 5. Graphic illustrating the three light-dark box protocols used in the current study.

Figure 6. Shank3 mice do not exhibit reduced social interaction incentive when presented with a less

aversive barrier to interaction.

Figure 7. BTBR mice do not exhibit reduced social interaction incentive when presented with a less

aversive barrier to interaction.

xi

Part I

Published in Trends in Neurosciences (2020), 24(11), 886-901.

Chapter I:

16p11.2 Copy Number Variations and Neurodevelopmental Disorders

Ben Rein* & Zhen Yan*

Department of Physiology and Biophysics, State University of New York at Buffalo, Jacobs School of

Medicine and Biomedical Sciences, Buffalo, NY 14214, USA

*: Correspondence should be addressed to B. R. (brein@buffalo.edu) or Z. Y. (zhenyan@buffalo.edu)

ABSTRACT

Copy number variations (CNVs) of the human 16p11.2 genetic locus are associated with a range

of neurodevelopmental disorders, including autism spectrum disorder, intellectual disability and epilepsy.

In this review, we delineate genetic information and diverse phenotypes in individuals with 16p11.2

CNVs, and synthesize preclinical findings from transgenic mouse models of 16p11.2 CNVs. Mice with

16p11.2 deletions or duplications recapitulate many core behavioral phenotypes, including social and

cognitive deficits, and exhibit altered synaptic function across various brain areas. Mechanisms of

transcriptional dysregulation and cortical maldevelopment are reviewed, along with potential therapeutic

intervention strategies.

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The Link between 16p11.2 CNVs and Neurodevelopmental Disorders

Genetic factors comprise a large proportion of the risk for neurodevelopmental disorders (NDDs)

such as autism spectrum disorder (ASD), schizophrenia, and intellectual disability (ID) [1]. Copy number

variations (CNVs, i.e. deletion or duplication) of various susceptible genetic loci predispose individuals to

these NDDs and other developmental abnormalities [2, 3]. The human 16p11.2 gene locus (chromosome

16, position 11.2) is a ~500-600 kb region containing 27-29 genes [4-7] located on the proximal short arm

of chromosome 16 [8]. Deletions and duplications of 16p11.2 have highly pleiotropic phenotypic effects,

with strong links to ASD [8-19], ID [8, 10, 12, 14-16, 19-21], motor/developmental delay [8-10, 12, 13,

15-19, 22], dysmorphic features [8, 12, 16, 17, 22], and epilepsy/seizures [10, 12, 17]. Deletions of

16p11.2 are associated with increased head circumference (macrocephaly) [10, 12, 17, 23] and obesity

[10, 12, 16, 20], whereas duplications often result in below average head size (microcephaly) [10, 17, 23,

24] and low body weight/BMI [10, 24]. Schizophrenia (SZ) appears to be associated more strongly with

16p11.2 duplications [9, 25-27].

The literature on 16p11.2 CNVs is large and growing. Here I provide a synthesis of the most

common neurodevelopmental phenotypes associated with 16p11.2 deletions/duplications (Figure 1).

Studies performed in animal models of 16p11.2 CNVs have also begun highlighting core neurobiological

mechanisms. Here we discuss key pathways and mechanisms of dysfunction identified through these

studies (Figure 2 and Figure 3), which may provide explanations for some of the phenomena observed in

human 16p11.2 CNVs.

Analysis of Clinical Data from Humans with 16p11.2 CNVs

Prevalence of 16p11.2 CNVs

Genetics Home Reference estimates that 16p11.2 deletions and duplications each affect about 3 in

every 10,000 individuals [28, 29]. A predictive algorithm estimated 16p11.2 deletions to affect 1 in every

3,021 live births, and duplications 1 in every 4,216 [30]. These estimates are supported by large genetic

screenings [20, 24, 31]. 16p11.2 deletions have been reported at rates of 0.028% - 0.043% in the general

population, while duplications have been reported between 0.035% - 0.053% (Table 1). Triplications of

16p11.2 have also been reported [32], though the frequency of this presumably rarer CNV is unknown.

Genetic screenings of individuals with NDDs find starkly higher rates of 16p11.2 CNVs. Data

compiled from eight studies [13, 15-17, 20, 24, 26, 33] screening individuals with either ASD, ID,

developmental delay (DD), dysmorphic features (DF), multiple congenital anomalies (MCA), obesity, or

seizures find 16p11.2 deletions at rates of 0.25-2.9%, and duplications at rates between 0.15-0.78%

(Table 1).

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Inheritance Patterns of 16p11.2 CNVs

We compiled data from 13 studies in which the rates of de novo vs. inherited cases of 16p11.2

deletions and duplications were reported [10, 12, 13, 15-17, 19, 20, 26, 33-36]. In studies with at least 50

subjects, between 60-76% of 16p11.2 deletions were reported as de novo events [10, 12, 34, 36], while

only 16-29% of 16p11.2 duplications occurred de novo [10, 19, 34, 36]. Both the deletion and duplication

appear to be preferentially maternally inherited.

Differing pathological severity of 16p11.2 deletions/duplications may underlie these dissimilar

inheritance patterns. One study estimates that the penetrance of any pediatric phenotype in deletion

carriers is 62.4%, relative to only 11.2% in duplication carriers [37]. The more severe outcomes

associated with 16p11.2 deletions preclude these patients from having children, resulting in lower rates of

inherited deletions [34]. However, de novo and inherited deletion carriers do not perform differently on

several cognitive tasks [35]. De novo and inherited duplication carriers also show no differences in most

clinical outcomes [19], and on cognitive tests [10]. Altogether, these results suggest that 16p11.2

deletions are more likely to be de novo events than duplications.

Neurodevelopmental Phenotypes Associated with 16p11.2 CNVs

Autism spectrum disorder (ASD): Genetic screenings of ASD patients repeatedly identify 16p11.2

deletions & duplications, placing them among the strongest genetic risk factors for ASD [11, 13, 16, 38].

Data accumulated from 14 studies on the penetrance of ASD in 16p11.2 deletion and duplication carriers

are shown in Table 2 [9, 10, 12, 14-17, 19-21, 23, 33-35]. In multiple studies of subjects with 16p11.2

deletion or duplication, autistic features or a formal ASD diagnosis were reported in 16.1% - 25.6% of

deletion carriers, and 20% - 33.9% of duplication carriers. Overall, ASD appears to be a highly penetrant

phenotype in both 16p11.2 duplications and deletions.

Intellectual Disability/Cognitive Impairment: Table 2 summarizes findings from 6 studies on the

penetrance of ID in 16p11.2 deletion and duplication carriers [10, 14, 19, 21, 23, 34]. In studies of at least

50 subjects, ID was reported in 10.3% - 28.1% of 16p11.2 deletion carriers, and 30.5% - 40.3% of

duplication carriers. Note that several of these studies were conducted in clinically ascertained

populations of 16p11.2 CNV carriers, so the estimate of ID penetrance may be exaggerated.

A study of 270 duplication carriers and 442 deletion carriers found that FSIQ was significantly

lower than intrafamilial controls in both 16p11.2 duplication carriers (18.0 points) and deletion carriers

(22.1 points) [10]. Several supporting studies also reported lower FSIQ scores in 16p11.2 deletion [12,

35] and duplication [19, 35] patients than intrafamilial controls. Higher variability in FSIQ scores is

observed among duplication carriers than deletion carriers [10, 19], suggesting a broader range of

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cognitive impairments in duplication patients. In one group of 16p11.2 duplication carriers, FSIQ scores

range from 33-129, with 17% displaying severe impairment (<55), while 33% fall within the average

range or better [19]. Interestingly, 16p11.2 duplication patients with ASD display more severe cognitive

impairments than those without ASD [10], indicating that the severity of cognitive phenotypes may

correlate with the pathogenesis of other neurodevelopmental disorders, particularly ASD.

Epilepsy: Epilepsy/seizures ranks among the most common phenotypes observed in both 16p11.2

deletions and duplications [10, 12, 16, 17, 33, 36] (Table 2). In studies of at least 50 subjects,

epilepsy/seizures has been reported in 21.8% - 26.8% of deletion carriers, and 19.4% - 29% of duplication

carriers. Epilepsy is thus a highly penetrant phenotype in 16p11.2 deletions and duplications.

Developmental Delay/Language Impairment: Motor delays are reported at rates of 57.1% [15] and 50%

[17] in deletion carriers, and in 60% of duplication carriers [17]. A very delayed (>24 mo.) age of first

walking is observed in 6.1% of deletion carriers and 15.9% of duplication carriers [10]. Balance

impairment and gait abnormalities are reported in both 16p11.2 CNVs [39]. Developmental coordination

disorder is reported at rates of 32% [23] and 57.7% [21] in deletion carriers, and 46.8% in duplication

carriers [19]. Speech articulation abnormalities [36], phonological processing disorder [21, 23], language

or communication disorder [14, 21, 23], speech or language deficits [15, 16], and speech delay [17, 33]

are present at high rates in deletion carriers. Duplications are similarly associated with speech articulation

abnormalities [36], speech delay [17, 19], and language deficits [16]. One study performed deep

phenotyping of speech and language skills in 16p11.2 deletion carriers, providing further insight into the

specific impairments present [40]. Interestingly, motor control of speech is impaired in 16p11.2 deletion

carriers [41], indicating that the motor and language deficits may be fundamentally linked.

Schizophrenia/Psychosis: In a 2009 study, 21 duplications and 1 deletion were detected among 5,877

schizophrenia (SZ) patients, indicating a 14.5-fold increased SZ risk in 16p11.2 duplication patients [9].

One follow-up study reported 6 duplications in 659 SZ patients [42]. An analysis of 22 screenings in

various populations found 98 duplications in 36,676 SZ patients (0.26%), with only 12 duplications in

48,331 controls (0.025%), indicating a 10.79-fold increase in risk for developing SZ [43]. Psychotic

symptoms have also been reported in duplication (7/114 [6.1%]) and deletion (5/217 [2.3%]) carriers

[34]. In one study, both 16p11.2 deletion and duplication carriers exhibited psychotic symptoms, though

only the duplication was a significant predictor of psychotic symptoms [44]. Thus, 16p11.2 duplications

predispose robustly to SZ and associated psychotic symptoms, with greater penetrance than 16p11.2

deletions. The link between 16p11.2 duplications and SZ is particularly interesting, considering the

5

growing perspective of SZ as a neurodevelopmental disorder [45]. The overlapping genetic predisposition

by 16p11.2 CNVs to both SZ and more typical NDDs like ASD and ID appears to support a

neurodevelopmental origin of SZ [46].

Other psychiatric phenotypes in 16p11.2 CNVs: 16p11.2 deletions and duplications are linked to several

other psychiatric conditions, including depression [14, 47] and anxiety [12, 14, 18, 19, 23, 34]. Attention-

deficit/hyperactivity disorder (ADHD) is also observed in 16p11.2 CNV carriers [12, 15-17, 19, 23, 48],

at rates as high as 29% (63/217) and 42% (48/114) in deletion and duplication carriers, respectively [34].

Bipolar disorder is also reported in 16p11.2 duplication carriers [9, 14, 24].

Dysmorphic features/congenital anomalies: Summarized data from published reports on dysmorphic

features/congenital anomalies (DF/CA) in 16p11.2 CNVs are shown in Table 2 [10, 12, 17, 24, 33]. In

studies of at least 50 subjects, DF/CA were reported in in 21.1% - 58.5% of deletion carriers, and 16.7% -

28.7% of duplication carriers, indicating high penetrance in both CNVs.

Obesity/Body Mass Index (BMI): Several reports indicate higher BMI in 16p11.2 deletion patients [10,

12, 23]. 16p11.2 deletions are present at higher-than-expected rates in obese individuals [31], and are

more prevalent in developmental delay (DD) patients with obesity than those without obesity [20].

Conversely, BMI of 16p11.2 duplication patients is significantly lower than controls [10, 24]. Birth

parameters for 16p11.2 duplication carriers are generally normal [24], indicating a postnatal effect.

Cephalic phenotypes & neurostructural changes: Certain physical phenotypes, such as head size, display

dosage-dependent effects in 16p11.2 CNVs. Deletions of 16p11.2 are associated with macrocephaly [12,

17, 23, 36], while duplications are associated with microcephaly [10, 17, 23]. Summarized data on

penetrance of microcephaly/macrocephaly in 16p11.2 CNVs are shown in Table 2 [10, 12, 17, 23, 36]. In

studies of at least 50 subjects, macrocephaly was reported in 17.1% - 17.3% of deletion carriers, while

microcephaly was reported in 17.1% - 22.3% of duplication carriers. Several studies also report increased

or decreased head circumference in deletion [10, 12, 24, 36] or duplication carriers [10, 24], respectively.

MRI studies show reciprocal brain volume changes, with increased gray/white matter in 16p11.2

deletions, and decreased gray/white matter in 16p11.2 duplications [14, 23, 49, 50]. The increased or

decreased axial diffusivity of white matter and the thickening or thinning of the corpus callosum have

also been found in deletion or duplication patients, respectively [51-53]. Regional volumetric differences

are also found in 16p11.2 CNV humans. Several brain areas exhibit the increased volume in deletion

carriers and reduced volume in duplications, including insula, calcarine cortex [49, 50], accumbens,

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pallidum [54], transverse temporal gyrus [49, 50], caudate, putamen [49, 54], and thalamus [23].

Increased or decreased cortical surface area has been reported in 16p11.2 deletions or duplications,

respectively [14, 23, 49]. 16p11.2 duplications also have the reduced cortical thickness [14, 23], reduced

hippocampal volume [49] and enlarged ventricles [50, 53, 55].

Sex differences in 16p11.2 CNVs: Several neurodevelopmental disorders display a sex bias where males

are at a higher risk [56]. In agreement with this, the ratio of males to females with either ASD or ID

shows a male predominance in both 16p11.2 CNVs [57], suggesting that being female is a protective

factor when predicting overall ASD severity in either 16p11.2 CNV [58]. However, another study found

that sex was not a significant predictor of psychosis in 16p11.2 CNV carriers [44]. Thus, the association

between 16p11.2 CNVs and ASD/ID, but not psychosis, may involve a sex bias.

Insights from Preclinical Studies in 16p11.2 CNV Mouse Models

Here we describe behavioral phenotypes in 16p11.2 CNV mouse models, and synthesize the

major biological takeaways and implications (Figure 2 and Figure 3). Three lines of 16p11.2 CNV mice

have been generated: 16p11.2 mice (Mills) carrying deletion (16p11.2+/-) or duplication (16p11.2dp/+) of

7F4 region (Slx1b-Sept1) syntenic to human 16p11.2 [5]; 16p11.2 mice (Dolmetsch) with deletion of the

Coro1a-Spn interval [4]; 16p11.2 mice (Herault) with deletion/duplication of the Sult1a1-Spn genetic

interval [59]. Behavioral phenotypes in 16p11.2 deletion or duplication mice are summarized in Table 3.

Behavioral Phenotypes in 16p11.2 Deletion or Duplication Mice Recapitulate Neurodevelopmental

Deficits of Human Carriers

All 3 lines of 16p11.2+/- mice display social deficits in various testing paradigms [59-64], and an

array of cognitive deficits [4, 59, 62-68]. Sleep abnormalities [69, 70] and anxiety [66] have been

reported in 16p11.2+/- mice (Mills). Startle response [4, 60] and pre-pulse inhibition (PPI) [60] are

severely impaired in the 16p11.2+/- mice (Dolmetsch). However, these mice are deaf [60], which likely

underlies startle/PPI deficits and may be related to altered ultrasonic vocalizations. Mild motor deficits

are also present in these models [4, 61, 68], consistent with human phenotypes. However, 16p11.2+/-

models show reduced body size/weight [4, 5, 60, 61, 63], in contrast to the human obesity phenotype.

Hyperlocomotion is also broadly reported in 16p11.2+/- mice [4, 5, 59, 61, 63, 64, 70], another behavioral

feature that does not coincide directly with human symptomology.

16p11.2+/- mice display impairments in cognition, sociability and motor function, coinciding with

phenotypes present in human 16p11.2 deletion carriers. However, several phenotypes, such as body

weight, are discordant between mice and humans. Another limitation is that many deficits present in

7

deletion carriers, such as apraxia of speech, cannot be tested or modeled in animals [71]. Thus, 16p11.2+/-

mice may represent a suitable preclinical model system for evaluating mechanisms of certain social and

cognitive dysfunctions, but several of the deficits associated with 16p11.2 deletions cannot be modeled in

these animals.

The 16p11.2dp/+ mice (Mills) exhibit social and cognitive deficits [72, 73] along with increased

repetitive self-grooming [5, 72], with the absence of motor coordination deficits [72]. Two behavioral

phenotypes associated with SZ, PPI of startle responses and MK-801-induced hyperlocomotion, were not

observed in 16p11.2dp/+ mice [72], despite a report on female-specific PPI deficits [73]. Based on these

results, the SZ-linked changes should be further investigated in 16p11.2dp/+ mice. 16p11.2dp/+ mice also

exhibit hypolocomotion [5, 72, 73], in direct contrast to 16p11.2+/- animals. Hypolocomotion and

enhanced object recognition memory are reported in 16p11.2dp/+ mice (Herault) on a C57BL/6N inbred

genetic background, whereas on a F1 C57BL/6N-C3B hybrid background, mice additionally exhibit

social approach deficits [59]. These studies indicate that 16p11.2dp/+ mice recapitulate several, but not all,

human 16p11.2 duplication phenotypes, including social deficits, repetitive behaviors, and cognitive

impairment.

Sex-Specific Behavioral Phenotypes in Mouse Models of 16p11.2 CNVs

Male, but not female, 16p11.2+/- mice (Mills) display reduced and altered pup isolation calls,

indicating sex-specific communication impairments in perinatal development [74]. Additionally, male

16p11.2+/- mice (Mills) exhibit sleep deficits [70], and impairments in a reward-directed learning task

[67], while females do not. In 16p11.2dp/+ mice (Mills), male-specific reductions in locomotion, time in

the center of an open field, and ratio of open arm/closed arm time in the elevated plus maze have also

been reported [73].

16p11.2 CNV Mouse Models Exhibit Synaptic Dysfunction in Distributed Brain Regions

The 16p11.2+/- mice (Mills) display deficient NMDA-receptor-mediated glutamatergic

transmission and reduced frequency of action potential firing in medial prefrontal cortex (mPFC)

pyramidal neurons [62], along with increased excitation-inhibition (E-I) ratio in somatosensory cortex

[75]. Additionally, GABAergic neurons in the ventral medulla display hyperpolarized resting membrane

potential and increased membrane resistance [69]. Long term potentiation and depression (LTP/LTD)

appear to be intact in hippocampal CA1 of 16p11.2+/- mice (Mills), though protein-synthesis-dependent

mGluR5 LTD is impaired [65]. Recordings from CA1 of 16p11.2+/- mice (Herault) reveal intact synaptic

transmission, along with modest but not statistically significant reductions in LTP [59]. Nucleus

accumbens (NAc) medium spiny neurons (MSNs) in 16p11.2+/- mice (Dolmetsch) display increased

8

AMPAR/NMDAR ratio and decreased paired pulse ratios (PPR), along with increased miniature EPSC

frequency [4]. The authors hypothesize that these results are due to increased release probability of

excitatory synapses on NAc MSNs [4]. Synaptic phenotypes in 16p11.2+/- mice evidently vary across

brain areas, suggesting that 16p11.2 deletion may cause region- or cell-type-specific impairments.

The 16p11.2dp/+ mice (Herault) show impaired LTP in CA1 [59]. However, these findings have

not yet been confirmed in other 16p11.2dp/+ models, and may benefit from behavioral validation with

corresponding memory tasks such as contextual fear conditioning [65]. The 16p11.2dp/+ mice (Mills)

display GABAergic synaptic deficits in mPFC pyramidal neurons and increased action potential firing

rates [72]. Restoring mPFC GABAergic synaptic activity is sufficient to reverse the social and cognitive

deficits [72], implicating prefrontal cortical synaptic dysfunction in 16p11.2 duplication pathology. In

addition, disrupted connectivity between hippocampal-orbitofrontal and hippocampal-amygdala circuits

has been found in 16p11.2dp/+ mice (Mills), as well as reduced expression of several GABAergic markers,

including parvalbumin, calbindin and somatostatin in frontal cortex [73].

16p11.2 CNVs Induce Broad Transcriptional Disruptions in Mice and Human Cells

Sequencing studies have revealed broad transcriptional disruptions in 16p11.2 CNVs. RNA-

sequencing identifies 2,344 and 1,504 significant differentially expressed genes (DEGs) in the cortex of

16p11.2+/- and 16p11.2dp/+ mice (Mills), respectively, as well as 908 and 1,290 nominally significant

DEGs in lymphoblastoid cell lines (LCLs) from human 16p11.2 deletion or duplication carriers,

respectively [76]. Gene ontology (GO) analysis of DEGs in mouse cortex indicates the strongest

disruption of genes related to “regulation of transcription”. The top pathways implicated in human LCLs

are “microtubule cytoskeleton organization” and “cell surface receptor-linked signal transduction”.

A separate RNA-seq experiment in mPFC of 16p11.2dp/+ mice (Mills) identifies 388 DEGs (277

downregulated, 111 upregulated), confirming broad transcriptional disruption extending far beyond the

genes located within the 16p11.2 region [72]. GO analysis indicates that the largest portion of

downregulated genes are classified as “Transcription Factors” (17.7%), consistent with prior RNA-seq

data [76]. The largest portion of upregulated DEGs (21.6%) are identified as “Enzyme Modulators”.

These findings highlight the wide transcriptional impacts of 16p11.2 CNVs, but further studies should

clarify the specific contributions of 16p11.2 genes to these far-reaching downstream transcriptional

disruptions and their links to the heterogeneous associated behavioral phenotypes.

Abnormal Cortical Development Driven by 16p11.2 CNVs Involves Several Genes in 16p11.2 Region

Abnormal cortical development has been proposed as a core mechanism in 16p11.2 CNVs. As

reviewed earlier, 16p11.2 deletions are associated with macrocephaly, whereas duplications are linked to

9

microcephaly. Human induced pluripotent stem cell (iPSC)-derived neurons from 16p11.2

deletion/duplication carriers display corresponding features, with increased soma size/dendrite length in

16p11.2 deletion neurons, and reduced size/dendrite length in duplication neurons [77]. In contrast,

16p11.2+/- mice display reduced brain size and reductions in upper layer cortical projection neurons,

driven by increased progenitor proliferation and premature cell cycle exit, resulting in depleted progenitor

pools and maldeveloped cortical structures [66]. The precise reasons for the reduced brain size remain

unclear, although the reduced brain size is associated with reduced body weight phenotype in 16p11.2+/-

mice, suggesting that at least in part, a broader developmental physiological impairment might be

involved.

Cortico-striatal circuits have been implicated in the pathophysiology of 16p11.2 deletion. Various

structural abnormalities are reported in the striatum and cortex of 16p11.2+/- mice (Dolmetsch), along with

an increased population of Drd2+ MSNs in the striatum, and reduced Drd1+ neurons in the cortex [4]. An

elevated number of MSNs and the increased expression of Drd2 in striatum are found in male 16p11.2+/-

mice (Mills) [4, 67]. Disrupted synaptic function in striatal MSNs is thought to underlie locomotion-

related behavioral phenotypes in 16p11.2+/- mice [4].

The MAPK3 gene located within the 16p11.2 region encodes the signaling molecule ERK1.

Human genetic screenings link MAPK3 signaling to ASD [78], and ERK dysregulation is associated with

ASD-related behavioral phenotypes in mice [79]. This pathway is also linked to ID [80]. Cultured

pyramidal neurons from 16p11.2dp/+ mice exhibit increased ERK1 phosphorylation, and greater dendritic

arborization, which can be reversed by ERK inhibition [81]. These findings support a role for Mapk3 in

cortical development abnormalities associated with 16p11.2 CNVs.

The 16p11.2 gene KCTD13 is also associated with cranial size phenotypes. In zebrafish, out of all

16p11.2 gene transcripts, only KCTD13 overexpression drives a microcephalic phenotype with reduced

brain cell counts, whereas its suppression produces a macrocephalic phenotype with increased brain cell

counts [6]. However, these findings do not generalize to mice: two follow-up studies reported normal

brain size in Kctd13-deficient mice [82, 83]. Kctd13-deficient mice display reduced dendritic

length/complexity and spine density, which is linked to downstream RhoA overexpression [82]. Kctd13-

deficient mice also display cognitive deficits in several memory tasks [83], raising the possibility that

similar mechanisms may contribute to ID pathogenesis in 16p11.2 CNVs.

Kctd13 is one of the adaptors of Cullin 3 (Cul3), a core component of the E3 ubiquitin ligase

complex mediating protein ubiquitination and degradation [84]. The Cul3 gene is one of the top-ranking

high-risk factors for autism and related neurodevelopmental disorders [85, 86]. In mice, Cul3 deficiency

in the forebrain or PFC induces social interaction impairment and sensory-gating deficiency, as well as

NMDA receptor hypofunction, whereas Cul3 loss in the striatum causes stereotypic behaviors, as well as

10

cell type-specific changes in neuronal excitability [87]. Abnormality in gene transcription or protein

translation may underlie the involvement of Cul3 in neurodevelopmental disorders [87, 88]. It has also

been suggested that dysregulation of the KCTD13-Cul3-RhoA pathway during the critical period for

establishing the connectivity of 16p11.2 proteins with their co-expressed partners determines the

abnormal brain sizes associated with 16p11.2 CNVs [89].

Knockout of the 16p11.2 gene Taok2 produces cognitive and social deficits in mice, and also

results in increased brain volume [90]. Taok2-deficient mice display reduced dendritic growth and

deficient excitatory synaptic transmission in PFC through a mechanism involving the reduced RhoA

expression [90], suggesting that the 16p11.2 genes Kctd13 and Taok2 may act upon convergent

downstream molecules. Taok2 has also been implicated in synaptic stabilization and spine maturation

[91].

In humans, mutations in MAPK3 [92] and TAOK2 [93, 94] but not in KCTD13 [85, 95] have been

linked to ASD. Missense variants in MAPK3 have been identified in ASD probands [85, 92], though no

loss of function (LoF) MAPK3 mutations have been reported in ASD, leaving the direct link to ASD

uncertain. LoF variants in TAOK2 have been identified in ASD probands [93, 94], and TAOK2 variants

were identified by whole-genome and exome sequencing of over 2,600 families with ASD [90]. However,

neither MAPK3 nor TAOK2 are definitively associated with neurodevelopmental disorders. Additionally,

results from rodent studies of individual genes need to be considered with caution, as the phenotypes of

16p11.2 CNVs may depend on the collective effects of multiple genes in 16p11.2 locus.

Neurostructural Changes in Mouse Models of 16p11.2 CNVs

In contrast to the human data [14, 23, 49, 50], 16p11.2+/- mice (Mills) do not exhibit changes in

gray matter [7], but they show male-specific increases in medial and peristriatal fiber tracts [7],

coinciding with the increased medial fiber tracts in human deletion carriers [14]. Increased volumes of

several brain areas, including the hypothalamus, midbrain, cerebellar cortex, striatum, nucleus accumbens

and globus pallidus, are observed in 16p11.2+/- mice (Mills, Dolmetsch) [4, 5, 96], concordant with the

increased volume of these structures in human deletion patients [54]. No significant differences were

found between 16p11.2dp/+ (Mills) and WT mice, though trends toward reduced volume were observed in

several regions [5].

Therapeutic Interventions: Targeting Glutamatergic and GABAergic Systems

Many valuable findings have been drawn from attempted intervention strategies in 16p11.2

mouse models. Inhibitory avoidance (IA) deficits are observed in 16p11.2+/- mice (Mills) [65]. In a mouse

model of Fragile X Syndrome, IA deficits are reversed via chronic post-adolescent administration of the

11

mGluR5 negative allosteric modulator (NAM) CTEP [97]. This approach was thus tested in 16p11.2+/-

mice and proved effective in ameliorating IA deficits [65].

The GABAB-receptor selective agonist R-baclofen was tested in 16p11.2+/- mice (Mills and

Dolmetsch), as R-baclofen displayed therapeutic efficacy in Fmr1-deficient mice with a similar synaptic

plasticity phenotype [64]. R-baclofen improves performance of 16p11.2+/- mice in several cognitive tasks,

while hyperlocomotion and USVs are unaffected [64]. However, R-baclofen and several mGluR5 NAMs

have produced negative results in clinical trials with Fragile X patients [98]. Thus, findings from

preclinical studies in animal models should be interpreted with caution. It has also been demonstrated that

GABAA receptor availability is unaffected in human 16p11.2 deletion carriers [99], though this does not

preclude the possibility of presynaptic GABAergic alterations or effects on other GABA receptor

subunits.

As reviewed earlier, 16p11.2+/- mice exhibit NMDAR deficits and hypoactivity in PFC pyramidal

neurons [62]. Both humans and mice carrying 16p11.2 deletions display reduced prefrontal cortical

connectivity [100], further suggesting a role for PFC disruption in 16p11.2 CNVs. Chemogenetic

activation of PFC leads to increased NMDAR phosphorylation and function, resulting in amelioration of

social and cognitive deficits [62]. This study suggests NMDAR hypofunction in PFC as a core

mechanism in 16p11.2 deletion-linked phenotypes, consistent with the significant involvement of

NMDARs in ASD [101-103].

As to the rescue of phenotypes in 16p11.2dp/+ mice (Mills), a recent study from our group [72]

revealed that restoring expression of Npas4, a key regulator of GABA synapses [104, 105] reverses

GABAergic deficits and ameliorates social and cognitive deficits in these mice, but not repetitive

grooming behavior. The therapeutic potential of pharmaceutical interventions to boost GABAergic

transmission by targeting Npas4 or related molecules for 16p11.2 duplication syndrome awaits to be

further explored.

Concluding Remarks and Future Perspectives

Clinical and preclinical investigations illustrate the diverse neurobiological impact of 16p11.2

CNVs. A number of highly penetrant developmental phenotypes are linked to both 16p11.2 deletions and

duplications (Figure 1). Given their effects on 27 genes, 16p11.2 CNVs have the capacity to cause broad

and severe downstream biochemical insults across various brain areas. The array of cellular changes in

16p11.2 models, including transcriptional and synaptic dysregulation, aberrant cell proliferation and

cortical development, provides a window into the molecular pathologies underlying behavioral

syndromes associated with 16p11.2 CNVs. Restoring E/I balance and synaptic plasticity by targeting

glutamate and GABA systems is suggested as a core intervention strategy (Figure 2).

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Characterizations of 16p11.2 CNV mouse models have illuminated several important

pathophysiological clues, though more work must be done before one could come up with a clear disease

mechanism (see Outstanding Questions). Future studies could expand electrophysiological, biochemical

and genomic investigations into stem cell-derived neuronal models from human 16p11.2 CNV carriers, to

test the existence of similar molecular and cellular aberrations. Additionally, optogenetic and

chemogenetic approaches could be used to investigate long-range and local circuits in 16p11.2 CNV

mice, which will help explain how the distributed physiological changes integrate to produce broad

behavioral phenotypes. To identify the genes in the 16p11.2 region that drive morphological and

functional alterations, future studies could use CRISPR-Cas9 technology to manipulate individual

16p11.2 genes or combinations of them in human iPSC-derived neurons or transgenic mice. Combined

transcriptomic analyses of 16p11.2 CNV mice and human RNAseq datasets have identified

overrepresentation of pathways related to histone methylation [76], thus future studies are encouraged to

explore the role of epigenetic modifications of gene expression in 16p11.2 CNVs. The ultimate goal is to

find mechanism-based treatment strategy for neurodevelopmental disorders related to 16p11.2 CNVs and

beyond. Key molecular targets or biological pathways will guide translational research for therapeutic

intervention.

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Figure 1. Common phenotypes in carriers of 16p11.2 CNVs. Both 16p11.2 deletions and duplications predispose individuals to ASD, ID, epilepsy/seizures, and DF/CA with high penetrance, in addition to several other common phenotypes. Numbers inside parentheses indicate ranges of reported penetrance within cohorts of 16p11.2 CNV patients. Inset: Genes in the human 16p11.2 region. ASD: autism spectrum disorder; ID: intellectual disability; DF/CA: dysmorphic features/congenital anomalies. (This figure was generated in collaboration with Dr. Zhen Yan).

14

Figure 2. Summary of main preclinical findings from mouse models of 16p11.2 deletion and duplication. Studies in 16p11.2 CNV transgenic mice have identified several core behavioral abnormalities, along with associated neurobiological disruptions. The deletion or duplication of genes within the 16p11.2 region drives transcriptional dysregulation of various downstream pathways. These transcriptional changes in turn lead to the altered developmental trajectories and synaptic changes across distributed brain regions. A range of behavioral phenotypes are reported in both 16p11.2 deletion and duplication models, likely driven by structural/functional neurological changes. Preclinical studies have identified several therapeutic approaches targeting selected disrupted systems. (This figure was generated in collaboration with Dr. Zhen Yan).

15

Figure 3. Schematic showing molecular and cellular mechanisms of 16p11.2 genes and interacting partners in nucleus, soma and synapses. Proteins encoded by genes in 16p11.2 region are labeled with yellow. At glutamatergic synapses, Prrt2 and Doc2a are involved in regulating presynaptic transmitter release. TaoK2 and Erk1 act on postsynaptic actin cytoskeleton via RhoA signaling, which contributes to the deficits of spine structure and synaptic excitation associated with 16p11.2 deletions. Kctd13 is an adaptor of the E3 ligase Cul3, controlling the degradation of RhoA and other protein substrates. The therapeutic agent CTEP acts on mGluR5 to modify glutamatergic responses. At GABAergic synapses, the therapeutic agent R-baclofen acts on presynaptic GABAB receptors to regulate GABA release. Erk1 regulates the phosphorylation of GABAA receptor channels. In the nucleus, 16p11.2 CNVs could induce the alterations of chromatin remodeling and gene transcription. The expression of Npas4, a key regulator of GABA synapses via BDNF, is downregulated in 16p11.2 duplication mice, which contributes to the deficits of synaptic inhibition. (This figure was generated in collaboration with Dr. Zhen Yan).

16

Table 1. Prevalence of 16p11.2 CNVs in the general population and within groups of clinical cohorts with various neurodevelopmental disorders.

Ref.16p11.2 deletion prevalence (%)

16p11.2 duplicationprevalence (%) Sample Group Description

General population data

[31] 110/396,725 (0.028) 138/396,725 (0.035) General population (UK Biobank)[24] 25/58,635 (0.043) 31/58,635 (0.053) General European population

[20] 4/11,856 (0.034) - General Swiss, Finnish, and Estonian population

Clinical cohorts

[17] 27/7,400 (0.36) 18/7,400 (0.24) DD1/MR2, DF3, seizures, CA4, ASD5, ADHD, or failure to thrive.

[26] 98/38,779 (0.25) 59/38,779 (0.15) Unexplained physical and/or intellectual disabilities, with or without DF.

[20] 9/312 (2.9) - CA and/or DD with obesity

[20] 22/3,947 (0.56) - CA and/or DD without obesity

[20] 11/2,772 (0.40) - Childhood/adult obesity (combined datasets)

[24] 119/31,424 (0.38) 73/31,424 (0.23) DD/ID6

[15] 20/3,450 (0.58) - DD, ID, DF or MCA

[33] 14/4,284 (0.33) - MR or multiple congenital anomalies

[16] 45/9,773 (0.46) 32/9,773 (0.33) ASD, DD, DF, CA or seizures

[13] 5/512 (0.98%) 4/512 (0.78%) DD, MR, or suspected ASD

1DD: developmental delay; 2MR: mental retardation; 3DF: dysmorphic features; 4(M)CA:(multiple) congenital anomalies; 5ASD: autism spectrum disorder; 6ID: intellectual disability

Table 2. Penetrance of neurodevelopmental disorders and physical abnormalities among 16p11.2 deletion and 16p11.2 duplication carriers. ASD: autism spectrum disorder; ID: intellectual disability; E/S:

17

epilepsy/seizures; Mac: macrocephaly; Mic: microcephaly; DF/CA: dysmorphic features/congenital anomalies.

# 16p11.2 deletion carriers # 16p11.2 duplication carriersRef. ASD ID E/S Mac. DF/CA ASD ID E/S Mic. DF/CA

[34] 41/217 61/217 - - - 26/114 36/114 - - -

[35] 3/62 - - - - 5/44 - - - -[23] 4/25 5/25 - 6/25 - 1/17 1/17 - 4/17 -[17] 3/11 - 5/16 11/16 5/16 0/10 - 3/10 6/10 5/10[14] 0/14 4/14 - - - 2/17 3/17 - - -[10] 51/317 - 69/317 - 67/317 36/180 47/154 35/180 48/215 30/180[15] 6/21 - - - - - - - - -[21] 20/78 8/78 - - - - - - - -[33] 0/14 - 3/14 - 9/14 - - - - -[16] 9/16 - 2/18 - - - - 1/10 - -[20] 4/24 - - - - - - - - -[12] 8/55 - 47/195 29/170 76/130 - - - - -[9] - - - - - 4/21 - - - -[19] - - - - - 21/62 25/62 - - -[36] - - 22/82 14/81 - - - 22/76 13/76 -[24] - - - - - - - - - 29/101

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Table 3. Behavioral phenotypes present in various mouse models carrying 16p11.2 deletion (16p11.2+/-) or duplication (16p11.2dp/+).

19

20

Ref.Mouse origin/genetic interval

Behavioral Phenotypes

16p11.2+/- mice[5] Mills lab

Slx1b-Sept1 Reduced body size/body weight Hyperlocomotion

[62] Mills labSlx1b-Sept1

Barnes maze deficits Reduced social approach

[61] Mills labSlx1b-Sept1

Reduced body weight More complex locomotor trajectory/shorter latency to approach

stimulus Deficits in righting from upside-down position Normal 3 chamber social preference/social novelty recognition Normal startle/pre-pulse inhibition (PPI)

[67] Mills labSlx1b-Sept1

Delayed learning in FR1 continuous reinforcement nose-poke task (males only)

Earlier response termination in a progressive ratio nose-poke task (males only)

Deficits in the five-choice serial reaction time test (5-CSRTT) No difference in sucrose preference test

[70] Mills labSlx1b-Sept1

Hyperlocomotion over 24-hour period (both sexes). Reduced sleep time (males only) and less time in NREM sleep

(REM normal)[66] Mills lab

Slx1b-Sept1 Reduced open arm time in the elevated plus maze Novel object recognition deficits

[65] Mills labSlx1b-Sept1

Deficits in contextual fear conditioning (reduced freezing). Impaired memory acquisition and extinction in inhibitory

avoidance task [64] Mills lab

Slx1b-Sept1 Impaired object recognition memory Reduced freezing in context-dependent aversive learning task Open field hyperlocomotion

[64] Dolmetsch labCoro1a-Spn

Deficient object location memory Reduced male-to-female nose-to-nose/nose-to-anogenital

sniffing, following time, following bouts, and ultrasonic vocalizations.

Normal open field locomotion[4] Dolmetsch lab

Coro1a-Spn Reduced body length/body weight Severely impaired startle response Lack of gait fluidity; frequent tremor Hyperlocomotion in home cage and in an activity chamber Increased hanging; reduced grooming; reduced resting;

increased circling behavior Impaired novel object recognition

[63] Dolmetsch labCoro1a-Spn

Reduced body weight Reduced male-to-female anogenital sniff, follow time, and

ultrasonic vocalizations (mixed-genotype housed only) Increased open arm time in elevated plus maze (mixed-genotype

housed only) Reduced immobility time in tail suspension test (mixed-

genotype housed only) Impaired novel object recognition (mixed-genotype housed

only) Impaired object location memory (mixed-genotype housed only) Increased arena exploration Normal 3 chamber social preference

Chapter II:

Published in Journal of Neuroscience (2018), 38(26), 5939-5948.

Chemogenetic Activation of Prefrontal Cortex Rescues Synaptic and Behavioral Deficits in a Mouse

Model of 16p11.2 Deletion Syndrome

Wei Wang#, Benjamin Rein#, Freddy Zhang, Tao Tan, Ping Zhong, Luye Qin, Zhen Yan*

Department of Physiology and Biophysics, State University of New York at Buffalo, Jacobs School of

Medicine and Biomedical Sciences, Buffalo, NY 14214, USA

*: Correspondence should be addressed to Z. Y. (zhenyan@buffalo.edu)

#: These authors contributed equally

21

ABSTRACT

Microdeletion of the human 16p11.2 gene locus has been linked to Autism Spectrum Disorder

(ASD) and intellectual disability, and confers risk for a number of other neurodevelopmental deficits.

Transgenic mice carrying 16p11.2 deletion (16p11+/-) display phenotypes reminiscent of those in human

patients with 16p11.2 deletion syndrome, but the molecular mechanisms and treatment strategies for these

phenotypes remain unknown. In this study, we have found that both male and female 16p11+/- mice

exhibit deficient NMDA receptor (NMDAR) function in the medial prefrontal cortex (mPFC), a brain

region critical for high level “executive” functions. Elevating the activity of mPFC pyramidal neurons

with a CaMKII-driven Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (Gq-

DREADD) led to the significant increase of NR2B subunit phosphorylation and the restoration of

NMDAR function, as well as the amelioration of cognitive and social impairments in 16p11+/- mice.

These results suggest that NMDAR hypofunction in PFC may contribute to the pathophysiology of

16p11.2 deletion syndrome, and that restoring PFC activity is sufficient to rescue the behavioral deficits.

22

INTRODUCTION

The 16p11.2 human gene locus, which spans ~550 kb and encompasses 26 genes [4], has been

identified as a hotspot for copy number variations (CNV) that confer risk for Autism spectrum disorder

(ASD) [9-13, 15-18], intellectual disability (ID) [10, 12, 15, 20, 21, 106], schizophrenia (SZ) [9], and

obesity [9, 12, 16, 20]. ASD, a group of neurodevelopmental disorders characterized by social deficits,

repetitive behaviors and intellectual dysfunction, has been reported in both 16p11.2 deletions and 16p11.2

duplications, but appears to be more strongly associated with 16p11.2 deletions [11, 16]. Around 19%-

27% of 16p11.2 deletion carriers were found to meet criteria for ASD diagnosis [15, 16, 21].

Furthermore, genetic screenings suggest that 16p11.2 deletions account for ~0.5% of all ASD cases,

positioning it among the strongest known genetic predictors of ASD [11, 16, 33, 107].

Human patients with 16p11.2 deletions commonly exhibit motor and developmental delays,

below average intelligence, speech and language impairments, attention deficits, autism-related

symptoms, macrocephaly, and dysmorphic features [15-17, 21]. Transgenic mouse models carrying

16p11.2 deletions (16p11+/-) have been developed, which display similar phenotypes, including

impairments in novel object recognition (NOR) memory [4, 68, 108], delayed learning in multiple

paradigms [65, 68], loss of social novelty recognition and disrupted ultrasonic vocalizations [60, 68, 108].

Molecular and cellular mechanisms underlying the autism-like behavioral abnormalities in 16p11+/- mice

are largely unknown.

Prefrontal cortex (PFC), a brain region critical for high-level executive functions, plays an

essential role in mediating social cognition [109]. Disrupted NMDAR function in PFC has been found to

underlie the autism-like social deficits in the Shank3-deficient model of autism [110]. In this study, we

sought to find out whether 16p11+/- mice also exhibit NMDAR hypofunction in PFC and whether

elevating PFC neuronal activity is able to rescue the synaptic and behavioral deficits in 16p11+/- mice.

One way to manipulate the activity of specific neuronal populations in vivo is to use the

chemogenetic tool, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) [111].

Gq-coupled DREADDs utilize a modified form of the human M3 muscarinic receptor (hM3Dq) to induce

an excitatory cellular response in the presence of their ligand, clozapine-N-oxide (CNO) [112, 113].

Activating hM3Dq DREADDs with CNO increases neuron excitability by mobilizing intracellular

calcium [112]. Here we investigated the rescuing effect of Gq-DREADD-induced activation of PFC

pyramidal neurons in 16p11.2 microdeletion mice.

RESULTS

16p11+/- Mice Exhibit NMDA Receptor Hypofunction in PFC.

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To determine whether deletion of the 16p11.2 region affects glutamatergic transmission in the

PFC, we performed whole-cell electrophysiological recordings in PFC pyramidal neurons from 16p11+/-

(8-10-week-old) and age-matched wild-type (WT) mice. NMDA receptor-mediated excitatory

postsynaptic currents (NMDAR-EPSC) evoked by multiple stimulation intensities were significantly

reduced in PFC neurons of 16p11+/- mice (Figure 1A, 39%-52% decrease, WT: n = 21 cells/4 mice,

16p11+/-: n = 26 cells/5 mice, F1,45 (genotype) = 22.5, p < 0.001, two-way ANOVA). The ratio of NMDAR-

EPSC to AMPAR-EPSC was also significantly lower in 16p11+/- mice, compared to WT mice (Figure

1B, WT: 0.62 0.02, n = 13 cells/4 mice, 16p11+/-: 0.47 0.05, n = 10 cells/3 mice, p < 0.01, t test).

However, the paired-pulse ratio (PPR) of NMDAR-EPSCs, a measurement of presynaptic vesicle release

[114], did not differ between WT and 16p11+/- mice (Figure 1C, WT: n = 23 cells/5 mice, 16p11+/-: n =

24 cells/4 mice, F1,45 (genotype) = 0.20, p > 0.05, two-way ANOVA), suggesting a lack of presynaptic changes.

In contrast to the reduction of NMDAR-EPSC, PFC pyramidal neurons from 16p11+/- mice had

unchanged AMPAR-EPSC amplitudes (Figure 2A, WT: n = 17 cells/5 mice, 16p11+/-: n = 14 cells/4

mice, F1,29 (genotype) = 2.43, p > 0.05, two-way ANOVA). Spontaneous excitatory postsynaptic currents

(sEPSC) also had no difference between 16p11+/- and WT mice (Figure 2B-2D, WT: 2.89 0.26 Hz,

12.2 0.42 pA, n = 14 cells/4 mice, 16p11+/-: 2.98 0.24 Hz, 12.5 0.29 pA, n = 11 cells/3 mice, p >

0.05, t test), indicating normal levels of presynaptic glutamate release and postsynaptic AMPAR

expression. Taken together, these results indicate that 16p11.2 deletion specifically induces NMDAR

hypofunction in PFC.

Given the observed NMDAR hypofunction and evidence implicating a post-synaptic mechanism,

we examined whether the synaptic expression of NMDAR subunits was reduced in PFC neurons from

16p11+/- mice. Subcellular fractions containing only synaptic membrane proteins were isolated from PFC

of 16p11+/- mice and WT controls. Protein expression levels of NMDAR and AMPAR subunits were

measured in total lysates and synaptic membrane fractions through Western blotting. No difference was

found between groups in total expression levels of NMDAR or AMPAR subunits (Figure 3A and 3C, n =

5-6 mice per group, p > 0.05, t test). Normal levels of NMDAR and AMPAR subunits were also detected

in the synaptic fraction (Figure 3B and 3C, n = 5-6 mice per group, p > 0.05, t test). Western blots for

Synaptophysin (a presynaptic vesicle protein) showed no differences between groups (Figure 3A and 3C,

n = 5-6 mice per group, p > 0.05, t test), suggesting a lack of changes in presynaptic components. Since

NMDARs can be regulated by GPCR signaling-mediated phosphorylation [115], it is likely that NMDAR

hypofunction in 16p11+/- mice is due to NMDAR dysregulation.

Chemogenetic Activation of PFC Pyramidal Neurons in 16p11+/- Mice Rescues NMDAR

Hypofunction.

24

Since 16p11+/- mice exhibited deficits in NMDAR-mediated synaptic responses in PFC, we next

tested whether elevating PFC neuronal activity via chemogenetic stimulation of the Gq pathway could

restore NMDAR function. The mCherry-tagged CaMKII-driven Gq-coupled DREADD AAV (hM3Dq)

was injected bilaterally to the medial PFC (mPFC) of 16p11+/- and WT mice (Figure 4A).

Immunohistochemical staining of CaMKII indicated that ~70% of mPFC pyramidal neurons in the

proximity of the injection site were DREADD-positive (Figure 4B). Selective activation of mPFC

pyramidal neurons was achieved by the systemic administration of CNO (3 mg/kg, i.p.). Considering that

systemically-injected CNO converts to clozapine [116], which could occupy endogenous GPCRs in the

brain, we also injected the GFP-tagged CaMKII-driven control virus to mPFC to determine the

requirement of DREADD for the effect of CNO.

In hM3Dq-DREADD-injected animals, the frequency of synaptic-driven, spontaneous action

potentials (sAP) was significantly lower in PFC pyramidal neurons from 16p11+/- mice than those from

WT mice, and CNO injection (i.p.) significantly increased sAP frequency in 16p11+/- mice, restoring PFC

neuronal activity to the normal level (Figure 4C, WT+saline: 1.87 0.09 Hz, 16p11+/-+saline: 0.99

0.06 Hz, 16p11+/-+CNO: 1.93 0.08 Hz, WT+CNO: 2.66 0.18 Hz, n = 18-32 cells/3-4 mice per

group, F1,93 (genotype) = 59.6, p < 0.0001, F1,93 (treatment) = 68.9, p < 0.0001, two-way ANOVA). In contrast, in

GFP-injected animals, systemic administration of CNO failed to restore sAP frequency in 16p11+/- mice

(Figure 4D, WT+saline: 1.96 0.25 Hz, 16p11+/-+saline: 1.06 0.17 Hz, 16p11+/-+CNO: 0.94 0.16

Hz, WT+CNO: 1.89 0.24 Hz, n = 7-9 cells/2 mice per group, F1,28 (genotype) = 16.5, p < 0.001, F1, 28 (treatment)

= 0.83, p > 0.05, two-way ANOVA).

Next, we examined the impact of chemogenetic elevation of PFC activity on NMDAR-EPSC in

PFC pyramidal neurons from 16p11+/- mice. In hM3Dq-DREADD-injected 16p11+/- mice, CNO injection

significantly increased NMDAR-EPSC amplitudes, compared to saline injection (Figure 5A, 45%-106%

increase, n = 15-17 cells/3 mice per group, F1,59 (genotype) = 10.37, p <0.01, F1,59 (treatment) = 8.95, p < 0.01, two-

way ANOVA), raising NMDAR-EPSC amplitudes to levels similar to WT mice. Such rescuing effect of

CNO was not observed in GFP-injected 16p11+/- mice (Figure 5B, n = 16-17 cells/3 mice per group, F1,63

(genotype) = 35.89, p <0.001, F1,63 (treatment) = 0.17, p > 0.05, two-way ANOVA).

We next sought to determine the potential mechanism by which hM3Dq-DREADD activation

restored NMDAR function in 16p11+/- mice. Since hM3Dq-DREADD employs Gq-coupled GPCR

activity to induce an excitatory cellular response, we examined the link between Gq signaling and NMDA

receptors. Protein Kinase C (PKC) and CaMKII, which are activated downstream of Gq signaling [117]

(Lu et al., 1999)[35](Lu et al., 1999) have been shown to phosphorylate NMDA receptors at multiple

residues [118]. Since NR2B phosphorylation at S1303 by PKC and CaMKII can potentiate NMDAR

currents [117-119], we focused on this phosphorylation site. PFC slices collected from hM3Dq

25

DREADD-injected WT or 16p11+/- mice were treated with either saline or CNO ex vivo for 20 minutes,

then probed for the level of phosphorylated NR2B at S1303 via western blotting. As shown in Figure 5C

and 5D, the basal level of S1303phos-NR2B was lower in 16p11+/- mice, compared to WT mice, and CNO

treatment led to a significant elevation of S1303phos-NR2B in DREADD-injected 16p11+/- mice, while the

effect of CNO in DREADD-injected WT mice was marginal (WT+saline: 1.00 0.12, 16p11+/-+saline:

0.74 0.14, 16p11+/-+CNO: 1.30 0.08, WT+CNO: 1.14 0.17, n = 6-10 slices/2-3 mice per group,

F1,25 (genotype) = 0.01, p = 0.72, F1,25 (treatment) = 7.8, p < 0.01, two-way ANOVA). These data suggest that Gq-

DREADD activation might restore NMDAR function in 16p11+/- mice via elevating the phosphorylation

of NR2B subunits.

Chemogenetic Activation of PFC Pyramidal Neurons in 16p11+/- Mice Ameliorates Cognitive and

Social Deficits.

Considering the loss of NMDAR function and synaptic-driven excitability of PFC neurons in

16p11+/- mice, we next examined whether chemogenetic activation of PFC neurons could ameliorate the

deficits in cognition and social behaviors exhibited in 16p11+/- mice [4, 60, 65, 68, 108]. WT and 16p11+/-

mice with PFC infection of the CaMKII-driven DREADD AAV (hM3Dq) were subject to a variety of

behavioral assays. As controls, another cohort of WT and 16p11+/- mice received mPFC injection of the

GFP virus before being subjected to the same behavioral assays. Both male and female WT and 16p11+/-

mice were used in behavioral tests.

The Barnes maze, a cognitive test assessing spatial memory [120], was first performed, because

NMDAR hypofunction in mPFC has been linked to impaired Barnes Maze performance by a previous

pharmacological study [121]. Barnes maze tests the animal’s spatial memory by recalling the location of

one correct hole (where an escape box was attached before) from 7 other incorrect holes on a round

platform. The spatial memory index is calculated by the time spent exploring the correct hole divided by

the time spent exploring all the other incorrect holes. We found that in Gq-DREADD-injected animals,

16p11+/- mice spent significantly less time in exploring the correct hole and more time exploring the

incorrect holes, compared to WT mice, suggesting diminished spatial memory, and after CNO

administration, 16p11+/- mice spent significantly more time in exploring the correct hole and less time

exploring the incorrect holes (Figure 6A, correct hole: WT+saline: 37.9 1.7 sec, 16p11+/- +saline: 26.9

2.7 sec, 16p11+/-+CNO: 35.9 2.2 sec, n = 6-8 per group, F1,23 (genotype) = 7.05, p < 0.05, F1,23 (treatment) =

4.85, p < 0.05, two-way ANOVA; incorrect holes: WT+saline: 27 1.0 sec, 16p11+/- +saline: 32.3 1.4

sec, 16p11+/-+CNO: 25.3 1.1 sec, n = 6-8 per group, F1,23 (genotype) = 4.9, p < 0.05, F1,23 (treatment) = 11.9, p <

0.01, two-way ANOVA). Accordingly, the spatial memory index was significantly lower in 16p11+/- mice

than WT animals, which was brought to the normal level by CNO (WT+saline: 1.43 0.12, 16p11+/-

26

+saline: 0.85 0.1, 16p11+/-+CNO: 1.43 0.1, n = 6-8 each group, F1,23 (genotype) = 8.8, p < 0.01, F1,23

(treatment) = 8.7, p < 0.01, two-way ANOVA).

In GFP-injected animals (Figure 6B), CNO administration did not affect the amount of time that

16p11+/- mice spent in exploring the correct hole (WT+saline: 35.2 1.3 sec, 16p11+/- +saline: 20.0 2.2

sec, 16p11+/-+CNO: 21.4 2.0 sec, n = 7 per group, F1,24 (genotype) = 48.53, p < 0.0001, F1,24 (treatment) = 0.32, p

> 0.05, two-way ANOVA) or incorrect holes (WT+saline: 24 2.5 sec, 16p11+/- +saline: 30.1 1.8 sec,

16p11+/-+CNO: 34.1 3.2 sec, n = 7 each group, F1,24 (genotype) = 11.2, p < 0.01, F1,24 (treatment) = 0.64, p > 0.05,

two-way ANOVA). The spatial memory index of 16p11+/- mice was also not restored after CNO injection

(WT+saline: 1.55 0.14, 16p11+/- +saline: 0.70 0.11, 16p11+/-+CNO: 0.67 0.09, n = 7 each group,

F1,24 (genotype) = 52.62, p < 0.0001, F1,24 (treatment) = 1.22, p > 0.05, two-way ANOVA).

Next, the social approach behavior was examined. In Gq-DREADD-injected animals (Figure

6C), 16p11+/- mice spent significantly less time interacting with a social stimulus and engaged in fewer

social interactions, compared to WT mice, and CNO injection significantly increased the amount of time

that 16p11+/- mice spent interacting with the social stimulus and the number of social interactions (social

time, WT+saline: 166 13.5 sec, 16p11+/-+saline: 108.3 13.8 sec, 16p11+/-+CNO: 165.1 11.5 sec, n

= 6-8 per group, F1,24 (genotype) = 5.0, p < 0.05, F1,24 (treatment) = 4.7, p < 0.05, two-way ANOVA; social number,

WT+saline: 28.5 2.2, 16p11+/-+saline: 20.3 1.2, 16p11+/-+CNO: 28.5 1.6, n = 6-8 per group, F1,24

(genotype) = 4.8, p < 0.05, F1,24 (treatment) = 4.8, p < 0.05, two-way ANOVA). Conversely, in GFP-injected

16p11+/- mice (Figure 6D), CNO injections failed to affect social interaction time (WT+saline: 134.2

12.04 sec, 16p11+/-+saline: 100.8 8.8 sec, 16p11+/-+CNO: 94.4 12.3 sec, n = 7 per group, F1,24 (genotype)

= 10.44, p < 0.01, F1,24 (treatment) = 0.03, p > 0.05, two-way ANOVA), and the number of social interactions

(WT+saline: 28.1 1.2, 16p11+/-+saline: 20.6 1.8, 16p11+/-+CNO: 21.1 2.1, n = 7 per group, F1,24

(genotype) = 17.6, p < 0.001, F1,24 (treatment) = 0.007, p > 0.05, two-way ANOVA). Collectively, these results

indicate that elevating PFC pyramidal neuronal activity in 16p11+/- mice is sufficient and necessary to

ameliorate the cognitive and social deficits exhibited by these animals.

DISCUSSION

Microdeletion of the 16p11.2 region is associated with several syndromes largely characterized

by intellectual and social deficits [4, 60, 65, 68, 108]. Previous reports have implicated dysfunction of

basal ganglia circuitry [4, 106] and widespread brain structure irregularities [5] in the etiology of 16p11.2

deletion syndrome. In the present study, we show that 16p11.2 deletion mice exhibit deficient NMDAR

function in the PFC. By using chemogenetic activation of PFC pyramidal neurons, we have demonstrated

that NMDAR function is restored, and cognitive and social deficits are ameliorated in 16p11+/- mice.

27

Dysfunctional glutamatergic activity in the PFC has not been previously reported in 16p11.2

deletion syndrome, although an increase in the AMPAR/NMDAR ratio was found in the nucleus

accumbens (NAc) of 16p11+/- mice [4]. This result is analogous to the decreased NMDAR/AMPAR ratio

observed in the PFC. Given the deficits in spatial memory shown previously [68] and replicated here,

disruptions in 16p11+/- hippocampal activity would also be conceivable, however, multiple studies have

indicated normal synaptic function in the hippocampus of 16p11+/- mice [65, 108]. It is possible that

NMDAR hypofunction in the PFC underlies the intellectual and social deficits associated with 16p11.2

deletion syndrome, as glutamatergic transmission in the PFC, and specifically NMDAR function, is a

critical component for working memory and cognitive processing, as well as for social behaviors [122,

123]. Moreover, the disrupted signaling observed in the NAc and PFC may be related, as the NAc

receives glutamatergic projections from the PFC [124], and NAc activity is regulated by PFC inputs

[125].

NMDAR function is highly regulated by GPCR signaling [115]. Activation of protein kinases

downstream of GPCR signaling have been shown to potentiate NMDAR currents in the PFC and other

brain areas through mechanisms including receptor phosphorylation [126-128]. Phosphorylation of NR1

subunit at S890 and S896 alters NMDAR clustering and surface expression [129, 130]. The lack of

changes in the level of synaptic NMDARs in 16p11+/- mice led us to focus on NR2B phosphorylation at

S1303, a substrate of PKC and CaMKII [118]. Interestingly, hM3Dq-DREADD significantly elevated the

diminished level of S1303-phosphorylated NR2B in 16p11+/- mice, which correlated well with the

DREADD-induced restoration of NMDAR function in these animals. It suggests that NMDAR

hypofunction in 16p11+/- mice is likely due to the dysregulation of NMDAR phosphorylation, which can

be reversed by chemogenetic stimulation of the GPCR signaling.

In conclusion, our results suggest that dysregulated NMDAR function in the PFC is strongly

implicated in the manifestation of cognitive and social impairments in 16p11.2 deletion syndrome.

Chemogenetically activating PFC pyramidal neurons in 16p11+/- mice is sufficient to ameliorate

behavioral deficits, potentially revealing a novel intervention strategy for the treatment of 16p11.2

deletion syndrome.

28

Figure 1. 16p11+/- mice exhibit diminished NMDAR-mediated synaptic responses in PFC. (A) Summarized input-output curves of NMDAR-EPSC in PFC pyramidal neurons from WT and 16p11+/- mice. Inset: representative NMDAR-EPSC traces. * P<0.05, *** P<0.001, two-way ANOVA. (B) Bar graphs (mean + s.e.m.) of NMDAR-EPSC to AMPAR-EPSC ratio in PFC pyramidal neurons from WT and 16p11+/- mice. Inset: representative traces. ** P<0.01, t-test. (C) Plot of paired-pulse ratio (PPR) of NMDAR-EPSC evoked by double pulses with various intervals in PFC pyramidal neurons from WT or 16p11+/- mice. Inset: representative traces. (All data presented in this figure were generated by Dr. Tao Tan and Dr. Wei Wang).

29

Figure 2. AMPAR-mediated synaptic transmission is unchanged in PFC of 16p11+/- mice. (A) Summarized input-output curves of AMPAR-EPSC in PFC pyramidal neurons from WT and 16p11+/- mice. Inset: representative AMPAR-EPSC traces. (B, C) Bar graphs (mean s.e.m.) showing sEPSC frequency (B) and amplitude (C) in PFC pyramidal neurons from WT and 16p11+/- mice. (D) Representative sEPSC traces. (All data presented in this figure were generated by Dr. Tao Tan and Dr. Wei Wang).

30

Figure 3. The total and synaptic levels of glutamate receptor subunits are unchanged in PFC of 16p11+/- mice. (A, B) Immunoblots showing the expression of NMDAR and AMPAR subunits in the total lysates (A) or the synaptic fraction (B) of PFC from WT vs 16p11+/- mice. (C) Quantification analysis of total and synaptic protein levels in PFC from WT and 16p11+/- mice. Total protein levels were normalized to tubulin. Synaptic protein levels were normalized to PSD-95.

31

Figure 4. Chemogenetic activation of PFC restores neuronal excitability in 16p11+/- mice. (A) Immunofluorescent image of a brain slice from a mouse showing the expression of Gq-DREADD (mCherry-tagged) in medial PFC. (B) High-resolution confocal images of CaMKII staining (Green) and Gq-DREADD (Red) in the viral infected mPFC region. (C, D) Bar graphs (mean s.e.m.) showing the frequency of synaptic-driven, spontaneous action potential (sAP) in PFC pyramidal neurons from saline- or CNO-injected WT or 16p11+/- mice with prior infection of Gq-DREADD AAV (C) or GFP-AAV (D). Inset (C): representative sAP traces. *** P<0.001, two-way ANOVA. (The data presented in this figure were generated by Dr. Luye Qin, Dr. Tao Tan and Dr. Wei Wang).

32

Figure 5. Chemogenetic activation of PFC restores NMDAR function and elevated NR2B phosphorylation in 16p11+/- mice. (A, B) Summarized input-output curves of NMDAR-EPSC in PFC pyramidal neurons from saline- or CNO-injected WT or 16p11+/- mice with prior infection of Gq-DREADD AAV (A) or GFP-AAV (B) *** P<0.001, two-way ANOVA. Inset: representative NMDAR-EPSC traces. (C) Immunoblots of S1303phos-NR2B and total NR2B in the lysates of saline- or CNO-treated PFC slices from WT vs. 16p11+/- mice with prior infection of Gq-DREADD AAV. (D) Quantification analysis of S1303phos-NR2B (normalized to total NR2B) levels in different groups. ** P<0.01, two-way ANOVA. (The data presented in Panels A & B were generated by Dr. Tao Tan and Dr. Wei Wang).

33

Figure 6. Chemogenetic activation of PFC ameliorates cognitive and social deficits in 16p11+/- mice. (A, B) Bar graphs (mean s.e.m.) showing the time spent in exploring the correct hole (T1) vs. the 7 incorrect holes (T2) in the memory phase of Barnes maze tests (left) and the spatial memory index (T1/T2) in Barnes maze tests (right) of saline- or CNO-injected WT or 16p11+/- mice with prior infection of Gq-DREADD AAV (A) or GFP-AAV (B). * P<0.05, ** P<0.01, *** P<0.001, two-way ANOVA. (C, D) Bar graphs (mean s.e.m.) showing the time (left) and number (right) of social interactions in social approach tests of saline- or CNO-injected WT or 16p11+/- mice with prior infection of Gq-DREADD AAV (C) or GFP-AAV (D). * P<0.05, two-way ANOVA. (The data presented in Panels A & C were generated by Dr. Wei Wang).

34

Chapter III

Published in Molecular Psychiatry (2020) [Epub ahead of print]

Reversal of Synaptic and Behavioral Deficits in a 16p11.2 Duplication Mouse Model via Restoration

of the GABA Synapse Regulator Npas4

Benjamin Rein1, Tao Tan1, Fengwei Yang1, Wei Wang1, Jamal Williams1, Freddy Zhang1, Alea Mills2,

and Zhen Yan1*

1Department of Physiology and Biophysics, State University of New York at Buffalo, Jacobs School of

Medicine and Biomedical Sciences, Buffalo, NY 14214, USA

2Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA

*: Correspondence should be addressed to Z. Y. (zhenyan@buffalo.edu)

35

ABSTRACT

The human 16p11.2 gene locus is a hot-spot for copy number variations which predispose carriers to a

range of neuropsychiatric phenotypes. Microduplications of 16p11.2 are associated with autism spectrum

disorder (ASD), intellectual disability (ID) and schizophrenia (SZ). Despite the debilitating nature of

16p11.2 duplications, the underlying molecular mechanisms remain poorly understood. Here we

performed a comprehensive behavioral characterization of 16p11.2 duplication mice (16p11.2dp/+) and

identified social and cognitive deficits reminiscent of ASD and ID phenotypes. 16p11.2dp/+ mice did not

exhibit the SZ-related sensorimotor gating deficits, psychostimulant-induced hypersensitivity or motor

impairment. Electrophysiological recordings of 16p11.2dp/+ mice found the deficient GABAergic synaptic

transmission and elevated neuronal excitability in the prefrontal cortex (PFC), a brain region critical for

social and cognitive functions. RNA-sequencing identified genome-wide transcriptional aberrance in the

PFC of 16p11.2dp/+ mice, including downregulation of the GABA synapse regulator Npas4. Restoring

Npas4 expression in PFC of 16p11.2dp/+ mice ameliorated the social and cognitive deficits and reversed

the GABAergic synaptic impairment and neuronal hyper-excitability. These findings suggest that

prefrontal cortical GABAergic synaptic circuitry and Npas4 are strongly implicated in 16p11.2

duplication pathology, and may represent potential targets for therapeutic intervention in ASD.

36

INTRODUCTION

The human 16p11.2 genetic locus (chromosome 16, position 11.2) constitutes a ~550 kb (26

gene) chromosomal region that is susceptible to copy number variations (CNVs; i.e. deletion or

duplication), which confer risk for a range of neurodevelopmental conditions [9, 13, 34].

Microduplications of 16p11.2 are estimated to affect 1 in every 4,216 live births [30], and often carry

broad and multifaceted phenotypic consequences due to frequent comorbidity among psychiatric,

physical/developmental and cognitive symptoms. 16p11.2 duplication carriers most commonly exhibit

neurodevelopmental deficits characterized by intellectual disability (ID), speech & language

deficits/autism spectrum disorder (ASD), and developmental/motor delays [8-10, 13, 17-19, 131].

16p11.2 duplications are also associated with schizophrenia and bipolar disorder [9, 25-27, 43]. In

addition, epilepsy, dysmorphic features, and microcephaly are often observed in 16p11.2 duplications [8,

16, 17].

Numerous clinical reports have substantiated the debilitating nature of 16p11.2 duplications.

Mice carrying duplication of the genomic region homologous to 16p11.2 (mouse chromosome 7F3)

exhibit neurocognitive and metabolic phenotypes [5, 59], however, it remains to be determined whether

16p11.2 duplication mice (16p11.2dp/+) thoroughly and accurately depict the clinical features present in

human patients, and what molecular mechanisms are underlying these behavioral abnormalities. We thus

performed a comprehensive behavioral examination of 16p11.2dp/+ mice, and report social and cognitive

behavioral deficits reminiscent of ASD and ID phenotypes, respectively.

Dysfunction of inhibitory gamma-aminobutyric acid (GABA) neurotransmission is highly

implicated in ASD [132], and the resulting imbalance of excitatory and inhibitory synaptic activity (E/I

imbalance) has been theorized to underlie ASD pathology [133, 134]. Moreover, brain GABA levels are

significantly reduced in human ASD patients [135], and numerous mouse models of ASD exhibit

disrupted E/I balance in cortical regions and specifically in the medial prefrontal cortex (mPFC) [75, 136-

139], a brain region critical for higher level executive functions and involved in social cognition [109]. In

the current study, we found that GABAergic synaptic transmission was disrupted and neuronal

excitability was elevated in the mPFC of 16p11.2dp/+ mice, an electrophysiological profile consistent with

existing explanations of ASD pathology, which may explain the social deficits in 16p11.2 duplication

carriers.

Our genome-wide search for gene alterations associated with the disrupted GABA signaling in

16p11.2dp/+ mice led to the discovery of the downregulated gene Npas4, an activity-dependent

transcription factor highly expressed in PFC [140]. Npas4 is induced in response to neuronal excitation

and subsequently regulates the formation of inhibitory GABAergic synapses onto pyramidal neurons

37

[104, 105, 141]. Npas4 expression in the PFC during adolescence appears to be critical for the proper

establishment of GABAergic synapse markers [142], and Npas4 deficiency is associated with cognitive

impairment and compromised memory formation [142-145] along with social deficits [144]. Here, we

found that restoring Npas4 expression in PFC of 16p11.2dp/+ mice was sufficient to reverse GABAergic

synaptic deficits and ameliorate the observed social and cognitive phenotypes, implicating Npas4 and the

prefrontal cortical GABA system in the pathogenesis of social and cognitive deficits in 16p11.2

duplication syndrome.

RESULTS

16p11.2dp/+ Mice Exhibit Social and Cognitive Behavioral Deficits Reminiscent of ASD and ID.

To determine whether mice carrying the 16p11.2 duplication (16p11.2dp/+) exhibit phenotypes

resembling the clinical features present in human patients, we performed an array of behavioral tests on

both male and female 7-9-week-old 16p11.2dp/+ mice and age-matched wild-type (WT) controls. Since

human 16p11.2 duplication carriers are strongly predisposed to ASD [8-10, 13, 14, 17-19], we first

evaluated social behavior in the three-chamber social preference test. When animals were exposed to a

social stimulus and a non-social stimulus, 16p11.2dp/+ mice spent significantly less time than WT mice

interacting with the social stimulus (Figure 1A, F1,38 (genotype x stimulus type) = 16.7, p = 0.0002, two-way

ANOVA), and correspondingly demonstrated a significantly lower social preference index (Figure 1B, U

= 9, p = 0.0006, Mann-Whitney U test). When animals were exposed to a novel social stimulus and a

familiar social stimulus, WT mice spent significantly more time interacting with the novel mouse,

whereas 16p11.2dp/+ mice did not display a clear preference for the novel mouse (Figure 1C, F1,38 (genotype x

stimulus type) = 2.91, p = 0.10, two-way ANOVA), resulting in a trend toward a lower social novelty

preference index in 16p11.2dp/+ mice (Figure 1D, t(19) = 1.67, p = 0.11, unpaired t-test). In the social

approach test, 16p11.2dp/+ mice spent significantly less time than WT controls interacting with the social

stimulus (Figure 1E, t(53) = 3.65, p = 0.0006, unpaired t-test). WT and 16p11.2dp/+ mice did not differ in the

total distance travelled during the three-chamber social preference test (n = 9-14 mice/group, t(21) = 0.27,

p = 0.79, unpaired t-test) or the social approach test (n = 8-11 mice/group, t(17) = 1.10, p = 0.29, unpaired

t-test), suggesting that differences in locomotion are not contributing to the observed social phenotypes.

Self-grooming, a rodent behavior thought to model repetitive behaviors observed in human ASD patients

[146], was also assessed. Relative to WT animals, 16p11.2dp/+ mice spent significantly more time self-

grooming (Figure 1F, U = 29, p = 0.02, Mann-Whitney U test). Collectively, these data indicate that

16p11.2dp/+ mice exhibit both social deficits and repetitive behaviors, the two core behavioral features of

ASD.

38

We next sought to assess whether 16p11.2dp/+ mice exhibit cognitive deficits reminiscent of ID,

another phenotype strongly associated with 16p11.2 duplications [8, 10, 14, 19]. Temporal Order

Recognition Memory (TORM), a task testing the animal's ability to remember which of two objects it was

more recently exposed to, was used to assess cognitive processes mediated by the medial prefrontal

cortex (mPFC) [147]. In the TORM task, 16p11.2dp/+ mice spent significantly less time than WT controls

interacting with the more novel (less recent) object (Figure 1G, F1,38 (genotype x object type) = 10.62, p = 0.002,

two-way ANOVA), and correspondingly exhibited a significantly lower discrimination ratio (Figure 1H,

t(19) = 2.55, p = 0.02, unpaired t-test), indicating PFC-dependent cognitive impairment. However, in the

Novel Object Recognition (NOR) task, which is mediated primarily by the perirhinal cortex [147, 148],

16p11.2dp/+ mice displayed unimpaired performance (Figure 1I, t(19) = 0.79, p = 0.44, unpaired t-test),

suggesting that the cognitive deficits afflicting 16p11.2dp/+ mice may be driven by brain region-specific

neurobiological changes.

Since several reports have linked 16p11.2 duplications to schizophrenia (SZ) [9, 25-27, 43], we

next examined SZ-related behaviors in 16p11.2dp/+ mice. Pre-pulse inhibition (PPI) is a measure of

sensorimotor gating which is disrupted in human SZ patients and animal models of SZ [149-151].

Abnormalities in startle-responses or PPI have also been reported in autism [152-154] and fragile X

patients [155-157], as well as in mouse models of ASD and fragile X syndrome [61, 156]. Compared to

WT counterparts, 16p11.2dp/+ mice displayed normal startle responses at multiple stimulus intensities

(Figure 1J, F1,17 (genotype) = 0.86, p = 0.36, two-way ANOVA), and intact pre-pulse inhibition at all pre-pulse

intensities (Figure 1K, F1,17 (genotype) = 0.11, p = 0.75, two-way ANOVA), suggesting the lack of SZ-related

sensorimotor gating deficits.

Based on the NMDAR hypofunction theory of SZ [158], NMDAR antagonists have been used to

evoke psychosis-related behaviors, including hyperlocomotion [159-162]. We tested whether a single

administration of the NMDAR antagonist MK-801 (2.0 mg/kg) could induce enhanced hyperlocomotion

in 16p11.2dp/+ mice. Prior to MK-801 injection, 16p11.2dp/+ mice exhibited significantly lower baseline

locomotor activity relative to WT mice. In contrast to WT animals, 16p11.2dp/+ mice failed to display

elevated locomotion after MK-801 injection (Figure 1L, F1,18 (genotype) = 20.41, p = 0.0003, two-way

ANOVA). These data indicate that 16p11.2dp/+ mice do not exhibit SZ-related hypersensitivity to

psychostimulants.

Motor deficits, which are associated with 16p11.2 duplications [8-10, 13, 17-19], were assessed

in 16p11.2dp/+ mice via the rotarod test. At both 4- and 8-weeks of age, latency to fall did not differ

between 16p11.2dp/+ and WT mice, suggesting a lack of motor coordination deficits (Figure 1M, 4 weeks:

t(16) = 0.22, p = 0.83, unpaired t-test; 8 weeks: t(15) = 0.16, p = 0.87, unpaired t-test). General anxiety has

also been reported in 16p11.2 duplication patients [19, 55]. In the elevated plus maze test, 16p11.2dp/+

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mice did not differ from WT animals in the amount of time spent exploring the open arms (Figure 1N,

t(20) = 0.33, p = 0.74, unpaired t-test), indicating the lack of anxiety-like behaviors. Collectively, our

behavioral characterization indicates that 16p11.2dp/+ mice exhibit many clinical features associated with

human 16p11.2 duplications, including ASD-related social deficits and repetitive behaviors, along with

cognitive deficits reminiscent of ID.

GABAergic Synaptic Transmission is Impaired in PFC of 16p11.2dp/+ mice.

Considering that 16p11.2dp/+ mice exhibited impaired sociability and cognition, two major

behavioral functions mediated by the PFC [109, 147], we next performed whole-cell patch clamp

recordings on WT and 16p11.2dp/+ medial PFC (mPFC; prelimbic and infralimbic) layer V pyramidal

neurons to identify synaptic transmission deficits which may underlie the observed behavioral

phenotypes. NMDA receptor (NMDAR)-mediated excitatory postsynaptic current (EPSC) amplitudes did

not differ between 16p11.2dp/+ and WT neurons at various stimulation intensities (Figure 2A, F1,29 (genotype) =

0.002, p = 0.96, two-way ANOVA). WT and 16p11.2dp/+ mPFC neurons also demonstrated comparable

AMPA receptor (AMPAR)-mediated EPSC amplitudes (Figure 2B, F1,25 (genotype) = 0.22, p = 0.64, two-way

ANOVA). In addition, 16p11.2dp/+ mPFC neurons exhibited normal paired-pulse ratios of NMDAR-EPSC

(Figure 2C, F1,40 (genotype) = 0.01, p = 0.90, two-way ANOVA) and AMPAR-EPSC (Figure 2D, F1,14 (genotype)

= 0.33, p = 0.57, two-way ANOVA). These data suggest that glutamatergic transmission is largely

unchanged in 16p11.2dp/+ mPFC neurons.

We next recorded GABAA receptor (GABAAR)-mediated inhibitory postsynaptic currents

(IPSCs). Relative to WT cells, 16p11.2dp/+ mPFC neurons displayed significantly reduced GABAAR-IPSC

amplitudes at multiple stimulation intensities (Figure 2E, F1,57 (genotype) = 24.41, p < 0.0001, two-way

ANOVA), indicating marked disruption of GABAergic synaptic transmission in 16p11.2dp/+ PFC. We then

measured action potential (AP) firing to assess neuronal excitability, which could be influenced by the

alteration of synaptic inhibition. Relative to WT cells, 16p11.2dp/+ mPFC neurons displayed significantly

increased frequencies of APs evoked by multiple current intensities (Figure 2F, F1,51 (genotype) = 13.03, p =

0.0007, two-way ANOVA). However, no changes were observed between WT and 16p11.2dp/+ neurons in

the resting membrane potential (Figure 2G, t(51) = 1.55, p = 0.13, unpaired t-test), action potential

threshold (Figure 2H, t(34) = 1.12, p = 0.27, unpaired t-test), or input resistance (Figure 2I, t(29) = 0.28, p =

0.78, unpaired t-test), suggesting that the intrinsic membrane properties of mPFC neurons from

16p11.2dp/+ mice are unchanged.

To determine whether the diminished GABAergic synaptic responses in PFC pyramidal neurons

was potentially caused by the loss of interneurons, we performed immunostaining for parvalbumin (PV)

in two regions of the prefrontal cortex, the prelimbic and cingulate areas. WT and 16p11.2dp/+ mice did not

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differ in the number of PV-expressing (PV+) cells in the cingulate cortex or the prelimbic cortex (Figure

2J, Cingulate: t(28) = 0.59, p = 0.56, unpaired t-test; Prelimbic: t(32) = 1.38, p = 0.18, unpaired t-test),

indicating that the observed GABAergic synaptic deficits are not due to the loss of parvalbumin-

expressing interneurons in the PFC. Collectively, these data indicate that 16p11.2dp/+ PFC neurons exhibit

selective impairments in synaptic inhibition, which may be mediated by the loss of GABAergic synapses.

Genome-wide Transcriptional Dysregulation in PFC of 16p11.2dp/+ mice.

In order to determine the genome-wide transcriptional impact of the 16p11.2 duplication, we next

performed RNA-sequencing (RNA-seq) with mPFC tissue. RNA-seq identified a total of 388 gene

transcripts with significantly altered expression levels in 16p11.2dp/+ PFC (>1.5-fold increase or decrease,

p < 0.05, and FDR < 0.3), with the majority of genes showing downregulation (Figure 3A), suggesting

that 16p11.2 duplication has a predominantly repressive impact on genome-wide transcriptional levels in

PFC. As shown in the heat map in Figure 3B, 111 gene transcripts demonstrated significant upregulation

in 16p11.2dp/+ mPFC (Table S1). Gene ontology (GO) analysis was performed to classify the upregulated

genes into 11 categories based on biological functions (Figure 3C). Enrichment was observed in

functional categories including enzyme modulator, nucleic acid binding and signaling molecule,

suggesting that transcriptional upregulation in 16p11.2dp/+ PFC occurs in diverse gene classes. The

interactome network demonstrated that the upregulated genes have rich interconnections (Figure 3D).

Quantitative PCR (qPCR) analysis was performed on WT and 16p11.2dp/+ mPFC tissue, and verified the

upregulation of several genes located in the duplicated 16p11.2 genomic region, including Mapk3, AldoA.

Doc2a, Mvp, and Cdipt (Figure 3E).

RNA-seq identified an additional 277 gene transcripts exhibiting significant downregulation in

16p11.2dp/+ PFC (Figure 4A, Table S2). GO analysis was performed to classify significantly

downregulated genes into 14 categories. Enrichment was observed in categories such as transcription

factors, signaling molecules, nucleic acid binding and cytoskeletal genes (Figure 4B), indicating that

transcriptionally repressed genes in 16p11.2dp/+ PFC assume a variety of functional roles. An interactome

network was also built to illustrate predicted interactions between the downregulated genes, along with

their respective ontological classifications (Figure 4C).

In order to verify the transcriptional reduction of the downregulated genes identified by our

RNA-seq experiments, we next performed qPCR analysis of selected genes from various ontological

classifications. Transcriptional levels were assessed for several histone modifiers/chromatin remodelers,

and significant downregulation was confirmed for the epigenetic enzymes Kmt2a, EP300, and Brd4,

while other genes such as Setd1b, Kmt2d, and Kdm6b failed to show significant reduction in mPFC of

16p11.2dp/+ mice (Figure 4D). Expression level of the synaptic genes Shank1 and Syngap1, both of which

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showed significant downregulation in RNA-seq, exhibited a trend of reduction in PFC of 16p11.2dp/+ mice,

while the sodium ion channel Scn9a was significant downregulated (Figure 4E). Additionally, the mRNA

level of other ASD- and/or ID risk genes identified by genomic screening, including Wdfy3, Bcl11a,

Ank3, and Asxl3 [85, 163, 164], was significantly reduced in PFC of 16p11.2dp/+ mice (Figure 4F).

Among the top 20 most strongly downregulated genes in 16p11.2dp/+ PFC identified by RNA-seq,

Npas4 (FC = -1.6, FDR = 0.0073, p < 0.0001, Table S2), a gene encoding the neuron-specific

transcription factor neuronal PAS domain-containing protein 4 (Npas4) [165], caught our attention.

Npas4 is a neuronal activity-dependent immediate early gene, which promotes GABAergic synapse

formation and plays a key role in maintaining homeostatic excitability [104, 105, 141]. In agreement with

RNA-seq data, qPCR found a significant reduction of Npas4 mRNA in 16p11.2dp/+ PFC (Figure 4G, t(35) =

2.92, p = 0.006, unpaired t-test). Western blotting revealed a significant loss of Npas4 protein expression

in the nuclear fraction of PFC from 16p11.2dp/+ mice (Figure 4H, t(17) = 2.59, p = 0.019, unpaired t-test).

Furthermore, qPCR analyses of human postmortem PFC tissue revealed that NPAS4 mRNA level was

significantly reduced in idiopathic human ASD patients compared to healthy controls (Figure 4I, U = 14,

p = 0.036, Mann-Whitney U test), suggesting that Npas4 dysregulation may be broadly involved in ASD.

Npas4 exhibits restricted regional expression in the brain, with the highest expression in cortical

areas. However, Npas4 is also expressed at relatively high levels in other areas including the striatum

[140]. To determine whether the observed loss of Npas4 expression is ubiquitous throughout the brain or

specific to PFC, we compared Npas4 mRNA in the striatum of WT and 16p11.2dp/+ mice. As shown in

Figure 4J, Npas4 mRNA level was unchanged in striatum of 16p11.2dp/+ mice, whereas the Mapk3 gene

which is located in the duplicated 16p11.2 region exhibited significant upregulation in striatum. This

suggests that Npas4 dysregulation in 16p11.2dp/+ mice is region-specific.

Other than Npas4, we also evaluated the expression level of various genes encoding GABAergic

synaptic components in PFC of WT and 16p11.2dp/+ mice. qPCR analyses indicated no change in mRNA

levels of Vgat, Gad65, Gabra1, Gabrb2, Gabrg2, and Pvalb (Figure 4K), consistent with our RNA-seq

data. This suggests that the observed GABAergic synaptic dysfunction in PFC of 16p11.2dp/+ mice is

unlikely caused by the direct transcriptional changes of GABA transporters, enzymes or receptors, but

may be due to dysregulation of GABA synapses by Npas4.

Restoring Npas4 Expression in 16p11.2dp/+ mPFC Ameliorates Synaptic and Behavioral Deficits.

Considering the GABAergic deficits observed in 16p11.2dp/+ PFC, we sought to further investigate

the role that Npas4 downregulation may play in 16p11.2dp/+ or ASD pathology. Since Npas4 plays a major

role in regulating GABAergic synapse development [104, 105] and is implicated in neurodevelopmental

disorders [142, 144, 166], we hypothesized that Npas4 downregulation in 16p11.2dp/+ PFC may underlie

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the observed GABAergic synaptic impairment and social/cognitive deficits. To test this, we examined

whether restoring Npas4 expression in 16p11.2dp/+ PFC could ameliorate the synaptic and behavioral

deficits. Either Npas4 CRISPR lentiviral activation particles or GFP control lentiviral particles were

stereotaxically injected into mPFC of WT and 16p11.2dp/+ mice (Figure 5A). The significant upregulation

of Npas4 mRNA level in Npas4-injected groups relative to GFP-injected groups was verified via qPCR

(Figure 5B, F1,23 (treatment) = 4.69, p = 0.041, two-way ANOVA). Additionally, immunostaining of Npas4

revealed the significantly increased Npas4 expression in mPFC of Npas4-injected 16p11.2dp/+ mice,

relative to GFP-injected 16p11.2dp/+ mice (Figure 5C, t(25) = 3.48, p = 0.002, unpaired t-test),

authenticating the viral upregulation of Npas4. Viral upregulation of Npas4 was detected in both

CaMKII-expressing pyramidal neurons and GAD67-positive interneurons (data not shown).

To determine whether Npas4 upregulation was driving GABA synapse formation in 16p11.2dp/+

mPFC, we next performed immunostaining for the vesicular GABA transporter VGAT. Relative to GFP-

injected WT mice, GFP-injected 16p11.2dp/+ mice displayed a marked reduction of VGAT expression in

PFC, and VGAT expression was rescued to the control level in PFC of Npas4-injected 16p11.2dp/+ mice

(Figure 5D, F1,105 (genotype x treatment) = 16.16, p = 0.0001, two-way ANOVA). The cellular expression level of

Npas4 was significantly correlated with the level of VGAT expression in the immediate proximity of the

soma (n = 77 cells/4 mice, R2 = 0.25, p < 0.0001).

This suggests that upregulating Npas4 expression in 16p11.2dp/+ PFC is sufficient to induce the

pronounced restoration of GABAergic synaptic density.

We next performed whole-cell patch clamp electrophysiology on mPFC pyramidal neurons to

assess whether the Npas4-driven induction of GABA synapse formation could reverse the observed

synaptic deficits in 16p11.2dp/+ PFC. Compared to GFP-injected WT neurons, GABAAR-IPSC amplitudes

were significantly diminished in GFP-injected 16p11.2dp/+ neurons, and this deficit was significantly

reversed by Npas4 injection into the PFC of 16p11.2dp/+ mice (Figure 5E, F3,54 (group) = 7.41, p = 0.0003,

two-way ANOVA). Furthermore, Npas4-injected 16p11.2dp/+ neurons exhibited significantly reduced AP

firing frequencies relative to GFP-injected 16p11.2dp/+ neurons (Figure 5F, F3,52(group) = 5.70, p = 0.002,

two-way ANOVA), collectively indicating that restoring Npas4 expression in 16p11.2dp/+ PFC is sufficient

to reverse the GABAergic synaptic deficits and restore homeostatic neuronal excitability.

We next tested whether restoring Npas4 expression in 16p11.2dp/+ PFC could ameliorate the ASD-

and ID-related behavioral phenotypes. In the 3-chamber social preference test, Npas4-injected 16p11.2dp/+

mice spent significantly more time than GFP-injected 16p11.2dp/+ mice interacting with the social stimulus

(Figure 5G, F1,107 (interaction) = 9.1, p = 0.003, three-way ANOVA), and exhibited a significantly elevated

preference for the social stimulus over the non-social stimulus (Figure 5H, F1,49 (interaction) = 21.78, p <

0.0001, two-way ANOVA). In the TORM task, Npas4-injected 16p11.2dp/+ mice spent significantly more

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time than GFP-injected 16p11.2dp/+ mice investigating the novel object (Figure 5I, F2,64 (object x group) = 9.56, p

= 0.0002, two-way ANOVA), and displayed a significant preference for the more novel object over the

more familiar object (Figure 5J, F2,32 (group) = 11.72, p = 0.0002, one-way ANOVA). However, viral

upregulation of Npas4 did not affect self-grooming behavior in 16p11.2dp/+ mice (Figure 5K, F1,48 (genotype x

treatment) = 0.01, p = 0.91, two-way ANOVA). Collectively, these data indicate that restoring Npas4

expression in 16p11.2dp/+ PFC is capable of ameliorating the social and cognitive deficits related to ASD

and ID.

DISCUSSION

The phenotypic impact of the 16p11.2 duplication has been thoroughly characterized in human

patients and the associated neurodevelopmental deficits are well-defined, though the underlying

molecular mechanisms remain almost completely unknown. Here we have demonstrated that transgenic

16p11.2dp/+ mice exhibit ASD- and ID-related behavioral phenotypes resembling neurodevelopmental

deficits in human 16p11.2 duplication patients, and discovered deficient GABAergic synaptic

transmission in the PFC of 16p11.2dp/+ mice. Furthermore, we observed the pronounced downregulation of

Npas4, a transcription factor responsible for the formation of GABAergic synapses in response to

neuronal excitation [104]. Restoring Npas4 expression in 16p11.2dp/+ PFC ameliorated the observed social

and cognitive deficits and restored GABAergic synaptic function and normal neuronal excitability,

suggesting a central role for Npas4 in 16p11.2 duplication pathology.

Our behavioral assays indicate that 16p11.2dp/+ mice exhibit social deficits and repetitive

behaviors reminiscent of ASD, PFC-dependent cognitive impairment, and hypolocomotion, with the

absence of schizophrenia-associated sensorimotor gating impairment, motor deficits, and anxiety. Thus, it

is evident that the behavioral profile of 16p11.2dp/+ mice recapitulates many, but not all,

neurodevelopmental deficits observed in human 16p11.2 duplication carriers. Importantly, the

performance of 16p11.2dp/+ mice in certain behavioral assays such as social approach and self-grooming

tests reflected heterogeneity within litters and specific batches, indicating that – like human 16p11.2

duplication carriers – individual 16p11.2dp/+ mice may present with variable behavioral phenotypes and at

different degrees of severity. Our results have confirmed the hypolocomotion, elevated self-grooming and

social deficits of 16p11.2dp/+ mice that were reported earlier [5, 59] and more comprehensively assessed

behavioral phenotypes related to ASD/SZ.

In addition to 16p11.2 duplication mice, 16p11.2 deletion mice (16p11.2+/-) also exhibit deficits in

sociability [59, 60, 64] and various cognitive impairments [59, 64, 68]. While 16p11.2-deletion and

16p11.2-duplication mice share similar behavioral phenotypes, it is notable that the two models exhibit

opposing electrophysiological profiles in PFC. Specifically, 16p11.2+/- PFC neurons exhibit hypoactivity

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[62], while 16p11.2dp/+ PFC neurons display abnormal hyper-excitability. Moreover, these divergent

phenotypes appear to underlie the shared behavioral abnormalities, as elevating PFC activity ameliorated

the social and cognitive deficits in 16p11.2+/- mice [62], whereas restoring inhibitory GABAergic

transmission in PFC of 16p11.2dp/+ mice gave similar therapeutic effects. These divergent phenotypes offer

an intriguing bidirectional explanation for the behavioral pathologies in 16p11.2 CNVs. The alteration of

excitation and inhibition has also been reported in the hippocampus of 16p11.2+/- mice [167]. Taken

together, these findings suggest that E/I imbalances across several implicated brain regions likely

contribute to the pathogenesis of neuropsychiatric phenotypes in mouse models of 16p11.2 CNVs.

Whole-cell patch clamp electrophysiology experiments revealed marked reductions in IPSC

amplitudes and elevated action potential firing frequencies in 16p11.2dp/+ mPFC pyramidal neurons,

indicating the disruption of GABAergic synaptic transmission and a potentially subsequent increase in

neuronal excitability. The electrophysiological phenotype of 16p11.2dp/+ PFC is consistent with extensive

evidence implicating GABAergic deficits and excitatory/inhibitory imbalance in both human ASD

patients and animal models of ASD [75, 132-137]. Additionally, the elevated excitability of 16p11.2dp/+

PFC neurons could provide a mechanism driving the epileptic phenotypes reported in some human

16p11.2 duplication patients [10, 17, 36].

Our RNA-seq experiments identified Npas4, a transcription factor with a key role in GABA

synapse formation, as one of the top 20 most strongly downregulated genes in 16p11.2dp/+ PFC.

Consistently, RNA sequencing of mice and humans have found that 16p11.2 CNV is associated with

altered expression of genes and networks that converge on synaptic function and transcriptional

regulation [76]. Npas4 knockout mice exhibit social anxiety [144] and impaired performance on various

cognitive and contextual learning tasks [142-144]. Considering the distinct role of Npas4 in GABAergic

synapse formation, we hypothesized that disruption of Npas4 may underlie GABAergic synaptic deficits,

which leads to social and cognitive deficits in 16p11.2 duplications and other forms of ASD. Indeed, we

found that Npas4 mRNA expression was significantly reduced in postmortem PFC tissue from idiopathic

ASD patients, suggesting that the dysregulation of Npas4 may be broadly implicated in ASD pathology.

Furthermore, restoring Npas4 expression in 16p11.2dp/+ PFC significantly increased sociability in the 3-

chamber social preference test and ameliorated the cognitive deficits in the temporal order recognition

memory task, indicating that Npas4 expression is functionally linked to the observed behavioral

phenotypes. In contrast, Npas4 upregulation in PFC did not affect self-grooming behavior in 16p11.2dp/+

mice, consistent with evidence suggesting that grooming behavior is controlled primarily by striatal

circuits [146]. Collectively, our findings suggest that PFC Npas4 expression is critical for the proper

development of social and cognitive functions, and that Npas4 dysregulation may broadly underlie the

behavioral features of ASD and ID.

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It has been extensively shown that Npas4 plays a key role in the formation of GABAergic

synapses [104, 105, 141]. Knockdown of Npas4 reduces GABAergic synapse density and disrupts

GABAergic synaptic transmission, whereas overexpressing Npas4 drives excessive GABA synapse

formation [104]. In the current study, we found that restoring Npas4 expression in 16p11.2dp/+ PFC

significantly elevated GABAR-mediated IPSCs and normalized action potential firing frequencies in

16p11.2dp/+ mPFC pyramidal neurons. Furthermore, Npas4 upregulation restored the downregulated

expression of the presynaptic GABA transporter VGAT in PFC of 16p11.2dp/+ mice, suggesting that

Npas4 expression may directly rescue the density of presynaptic GABAergic synaptic terminals.

Furthermore, since viral upregulation of Npas4 was observed in both pyramidal neurons and interneurons,

and Npas4 expression in either cell type promotes GABAergic input onto pyramidal neurons [141], it is

likely that the observed VGAT upregulation represents an Npas4-induced increase of GABAergic

synaptic input to pyramidal neurons, which is mediated through both pre- and post-synaptic mechanisms.

The current study presents strong evidence for the involvement of Npas4 and prefrontal cortical

GABA dysregulation in 16p11.2 duplication pathology. We propose that Npas4 dysregulation yields E/I

imbalances in prefrontal cortical synaptic circuitry, resulting in social and cognitive deficits in 16p11.2

duplications, a mechanism that may be more broadly implicated in ASD and ID.

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47

Figure 1. 16p11.2dp/+ mice exhibit social deficits, repetitive behaviors, and cognitive impairment reminiscent of ASD and ID symptoms. A, B, Bar graphs comparing the amount of time spent interacting with the social (Soc) vs. non-social (NS) stimuli (A) and the social preference index (B) in the 3-chamber social preference test of WT and 16p11.2dp/+ mice. n = 10-11 mice/group. C, D, Bar graphs showing the amount of time spent exploring the novel social stimulus (Nov) vs. the familiar social stimulus (Fam) (C) and the social novelty index (D) in the 3-chamber preference test of WT and 16p11.2dp/+ mice. n = 10-11 mice/group. E, Bar graphs showing the amount of time spent interacting with the social stimulus in the social approach test of WT and 16p11.2dp/+ mice. n = 23-32 mice/group. F, Bar graphs showing self-grooming time for WT and 16p11.2dp/+ mice. n = 12-13 mice/group. G, H, Bar graphs showing the amount of time spent exploring the novel (Nov) vs. familiar (Fam) objects (G) and the discrimination ratio (H) in temporal order recognition memory (TORM) test of WT and 16p11.2dp/+ mice. n = 10-11 mice/group. I, Bar graphs showing the discrimination ratio in the novel object recognition (NOR) test of WT and 16p11.2dp/+ mice. n = 11 mice/group. J, K, Bar graphs showing startle responses at various stimulus intensities (J) and pre-pulse inhibition levels at various pre-pulse intensities (K) for WT and 16p11.2dp/+

mice. n = 9-10 mice/group. L, Plot showing the distance travelled (in 5-minute bins) by WT and 16p11.2dp/+ mice at baseline (0-30 min), after saline injection (30-60 min), and after injection of the NMDAR antagonist MK-801 (2 mg/kg, i.p., 60-150 min). n = 9-11 mice/group. M, Bar graphs showing the latency to fall in the rotarod test of WT and 16p11.2dp/+ mice at different ages. n = 7-11 mice/group. N, Bar graphs showing the total amount of time spent exploring the open arms in the elevated plus maze test of WT and 16p11.2dp/+ mice. n = 11 mice/group. All data are presented as mean SEM. In all figures, n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.0001.

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49

Figure 2. 16p11.2dp/+ mPFC pyramidal neurons exhibit GABAergic synaptic deficits and elevated excitability. A, B, Summarized input-output curves of NMDAR-EPSC (A) and AMPAR-EPSC (B) in WT and 16p11.2dp/+ PFC neurons. Inset: representative NMDAR-EPSC and AMPAR-EPSC traces. NMDA: n = 14-17 cells, 3-4 mice/group; AMPA: n = 12-15 cells, 3 mice/group. C, D, Plot of paired-pulse ratio (PPR) of NMDAR-EPSC (C) and AMPAR-EPSC (D) evoked by double-pulses with various intervals in PFC pyramidal neurons from WT and 16p11.2dp/+ mice. Inset: representative traces. NMDA: n = 16-24 cells, 3-5 mice/group; AMPA: n = 8 cells, 2 mice/group. E, Summarized input-output curves of GABAAR-IPSC in WT and 16p11.2dp/+ mPFC pyramidal neurons. Inset: representative GABAR-IPSC traces. n = 28-31 cells, 7-8 mice/group. F, Plot of AP firing frequencies evoked by different depolarizing current injections in WT and 16p11.2dp/+ PFC neurons. Inset: representative eAP firing traces. n = 26-27 cells, 4 mice/group. G, Bar graph showing resting membrane potential (RMP) in PFC pyramidal neurons from WT and 16p11.2dp/+ mice. n = 26-27 cells, 4 mice/group. H, Bar graph showing action potential (AP) threshold in PFC pyramidal neurons from WT and 16p11.2dp/+ mice. n = 18 cells, 4 mice/group. I, Bar graph showing input resistance in PFC pyramidal neurons from WT and 16p11.2dp/+ mice. n = 15-16 cells, 4 mice/group. J, Bar graph showing the number of Parvalbumin expressing (PV+) cells in the cingulate cortex and prelimbic cortex of WT and 16p11.2dp/+ mice. Inset: representative immunostaining images; scale bars = 200 M. Cingulate cortex: n = 11-19 slices, 4 mice/group; Prelimbic cortex: n = 15-19 slices, 4 mice/group. All data are presented as mean SEM. In all figures, *p < 0.05, **p < 0.01, ***p < 0.0001. (The data presented in Panels A & I were generated by Dr. Tao Tan and Dr. Wei Wang).

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Figure 3. RNA-sequencing identifies numerous upregulated genes in PFC of 16p11.2dp/+ mice. A, Volcano plot illustrating gene distributions based on expression levels in 16p11.2dp/+ mice relative to WT animals; black dots represent genes not significantly altered, red dots represent differentially expressed genes in 16p11.2dp/+ (>1.5-fold change, p < 0.05, FDR < 0.3). B, Heat map representing expression (row z-score) of 111 significantly upregulated genes in PFC from 16p11.2dp/+ mice relative to WT values. C, Pie chart displaying the biological function classification of the upregulated genes in 16p11.2dp/+ PFC based on Gene Ontology. D, Interactome network showing predicted interactions between the upregulated genes in various ontological classifications. Genes located within the duplicated 16p11.2 chromosomal region are designated in red. E, Bar graph comparing mRNA level of five upregulated genes located in the 16p11.2 region between WT and 16p11.2dp/+ PFC. n = 12-15 mice/group. All data are presented as mean SEM. In all figures, *p < 0.05, **p < 0.01. (The figures presented in Panels A-C were generated by Fengwei Yang).

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Figure 4. RNA-sequencing identifies downregulated genes from diverse classes in 16p11.2dp/+ PFC, including the GABA-synapse regulator Npas4. A, Heat map representing expression (row z-score) of 277 significantly downregulated genes in PFC from 16p11.2dp/+ mice relative to WT values. B, Pie chart displaying the biological function classification of the downregulated genes in 16p11.2dp/+ PFC based on Gene Ontology. C, Interactome network showing predicted interactions between the downregulated genes in various ontological classifications. Genes assessed via qPCR are designated in yellow. D, Bar graph comparing WT and 16p11.2dp/+ PFC mRNA expression level for several genes encoding chromatin remodelers identified as significantly downregulated via RNA-seq. n = 7-20 mice/group. E, Bar graph comparing WT and 16p11.2dp/+ PFC mRNA level of genes encoding synaptic components/ion channels identified as significantly downregulated via RNA-seq. n = 10-17 mice/group. F, Bar graph comparing WT and 16p11.2dp/+ PFC mRNA level of genes related to ASD/ID identified as significantly downregulated via RNA-seq. n = 6-20 mice/group. G, Bar graph comparing WT and 16p11.2dp/+ PFC mRNA expression level of the GABA synapse regulator Npas4. n = 15-22 mice/group. H, Bar graph showing NPAS4 protein expression level in nuclear fractions isolated from WT and 16p11.2dp/+ PFC. Inset: representative immunoblot images. n = 9-10 mice/group. I, Bar graph showing Npas4 mRNA expression in human postmortem PFC tissue from healthy controls and ASD patients. n = 8-9/group. J, Bar graph comparing WT and 16p11.2dp/+ PFC mRNA expression level of Npas4 and the 16p11.2 gene Mapk3 in striatum. n = 4-7 mice/group. K, Bar graph comparing WT and 16p11.2dp/+ PFC mRNA level of genes related to GABAergic synaptic transmission. n = 6-19 mice/group. All data are presented as mean SEM. In panel E, *p < 0.05, **p < 0.01. (The data presented in Panels A-C were generated by Fengwei Yang).

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Figure 5. Restoring Npas4 expression in PFC ameliorates the social and cognitive deficits and restores GABAergic synaptic transmission in 16p11.2dp/+ mice. A, Immunofluorescent image showing the location of GFP expression in a viral-injected mouse. Scale bar = 50 m. B, Bar graph showing Npas4 mRNA levels in PFC of WT or 16p11.2dp/+ mice injected with GFP or Npas4 virus. n = 4-9 mice/group. C, Bar graph showing Npas4 fluorescence intensity in mPFC of GFP-injected and Npas4-injected 16p11.2dp/+

mice. Inset: representative images showing Npas4 expression in mPFC of both groups. Scale bar = 100 m. n = 13-14 slices, 3-4 mice/group. D, Bar graph showing VGAT immunostaining fluorescence intensity in mPFC (prelimbic area) of WT and 16p11.2dp/+ mice injected with GFP or Npas4 virus. Inset: representative images showing VGAT (red) and DAPI (blue) staining. Scale bar = 20 m. n = 18-38 slices, 2-3 mice/group. E, F, Plot of input-output curves of GABAAR-IPSC (E) and AP firing frequencies (F) in mPFC pyramidal neurons from WT or 16p11.2dp/+ mice injected with GFP or Npas4 virus. Insets: representative GABAAR-IPSC and AP firing traces. GABAAR-IPSC: n = 9-25 cells, 3-4 mice/group; eAP: 11-17 cells, 3-4 mice/group. G, H, Bar graphs showing the amount of time spent interacting with Soc vs. NS stimuli (G) and the social preference index (H) in the 3-chamber social preference test of WT or 16p11.2dp/+ mice injected with GFP or Npas4 virus. n = 11-17 mice/group. I, J, Bar graphs showing the amount of time spent interacting with the novel (Nov) vs. familiar (Fam) objects (I) and the discrimination ratio (J) in the TORM test of WT or 16p11.2dp/+ mice injected with GFP or Npas4 virus. n = 10-13 mice/group. K, Bar graphs showing self-grooming time in WT or 16p11.2dp/+ mice injected with GFP or Npas4 virus. n = 11-16 mice/group. All data are presented as mean SEM. In all figures, *p < 0.05, **p < 0.01, ***p < 0.0001. In figures D/E, * indicates 16p+GFP vs. 16p+Npas4 comparisons; + indicates 16p+GFP vs. WT+GFP comparisons. (The data presented in Panels E & F were generated by Dr. Tao Tan and Dr. Wei Wang).

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Part II

Chapter IV

Published in Nature Protocols (2020) 15, 3464-3477.

A Standardized Social Preference Protocol for Measuring Social Deficits in Mouse Models of

Autism

Benjamin Rein, Kaijie Ma, Zhen Yan*

Department of Physiology and Biophysics, State University of New York at Buffalo, Jacobs School of

Medicine and Biomedical Sciences, Buffalo, NY 14203, USA

*Correspondence should be addressed to Z.Y. (zhenyan@buffalo.edu)

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ABSTRACT

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social

communication deficits and other behavioral abnormalities. The three-chamber social preference test is

often used to assess social deficits in mouse models of ASD. However, varying and often contradicting

phenotypic descriptions of ASD mouse models can be found in the scientific literature, and the substantial

variability in the methods used by researchers to assess social deficits in mice could be a contributing

factor. Here we describe a standardized three-chamber social preference protocol, which is sensitive and

reliable at detecting social preference deficits in several mouse models of ASD. This protocol comprises

three phases that can all be completed within one day. The test mouse is first habituated to the apparatus

containing two empty cups in the side chambers, followed by the pre-test phase in which the mouse can

interact with two identical inanimate objects placed in the cups. During the test phase, the mouse is

allowed to interact with a social stimulus (an unfamiliar wild-type mouse) contained in one cup, and a

novel non-social stimulus contained in the other cup. The protocol is thus designed to assess preference

between social and non-social stimuli under conditions of equal salience. The broad implementation of

the 3-chamber social preference protocol presented here should improve the accuracy and consistency of

assessments for social preference deficits associated with ASD and other psychiatric disorders.

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INTRODUCTION

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by reduced or

impaired social interaction, repetitive behaviors and/or restricted interests. ASD has been linked to a

broad range of etiologies; >50% of ASD cases are thought to be caused by genetic variation[168], and

genetic screenings have led to the identification and implication of numerous high-risk genes in ASD

pathogenesis[95]. Transgenic mouse models carrying mutations in high-risk ASD genes or genetic loci,

such as Shank3-deficiencies[169, 170] or 16p11.2 copy number variations (CNVs; i.e.

deletion/duplication)[11, 13], display ASD-related behavioral phenotypes[62, 72, 102, 110, 171], and

represent powerful tools for elucidating neurobiological mechanisms that drive ASD pathogenesis.

Behavioral assays sensitive to social deficits are necessary for phenotypic verification of ASD

models and for evaluation of therapeutic intervention strategies. The three-chamber social preference test,

which assesses the animal’s preference for a social stimulus over a non-social stimulus, is one of the most

commonly used methods for evaluating sociability in mouse models of ASD[172]. However, numerous

modifications have been made to this assay since it was initially described, resulting in an array of

separate and distinct protocols across the ASD literature with dispersed usage[101, 102, 173-182]. The

variety of testing methods has contributed to discrepancies between studies in the phenotypic descriptions

of several ASD mouse models, with varying conclusions depending upon the protocol used. For example,

opposing phenotypes (i.e. the presence or absence of social preference deficits) have been reported in

Shank2-/- mice16,17, Shank3e4-9 mice[179, 183], and Shank3C/C mice[110, 177, 178], with differing protocols used

across studies. These findings suggest that the different three-chamber social preference test

methodologies used may have differing sensitivities for detection of social deficits. To encourage

consistent and reliable phenotyping of ASD-related social deficits in mice, we describe here a 3-chamber

social preference test protocol that offers robust detection of social preference deficits, and demonstrates

enhanced sensitivity relative to a commonly used alternative approach.

Comparison between the social preference test variants

The three-chamber social preference assay we describe here evaluates the test mouse’s preference

for interacting with a social (S) stimulus versus a non-social (NS) stimulus (termed as “3-phase S-NS”

protocol). First, the test mouse is habituated to a 3-chamber apparatus containing two empty cups, to

reduce the salience of these objects (Habituation Phase). Next, two identical objects (paper balls) are

placed within the cups, to familiarize the animal with the presence of objects contained within the cups

(Pre-test Phase). Finally, a social stimulus (an age- and sex-matched wild-type mouse) is introduced

under one cup and a novel non-social stimulus (wooden block) is placed under the other cup (Test Phase).

The amount of time spent interacting with either stimulus is recorded in order to assess the animal’s level

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of preference for the social stimulus over the non-social stimulus. This “3-phase S-NS” method is

designed with the intention to minimize variability caused by novelty-driven interactions with the cup and

reliably isolate the animal’s interest level towards a social stimulus versus a non-social stimulus. This

protocol and similar protocols have been effective in identifying social preference deficits in several ASD

mouse models, including Shank3-deficient mice, such as Shank3+/C mice[102, 110], Shank3C/C

mice[178] and Shank3e4-9 mice[179], Shank2 knockout mice[101], forebrain Cul3-deficient mice (Cul3f/-)

[87], and 16p11.2 duplication mice (16p11.2dp/+)[72].

The “3-phase S-NS” protocol represents a modification of a widely used 3-chamber social testing

method[172, 184]. In this alternative variant, the test mouse is first habituated to an empty 3-chamber

apparatus (Habituation Phase). In the subsequent Test Phase, two cups are placed on opposing sides of the

apparatus, one containing an age- and sex-matched wild-type mouse and the other being empty. Since this

protocol compares the mouse’s interaction with a social stimulus (S) versus an empty cup (E), it is

referred as the “2-phase S-E” method. Numerous variations of this method also exist. One commonly

used method[61, 183, 185-187] is identical to the “2-phase S-E” method, but includes an additional

habituation phase to only the center chamber before habituation to the entire apparatus. In another

protocol[173, 175, 181], the test mouse is only habituated the center chamber before the test phase.

Another method habituates the test mouse to the apparatus containing two empty cups, and in the

subsequent test phase, one cup contains a social stimulus, while the other remains empty[174, 176, 177].

A number of papers have been published indicating that the “3-phase S-NS” protocol displays

greater sensitivity to detect social deficits in ASD models than the “2-phase S-E” approach. Schmeisser et

al.[176] reported normal social interaction time and social preference in Shank2-/- mice tested with the S-E

approach. In contrast, Won et al.[101] used an S-NS 3-chamber social preference protocol, and found that

Shank2-/- mice spent significantly less time than WT animals interacting with the social stimulus.

Additionally, homozygous mice with the deletion of Shank3 exon 4-9 (Shank3e4-9) were reported to

display significant deficits in social preference when tested with the S-NS protocol[179]; however, a

separate study reported normal social preference in Shank3e4-9 mice when tested with an S-E

protocol[183]. Furthermore, one study using an S-E approach reported the lack of 3-chamber social

preference deficits in Shank3C/C mice[177], whereas multiple studies using the 3-phase S-NS approach

did find robust social deficits in Shank3C/C mice[110, 178] and male Shank3+/C mice[102, 110, 138].

The findings that disruption of Shank3 is linked to autism in humans[169, 170, 188, 189] and

leads to social impairments in macaques[190, 191] are consistent with the social deficits phenotypes in

Shank3-deficient mice[102, 110, 138, 175, 178, 179, 192], but are in disagreement with the normal

sociability in several lines of Shank3 mutant mice detected with the S-E approach[177, 183, 193]. These

results indicate that 3-chamber social preference protocols utilizing a novel object placed under a cup,

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rather than an empty cup alone, are more sensitive to social preference deficits in ASD models. The “3-

phase S-NS” protocol can also be used to examine social abnormalities relevant to the negative symptoms

of schizophrenia, as shown previously with a three-armed platform containing empty wire cages on two

arms[194]. The design of this method coincides with the protocol described here, in which the wire cup

functions only as a component of the testing apparatus, while an inanimate object (a Lego mouse) was

used as the non-social cue during the test phase. The results of ourselves and others suggest the "3-phase

S-NS" 3-chamber social preference protocol has much improved sensitivity and robustness in revealing

ASD-related social deficits.

Using the 3-chamber S-NS approach, we did not observe social preference deficits in the mouse

model of Phelan-McDermid Syndrome (PMS) with a complete deletion of Shank3, consistent with prior

reports on its normal social interest[195] and social preference[196]. While it is hard to explain this

apparently distinct mouse phenotype from human and monkey studies, one possibility is the

compensatory effects of other Shank family members in Shank3-deleted mice. While behavioral methods

are critical in phenotypic characterization, the choice of mouse lines is also a key determining factor[197].

In this study, we found that the "2-phase S-E" protocol failed to detect social deficits in several

mouse models of ASD. Nevertheless, we do not claim that this method is completely ineffective. Several

studies following the S-E method or similar protocols have identified social preference deficits in ASD

models, such as Shank3B-/- mice[175], Pten conditional knockout mice[198], and mice with homozygous

deletion of the ASD-associated genes Neuroligin-4[173] and Cntnap2[185]. However, another study

using the same S-E protocol reported the lack of social deficits in Cntnap2 knockout mice[61], suggesting

that the S-E protocol may be prone to substantial variability. Thus, despite the capability of the S-E

method to detect social preference deficits, it may be less sensitive to social deficits in mouse models that

recapitulate ASD humans with haploinsufficiency of risk genes.

The negative results seen with the “2-phase S-E” method could be due to inherent design

problems. The empty cup presented as the non-social stimulus also serves as a component of the social

stimulus, as an identical cup is used to house the wild-type mouse. This may result in an inherent bias in

favor of the social stimulus that contains both a novel social stimulus (mouse) and a novel non-social

stimulus (cup), and is thus more salient than the non-social stimulus containing a cup alone. This inherent

bias for the social stimulus driven by the design of the “2-phase S-E” protocol may mask the presence of

social deficits in ASD models tested with this method. In addition, due to the lack of habituation to the

empty cup, the test mouse is prone to engage in extended investigation of the cup, which may affect

interaction time with either the social or non-social stimulus, promoting unplanned and unpreventable

variability in sociability tests. We therefore encourage the use of the “3-phase S-NS” protocol, in order to

improve the sensitivity, robustness and consistency of phenotypic screenings in mouse models of ASD.

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Applications

To date, the three-chamber social preference protocol presented here has been used

predominantly for phenotyping of social deficits in transgenic mouse models of ASD[72, 87, 101, 110,

178, 179], and evaluating the effectiveness of treatment strategies[102, 138, 192]. However, this protocol

may be appropriately applied in other contexts, including environmentally-induced models of ASD[199,

200], animals affected by physical or emotional stress[201], functional studies of neurocircuitry

controlling sociability[202], etc.

Limitations

The described three-chamber sociability test (“3-phase S-NS”) offers robust sensitivity to the

measurement of social preference, however, not all socially-affected animals are guaranteed to exhibit

deficits. Mice carrying deletion of 16p11.2 fail to display three-chamber social preference deficits[61],

despite impairments in several other measurements of sociability, including social approach[59, 62],

male-female reciprocal social interactions[64], and ultrasonic vocalizations[60]. Therefore, this approach

appears to be sensitive to context-specific deficits in preference for a social over a non-social stimulus,

and should not be considered as a definitive indicator of the overall presence or absence of social deficits.

Sensory abnormalities are present in a large portion of children with ASD[203], and several

mouse models of ASD exhibit various sensory phenotypes[204]. It is possible that sensory deficits may

affect performances in social behavioral assays. However, 16p11.2 deletion mice, which are deaf and

have reduced ultrasonic vocalizations[60], display normal social preference in 3-chamber sociability

tests[61], consistent with our findings here. Therefore, the presence of sensory deficits is not ensured to

affect the social preference test. Nevertheless, it is encouraged to examine visual, auditory and olfactory

integrity.

Relative to “2-phase S-E” protocols, the method described here ("3-phase S-NS") involves more

rodent handling. However, taking the test mouse out of the apparatus while cleaning and replacing objects

between trials will be less disruptive to the animal’s behavior. Nevertheless, all animals should be

handled gently to minimize stress.

EXPERIMENTAL DESIGN

Choice of mouse strain. This protocol is suitable for assessing social preference in all strains of

mice. However, controls should be wild-type animals of the same strain, as baseline sociability may differ

between mouse strains. Locomotion differences or motor deficits could be a confounding factor

impacting test results. Animals of all ages may be tested, but controls must be age-matched, as sociability

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declines when animals get older[205, 206]. We have reliably used this protocol on juvenile to adult

animals (5-6 weeks old to 4-5 months old). For all experiments, WT littermates should be used as control

groups. Unfamiliar age-, strain-, and sex-matched WT mice should be used as the social stimulus. The use

of genetically-altered or otherwise socially-impaired mice as the social stimulus may affect the sociability

of the test mouse. Generally, two separate groups of mice should be used as the test mice and the stimulus

mice. However, if mouse availability is limited, test mice (only WT) may be used as the social stimulus

after they have completed their testing. All animals should be group-housed before testing of sociability

as single housing of animals will induce isolation stress and affect sociability. If highly variable social

behavior is observed among animals within a single genotype, experiments should include more than 10

animals in the group, from at least three cohorts, in order to draw accurate conclusions regarding social

preference differences between groups. Group sizes should be properly determined to avoid using too few

or too many mice – see ARRIVE guidelines for details[207].

The protocol described here has been designed and optimized for use in mice. However, the

three-chamber social preference test has also been performed in rats[208]. We thus think this protocol

could be adapted for rats, with the use of an appropriately sized, larger apparatus.

MATERIALS

Animals

- Test mice: This protocol may be used for testing commercially obtained mice (e.g. purchased from

the Jackson Laboratory) or newly created mouse lines by research laboratories. CAUTION: Mice

used as controls must be of the same strain as the test group. All animals tested and compared must be

similar in age, as interaction time with the social stimulus typically decreases as animals age. This

protocol has been used reliably in juvenile to adult animals (5-6 weeks old to 4-5 months old). Mice

are maintained on a 12-hour light (6:00 am – 6:00 pm)/dark (6:00 pm – 6:00 am) cycle. They should

be group-housed with gender-matched conspecifics (2-4 mice per cage) and provided with standard

enrichment. All experiments must receive approval from the relevant institutional review board and

be conducted in accordance with local and national regulations. We obtained permission from State

University of New York at Buffalo Institutional Animal Care and Use Committee (IACUC) to

undertake the studies shown here. CRITICAL: Animals that display major deficits in locomotion

should not be used for this assay.

- Social stimulus mice: The mice used as the social stimulus must be age-, sex-, and strain-matched to

test mice. CRITICAL: Using social stimulus mice that differ from test mice on any of these

parameters could impact test results. CRITICAL: Do not use socially-impaired mice (such as

transgenic ASD models) as the social stimulus - this may reduce the amount of time the test mouse

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spends interacting with the social stimulus. The stimulus mouse should be unfamiliar to the test mice;

do not use cage-mates of the test mice.

Reagents

- 75% ethanol (Decon Laboratories; #DSP-MD.43) diluted in ddH2O. CRITICAL: We use ethanol for

cleaning the testing apparatus and objects between tests because it evaporates quickly and effectively

removes odor. CAUTION: Ethanol can carry some odors and may have a fixative action on the

molecules that are present in urines and feces. A more thorough solution is to soak and wash with

soap, rinse and dry.

Equipment

- Three-chambered apparatus. The apparatus we use has the following specifications: 102 cm (L) x 47

cm (W) x 45 cm (H). The walls of the apparatus are made of transparent plexiglass. The two side

chambers that the stimuli are placed in measure 33 cm (L) x 47 cm (W). CRITICAL: The three-

chamber apparatus with side chambers should be large enough to permit the test mouse to explore the

area outside of the cup is used. The use of a small three-chamber apparatus with limited exploration

space in each side chamber may affect the measurement of the test animal’s social behaviors. Our

apparatus is larger than many commercially available 3-chamber apparatuses (e.g. Ugo basile; San

Diego Instruments), which have side chambers of approximately 20 cm (L) x 40 cm (W). This shorter

chamber permits less space for exploration in the outside area surrounding the cup, which may

interfere with accurate measurement of the time spent interacting with the social stimulus contained

inside the cup.

- Cup or capsule to house the social or nonsocial stimulus. We use a wire pencil cup (color: chrome,

made of sturdy steel), 10.2 cm (diameter) x 10.8 cm (height), with approximately 1 cm gaps between

bars, sufficient for animal interaction and sniffing (Spectrum Diversified Galaxy Pencil Holder;

Spectrumdiversified.com), for containing the social and nonsocial stimuli. CRITICAL: We

recommend users keep an extra set of cups, so that one set may be cleaned, while the other is in use.

- Glass bottle or other object to be placed on top of the cup to prevent the test mouse from climbing.

We use the 250 mL glass bottle (PYREX Reusable Media Storage Bottles; Fishersci.com).

- Inanimate object to be placed within the cup as the non-social stimulus. We use a square wooden

block (L: 2.5 cm). Other objects, such as Lego structures of simple shapes can also be used.

- Two identical inanimate objects to use in the pre-test phase. Paper balls, which are simple to prepare

and readily available, can be used. CRITICAL: The paper towel is crumpled by hand with clean

gloves to avoid transferring animal odor to the paper ball.

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- Digital camcorder to record for subsequent scoring or re-scoring of the test animal’s behavior.

- Video tracking and analysis softwares. We use Anymaze (Stoelting Co, Wood Dale, IL). Other

animal tracking softwares, such as EthoVision XT by Noldus, idTracker, can also be used.

PROCEDURE

Habituation (10 minutes)

1. Bring the test mice to the behavioral room and allow to habituate for at least one hour, with the room

set to the testing conditions. CRITICAL: Overhead lighting should be minimized to avoid anxiogenic

effects that may affect social interaction time. Brightness should be measured in the center of all three

chambers to ensure that the apparatus is evenly lit. Brightness should ideally be maintained at <50

lux. TROUBLESHOOTING.

2. Place two clean, empty inverted pencil cups into the three-chamber apparatus, each centered

approximately halfway between the midline and the far wall. CRITICAL The testing apparatus and

cups should be cleaned and free of debris prior to starting any new test. CRITICAL: Place a clean,

empty 250mL glass bottle upright atop each cup to prevent the test mouse from climbing the cup. The

bottle placed atop each cup should be identical in size, shape and color.

3. Gently place the test mouse into the center of the apparatus. Start a timer and allow 10 minutes for the

animal to explore freely while habituating to the apparatus and empty cups. CAUTION: When

transferring the test mouse from its home cage to the testing apparatus, the animal should be handled

gently, preferably carried on one arm or the home cage lid. Do not suspend the animal by its tail

while carrying. Tests preceded by rough handling may be affected by animal stress.

4. Remove the test mouse from the apparatus and gently return to its home cage for 5-min break.

5. Wipe down the apparatus and cups/bottles with 75% ethanol to remove any residual odors that may

affect subsequent tests.

PAUSEPOINT At this stage, the animals can be returned to their home cages and the remaining trials

may be optionally carried out on the following day. If this is done, on the next day, repeat step 1 before

proceeding with the following procedure.

Pre-test (10 minutes)

6. Prepare two clean paper balls and place one under each inverted pencil cup. The two paper balls used

should be of the same variety, as they are intended to represent identical objects. The paper balls

should be placed in the center of the cup. When placing the cups into the chambers, leave enough

space between the cup and the outer wall of the apparatus for the test mouse to explore the full

periphery of the cup. CAUTION: Use clean gloves when crumpling and placing the paper balls under

the pencil cups. Transferring odors onto the paper balls may affect the pre-test trial.

69

7. Gently place the test mouse into the apparatus. Start a timer and allow 10 minutes for the animal to

familiarize with the presence of the objects contained within the cups.

8. Remove the test mouse from the apparatus and gently return to its home cage for 5-min break.

9. Remove the paper balls and wipe down the apparatus, cups and bottles with 75% ethanol to remove

any residual odors that may affect subsequent tests.

Social Preference Test (10 minutes)

10. Place an age-, sex-, and strain-matched unfamiliar WT mouse under one cup, to serve as the social

stimulus. Rough handling of the stimulus mouse may negatively affect social interactions with the

test mouse; handle gently when placing the stimulus mouse into the cup. CRITICAL: The stimulus

mouse must be unfamiliar to the test mouse; do not use cage-mates. The mouse used as the social

stimulus should be interchanged regularly when conducting multiple tests to avoid exhaustion or

social fatigue of the stimulus mouse.

11. Place a wooden block or another unfamiliar, inanimate object under the other cup to serve as the non-

social stimulus. CRITICAL: The location of the social or non-social stimulus in either side chamber

should be counterbalanced between tests.

12. Place the test mouse into the apparatus containing the social- and non-social stimuli. Start a timer and

allow the mouse to explore for 10 minutes. The amount of time spent interacting with the social

stimulus and the non-social stimulus should be recorded. This can be done manually by an

experimentally blind researcher, or automatically by video tracking software such as Anymaze.

13. Return the test mouse and stimulus mouse to their respective home cages.

14. Remove the object and wipe down the apparatus and cups/bottles with 75% ethanol to remove any

residual odors that may affect subsequent tests.

(Optional) Social Novelty Test (10 minutes)

15. Replace the non-social object from the previous trial with an unfamiliar WT mouse (age-, sex-, and

strain-matched) as the “novel” social stimulus.

16. Place the test mouse into the apparatus containing the novel and familiar social stimuli. Start a timer

and allow the animal to explore for 10 minutes. Record the amount of time spent interacting with

each stimulus either manually or digitally.

17. Return the test mouse and both stimulus mice to their respective home cages.

18. Wipe down the apparatus and cups/media bottles with 75% ethanol to remove any residual odors

which may affect subsequent tests.

Timing

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Animals need to be transferred to the behavioral room at least 60 minutes prior to testing. This

protocol requires ~40 minutes to perform per animal if all 3 trials are undertaken (three 10-min testing

trials, with two 5-min intervals between trials for animal resting and apparatus cleaning). The 10-min

habituation trial may optionally be completed one day before the pre-test and social preference test.

When performing repeated measures on the same animals, allow at least three days between assays. It

takes ~25 minutes for each animal in subsequent days (habituation phase omitted, two 10-min testing

trials, with one 5-min interval).

Troubleshooting

Lighting. The lighting of the testing room may affect sociability. Social interaction time is

typically reduced when animals are tested in brighter conditions. If overhead lighting cannot be dimmed,

a standing lamp may be used to light the room. However, the lamp must be kept at a safe distance from

the testing chamber so as not to induce anxiogenic effects. The lighting must be consistent across all areas

of the 3-chamber apparatus to prevent animal preference for darker locations or chambers. In unevenly lit

testing conditions, the animal will prefer dimmer areas, which could affect testing results.

Animal testing and scoring. Sometimes the test animal climbs the cup and remains at the top of

the cup without interacting with the stimulus. Additionally, the software may fail to constantly track the

test animal. In such cases, manual counting is more accurate (see Scoring Methods for details).

Expected values. When tested with the “3-phase S-NS” protocol, the average social interaction

time for WT mice (of either sex, 6-8 weeks old) typically falls between ~125-150 seconds for a 10-minute

testing session, though this may vary between 100-200 seconds depending on the strain and age of

animals tested. The average non-social interaction time is typically between ~25-50 seconds. The average

social preference index for WT mice (C57BL6 background) should be 0.4-0.8. However, mice commonly

exhibit natural variability in behavioral tendencies, even within a single strain or genotype, so values may

fall within a broader range than this. Due to this expected spectrum of social behaviors, it is emphasized

that comparisons must be made between group averages, which include data from a sufficiently large

number of mice of either genotype, and from several litters. If any animal presents a value that is

determined to be a statistically significant outlier, this animal may be removed from the analysis.

Housing Effects. All test mice should be group-housed, as single-housing can produce severe

detrimental effects on sociability and other behaviors due to social isolation stress[209]. Furthermore,

housing mice with conspecifics of different genotypes can affect social behavior. Mouse models of ASD

may be more likely to assume submissive roles in social hierarchies, as demonstrated in neuroligin-3

deficient (Nlgn3y/-) mice[210], which may produce defeat-related social deficits. Indeed, male Nlgn3y/-

mice housed with WT animals display more severe social deficits than those housed with genotype-

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matched conspecifics[210]. Interestingly, raising WT mice with Nlgn3y/- mice also compromises

sociability of WT. The negative impact of mixed-genotype housing on social behavior has been similarly

reported in 16p11.2+/- mice[63]. There is also a report showing that enhancing environmental enrichment

within animal housing improves sociability in valproic acid-exposed autism model mice[211]. These

findings highlight the importance of carefully controlling housing conditions in order to produce accurate

measurements of social behavior in ASD models.

Scoring Methods

Scoring can be undertaken manually or using automated behavior tracking software. The key

information is the duration of direct interactions of the test mouse with the social or non-social stimulus.

We usually use automated scoring of the three-chamber social preference test with Anymaze behavior

tracking software (Stoelting, Wood Dale, IL). The area directly surrounding the cup is designated as a

zone of interest, and the amount of time spent in the zone by the test mouse is measured. This method of

scoring therefore measures the amount of time the test animal spends in close proximity to the cup

(distance of animal head to cup edge: ≤3.5 cm), rather than specifically measuring time spent sniffing or

engaging with the social stimulus. Automated scoring may produce inaccurate conclusions, for example if

animals remain in the vicinity of the cup without interacting with the social or non-social stimulus. For

this reason, manual scoring may be required to verify the scores. Automated scoring is also susceptible to

software errors if the animal is not properly tracked. All videos should therefore be reviewed to verify

that the animal has been tracked well.

If manual scoring is performed, all scoring should be performed by a researcher blinded to animal

genotype and/or treatment. For manual scoring purposes, behaviors that are typically counted as

interactions include: directly interacting with the stimulus mouse or non-social object between the wire

bars of the pencil cup; sniffing the base of the cup containing the stimulus; interacting with parts of the

stimulus that are protruding from the cup, such as the tail of the stimulus mouse; actively attending to

(sniffing/facing) the stimulus while climbing the cup. Behaviors that are not counted include: interacting

with the bottle on top of the cup; standing near the cup without attending to (sniffing/facing) the cup or

the contained stimulus; self-grooming in the proximity of the cup. While it is helpful to use clearly

defined scoring parameters, experimenters may differ in their assessment of behavior and therefore

produce different values. Thus, all videos generated within a single experiment should be scored by the

same experimenter to minimize human error. We recommend automated scoring followed by manual

correction, which gives the most accurate results. Supplemental Videos 1 and 2 show examples of a WT

and a Shank3-deficient mouse in the Social Preference Test phase, with added commentary. Additionally,

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Supplemental Table 1 provides a list of various observable behaviors throughout these two videos, and

indicates how they should be manually scored.

Statistical analysis

All behavioral testing should be performed on at least 3 independent cohorts. Interaction time

with the social stimulus (TS) and non-social stimulus (TNS) is quantified. For comparisons between WT vs

mutant, a two-way ANOVA should be performed with comparisons between all four values (TS in WT,

TNS in WT, TS in mutant, TNS in mutant), followed by post hoc Bonferroni tests for multiple comparisons

within and between groups. In addition, social preference indexes, ISP = (TS-TNS)/(TS+TNS), are compared

between groups using two-tailed Student’s t tests. All datasets should be tested for normality using

Shapiro-Wilk tests, and data that fail normality tests are compared with nonparametric tests, such as the

Mann-Whitney U test.

For a genotype where TS is significantly greater than TNS, this represents the existence of social

preference. A genotype showing the significant reduction of both TS and ISP relative to the WT group

warrants the interpretation that social deficits, including the impairment in social engagement, social

interest, social interaction, and social preference, are manifested. If TS is unchanged, and only ISP is

significantly reduced (due to the increased TNS) in the mutant group, it suggests the presence of relatively

mild social abnormality, reflected by the impairment in social preference.

Repeated Measures

The “3-phase S-NS” three-chamber social preference protocol can be performed repeatedly on the

same mice with consistent results. Several of our studies have included repeated testing at progressive

intervals in control and treatment groups across time points, to test the longitudinal therapeutic

efficacy[102, 138, 192]. The “3-phase S-NS” protocol is well suited for repeated testing, as a new object

may be placed inside the cup in the test phase during repeated measurements, thereby preserving the

novelty of the non-social stimulus. This represents an additional advantage of the “3-phase S-NS”

protocol over the “2-phase S-E” protocol, as the empty cup becomes familiar after a single test and cannot

be considered a novel non-social stimulus in subsequent testing unless different types of cups are used

each time.

When performing repeated testing with the 3-phase S-NS method, use a novel social stimulus in

subsequent tests following the initial assessment. Additionally, in subsequent days following the initial

testing, the habituation phase to the empty cups may be omitted, and performing only two phases (pre-test

and social preference) is sufficient.

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Typical results seen using the “3-Phase S-NS” Social Preference Protocol

In this section we discuss examples of results that have been obtained by following “3-phase S-

NS” protocol (Figure 1a), and demonstrate its sensitivity in detecting ASD-related social preference

deficits in several distinct transgenic mouse models of ASD. Detailed statistical information for all data

are described in Supplemental Table 2.

Shank3, which encodes a postsynaptic scaffolding protein located at glutamatergic synapses, is

among the strongest genetic risk factors for ASD[169, 170] and plays a causal role in Phelan-McDermid

Syndrome (PMS)[212]. Exon 21, the largest coding region of SHANK3, has the most variants and

mutations in humans with ASD[169, 170, 188, 189]. We tested heterozygous mice carrying exon 21-

deleted Shank3 gene, which results in the truncated form of Shank3 protein lacking the C-terminal region

(Shank3+/C), mimicking the human ASD-linked disruption of SHANK3 exon 21[188]. The 6-8-week-old

male Shank3+/C mice spent significantly less time than WT littermates investigating the social stimulus,

and did not exhibit a significant preference for the social stimulus over the nonsocial stimulus (Figure 1b,

WT: n = 8; Shank3+/C: n = 14, F 1,40 (interaction) = 10.0, p < 0.01, two-way ANOVA). Shank3+/C mice

correspondingly displayed a significantly lower social preference index than WT mice (Figure 1c, t(20) =

3.94, p < 0.01, unpaired t-test), indicating social interaction deficits. Two videos showing one male WT

and one male Shank3+/C mouse in the social preference test phase with the “3-phase S-NS” method are

included as Supplementary Videos 1 and 2.

We then tested 6-8-week-old female Shank3+/C mice with the “3-phase S-NS” method. Unlike

male Shank3+/C mice, female Shank3+/C spent significantly more time interacting with the social stimulus

than the non-social stimulus (Figure 1e, n = 9 mice/group, F 1,32 (interaction) = 0.4, p > 0.5, two-way ANOVA),

and exhibited a social preference index similar to female WT animals (Figure 1f, t(16) = 1.1, p > 0.2,

unpaired t-test). This suggests that heterozygous Shank3 exon 21-deletion confers sociability deficits that

are restricted to male mice, and the “3-phase S-NS” method is capable of isolating sex-specific deficits

within a single genotype.

Copy number variations (CNVs) of the human 16p11.2 gene locus are among the strongest

genetic risk factors for ASD [11, 13, 19]. Mice carrying deletion or duplication of the 16p11.2 murine

ortholog exhibit behavioral features of neurodevelopmental disorders including ASD-related social

deficits [4, 5, 59, 60, 62, 72]. We tested male and female 6-8-week-old 16p11.2 duplication mice

(16p11.2dp/+) and WT littermates using the 3-phase S-NS method, and found that 16p11.2dp/+ mice spent

significantly less time than WT animals interacting with the social stimulus, and failed to display a

significant preference for the social stimulus over the non-social stimulus (Figure 1h, WT: n = 10;

16p11.2dp/+: n = 12, F 1, 40 (interaction) = 11.5, p < 0.01, two-way ANOVA). Correspondingly, the social

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preference index for 16p11.2dp/+ mice was significantly reduced relative to WT mice (Figure 1i, t(20) = 2.5,

p < 0.05, unpaired t-test).

A previous characterization of 16p11.2 deletion mice (16p11.2+/-) found that they display normal

sociability when tested with the 2-phase S-E method [61]. We thus tested 16p11.2+/- mice (male and

female 6-7-week-old) with the 3-phase S-NS method to determine whether they may exhibit social

deficits with this more sensitive approach. Similar to WT mice, 16p11.2+/- mice spent significantly more

time interacting with the social stimulus than the non-social stimulus (Figure 1k, n = 8 mice/group, F 1, 28

(interaction) = 0.6, p = 0.4, two-way ANOVA), and the social preference index was not significantly altered

(Figure 1l, n = 8 mice/group, U = 24, p = 0.43, Mann-Whitney U test). These findings confirm that

16p11.2+/- do not display 3-chamber social preference deficits, despite exhibiting impairments in various

other sociability assays [4, 60, 62, 64].

We next tested 6-8-week-old male and female mice with forebrain-specific deletion of the high-

risk ASD gene Cul3 (Cul3f/-) [87]. Unlike Cul3f/f controls, Cul3f/- mice failed to show a significant

preference for the social over the nonsocial stimulus (Figure 1n, Cul3f/f: n = 10; Cul3f/-: n = 12, F 1, 40

(interaction) = 16.2, p < 0.001, two-way ANOVA), and exhibited a significantly reduced social preference

index (Figure 1o, t(20) = 7.2, p < 0.0001, unpaired t-test), indicating the presence of social deficits.

The dopamine D4 receptor (D4R) is implicated in schizophrenia (SZ) [213], and D4 receptor

knockout mice (D4KO) display hypersensitivity to psychomotor stimulants [214] and stress-induced SZ-

related phenotypes [215]. However, sociability is unimpaired in these animals [215]. We thus utilized

D4KO mice as a negative control to verify the reliability of the 3-phase S-NS method in detecting social

deficits without yielding false positives in socially unaffected transgenic models. Similar to WT mice,

D4KO mice spent significantly more time interacting with the social stimulus than the non-social

stimulus (Figure 1q, n = 6 mice/group, F 1, 20 (interaction) = 0.2, p = 0.6, two-way ANOVA), and did not differ

from WT animals in their social preference index (Figure 1r, t(10) = 0.2, p = 0.9, unpaired t-test),

confirming the lack of social deficits in D4KO mice.

Collectively, these results indicate that the 3-phase S-NS protocol has robust sensitivity in

revealing social deficits in distinct mouse models of ASD (male Shank3+/C, 16p11.2dp/+, and Cul3f/-).

Moreover, this method retains high reliability in confirming the lack of social preference deficits in

multiple mouse lines (female Shank3+/C, 16p11.2+/- and D4KO).

As mentioned earlier, the “3-phase S-NS” protocol may optionally be augmented to assess

preference for a novel social stimulus over a familiar social stimulus. We do not include example data

from the social novelty preference phase here, and interested readers are encouraged to refer to our

previous papers on social novelty preference data for Shank3+/C and 16p11.2dp/+ ASD mouse models[72,

110].

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Comparison with the “2-Phase S-E” Social Preference Protocol

To compare differences in sensitivity between the “3-phase S-NS” protocol and the widely-used

“2-phase S-E” protocol, we also tested the same mouse models of ASD with the “2-phase S-E” protocol

(Figure 2a). Using this testing method, male Shank3+/C mice did not differ from WT animals in the

amount of time spent interacting with the social stimulus, and showed a significant preference for the

social stimulus over the empty cup (Figure 2b, WT: n = 8; Shank3+/C: n = 14, F 1, 40 (interaction) = 2.4, p =

0.13, two-way ANOVA). Additionally, the social preference index did not differ between male WT and

Shank3+/C mice (Figure 2c, t(20) = 1.7, p = 0.10, unpaired t-test). These findings indicate that the “2-phase

S-E” protocol fails to reveal social preference deficits in male Shank3+/C mice, contrary to the findings

from “3-phase S-NS” protocol (Figure 1b-d). Our results suggest that contradicting phenotypic

descriptions of Shank3-deficient mice in the existing literature may be due to different testing methods.

Further testing of 16p11.2dp/+ and Cul3f/- mice (male and female 6-8-week-old) with the “2-phase

S-E” protocol indicated that they spent significantly more time interacting with the social stimulus than

the empty cup (Figure 2e, WT: n = 10, 16p11.2dp/+: n = 12, F 1, 40 (interaction) = 1.7, p = 0.2, two-way ANOVA;

Figure 2h, Cul3f/f: n = 7, Cul3f/-: n = 11, F 1, 32 (interaction) = 0.4, p = 0.5, two-way ANOVA), and their social

preference indexes did not differ from WT controls (Figure 2f, t(20) = 0.07, p = 0.9, unpaired t-test; Figure

2i, U = 33, p = 0.65, Mann-Whitney U test).

Collectively, these findings indicate that three distinct mouse models of ASD, which display clear

social deficits using “3-phase S-NS” protocol (male Shank3+/C, 16p11.2dp/+, and Cul3f/-), fail to show

social preference deficits using the “2-phase S-E” protocol, suggesting that the “3-phase S-NS” protocol

offers higher sensitivity to detect social deficits in ASD models. We thus propose that the adoption of this

method should be prioritized to maximize the accuracy of phenotypic behavioral screenings.

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Figure 1. Social behavioral data obtained from several transgenic mouse models using the 3-phase S-NS protocol. a, Graphic depicting the 3-phase S-NS protocol, consisting of a 10-minute habituation phase to the apparatus containing two empty cups, a 10-minute pre-test phase in which two identical objects (paper balls) are placed under the cups, and a 10-minute social preference test phase in which one cup contains a social (S) stimulus (age- and sex-matched WT mouse) and the other contains a non-social (NS) stimulus (wooden block). b, e, h, k, n, q, Bar graphs showing the amount of time spent interacting with the social stimulus (S) or non-social stimulus (NS) in male WT vs. Shank3+/C mice (b), female WT vs. Shank3+/C

mice (e), WT vs. 16p11.2dp/+ mice (h), WT vs. 16p11.2+/- mice (k), Cul3f/f vs. Cul3f/- mice (n), and WT vs. D4KO mice (q). Both sexes were used in g, j, m, p. c, f, i, l, o, r, Bar graphs comparing the social preference index of individual mouse lines. d, g, j, m, p, Representative heat maps illustrating the topographical time distribution in social preference tests of individual mouse lines. All data are presented as mean ± S.E.M. For all figures, n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.0001, S vs. NS; #p < 0.05, ##p < 0.01, WT vs. mutant (social time). Note, the results in panels b-d, h-j, and n-p are consistent with prior findings in references 7, 8, 11, 25, 31 and 36. (Some of the data in this figure were generated by Kajie Ma).

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Figure 2. Social behavioral data obtained from several ASD mouse models using the 2-phase S-E protocol. a, Graphic depicting the 2-phase S-E protocol, consisting of a 10-minute habituation phase to the empty apparatus and a 10-minute social preference test phase in which a social stimulus (age- and sex-matched WT mouse under cup) and non-social stimulus (empty cup) are introduced. b, e, h, Bar graphs showing the amount of time spent interacting with the social stimulus (S) or empty cup (E) in male WT vs. Shank3+/C mice (b), WT vs. 16p11.2dp/+ mice (e), and Cul3f/f vs. Cul3f/- mice (h). Both sexes were used in e and h. c, f, i, Bar graphs comparing the social preference index of individual mouse lines. d, g, j, Representative heat maps illustrating the topographical time distribution in social preference tests of individual mouse lines. All data are presented as mean ± S.E.M. For all figures, n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.0001. (Some of the data in this figure were generated by Kajie Ma).

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Chapter V

Published in Genes, Brain and Behavior (2019), 19(1): e12610.

Diminished Social Interaction Incentive Contributes to Social Deficits in Mouse Models of Autism

Spectrum Disorder.

Benjamin Rein, Zhen Yan, Zi-Jun Wang*

Department of Physiology and Biophysics, State University of New York at Buffalo, Jacobs School of

Medicine and Biomedical Sciences, Buffalo, NY 14214, USA

*: Correspondence should be addressed to Z.-J. W. (zijunwan@buffalo.edu)

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ABSTRACT

One of the core symptoms of Autism spectrum disorder (ASD) is impaired social interaction. Currently,

no pharmacotherapies exist for this symptom due to complex biological underpinnings and distinct

genetic models which fail to represent the broad disease spectrum. One convincing hypothesis explaining

social deficits in human ASD patients is amotivation, however it is unknown whether mouse models of

ASD represent this condition. Here we used two highly trusted ASD mouse models (male Shank3

deficient [Shank3+/C] mice modeling the monogenic etiology of ASD, and inbred BTBR mice [both male

and female] modeling the idiopathic and highly polygenic pathology for ASD) to evaluate the level of

motivation to engage in a social interaction. In the behavioral paradigms utilized, a social stimulus was

placed in the open arm of the elevated plus maze (EPM), or the light compartment of the light-dark box

(LDB). To engage in a social interaction, mice were thus required to endure innately aversive conditions

(open areas, height, and/or light). In the modified EPM paradigm, both Shank3+/C and BTBR mice

demonstrated decreased open-arm engagement with a social stimulus but not a novel object, suggesting

that the incentive to engage in a social interaction is reduced in these models. However, these deficits

were not expressed by Shank3+/C or BTBR mice under the less severe aversive pressures of the light-dark

box. Collectively, we show that ASD mouse models exhibit diminished incentive for social interaction,

and provide a new investigation strategy facilitating the study of the neurobiological mechanisms

underlying social reward and motivation deficits in neuropsychiatric disorders.

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INTRODUCTION

Autism spectrum disorder (ASD) is a complex heterogeneous neurodevelopmental disorder

characterized by impaired communication and social interaction, repetitive behaviors, and restricted

interests. ASD is a collection of clinically described disorders that affects 1 in every 59 children in the

United States [216] and presents an urgent public health need, with the annual estimated cost of treatment

and care for individuals with ASD in the US reaching $11.5-60.9 billion[217, 218]. Although social-skill

interventions have been used among Asperger’s Syndrome and high functioning ASD patients [219, 220],

no effective pharmacotherapeutic strategies exist for the social deficits in ASD[221] due to the broad

genetic etiology and resulting limitations of existing animal models.

Strong genetic factors contribute to the etiology of ASD[222]. Large-scale genetic studies have

revealed several susceptibility genes or copy number variations that are highly associated with the

diagnosis of ASD cases, such as SHANK-family genes[169, 223] and 16p11.2 deletions[224]. These

studies promote our understanding of the genetic basis of autism and facilitate the development of animal

models that reflect genetic polymorphisms linked to autism[225, 226]. Many animal models focus on the

monogenic heritable ASD conditions caused by loss-of-function mutations, such as the SHANK3 gene

that encodes a scaffolding protein at glutamatergic synapses[197]. Shank3 haploinsufficiency is one of

the most penetrant monogenic causes of autism[170]. Mice lacking the murine ortholog of the human

SHANK3 gene exhibit selective deficits in social interactions and repetitive behaviors reminiscent of

ASD in humans. In the 3-chamber social preference test, Shank3-deficient (Shank3+/C) mice demonstrate

a significantly lower preference for social stimuli than wild-type (WT) mice[102, 138, 227]. Additionally,

the BTBR T+Itpr3tf/J (BTBR) inbred mouse strain exhibits a variety of behavioral abnormalities that

model ASD symptoms[228, 229], including impaired social behavior and pronounced repetitive

behaviors. Although the genetic background of BTBR mice is complex and poorly understood, BTBR

mice carry single nucleotide polymorphisms in several autism candidate genes[228], and are a trusted

ASD model which imitates the idiopathic and highly polygenic pathology for ASD.

While both Shank3+/C and BTBR mice exhibit decreased social interaction, it remains unclear

whether this is related to changes in the hedonic impact of social reward, or rather impaired incentive

motivation, which has been implicated in rewarding processing[230]. In the current study, we aimed to

assess whether the incentive of social interaction is impaired in these ASD mouse models by merging the

elevated plus maze (EPM) and light-dark box (LDB) behavioral tests with elements of social interaction

tests. Since rodents prefer dark, enclosed areas, the elevated and well-lit open arms of the EPM and the

light-chamber of the LDB represent highly aversive environments for mice. Thus, mice typically exhibit a

strong preference for the enclosed arms in the EPM, and the dark compartment of the LDB[231, 232].

Here, we placed a social stimulus (age- and sex-matched WT mouse) into the aversive component of each

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behavioral paradigm - the open arm of the EPM, or the light chamber of the LDB - to assess whether WT

mice and ASD mouse models were equally motivated to enter the aversive environment in order to

engage in a social interaction. We found that both Shank3+/C and BTBR mice exhibited selective

reductions in social engagement in the modified EPM protocol, suggesting that the widely observed social

deficits in these ASD models are partially mediated by a reduction of social interaction incentive.

Furthermore, these deficits were not expressed in Shank3+/C or BTBR mice in the modified LDB

protocol, which presents a less aversive barrier to interaction, indicating that the relative aversive strength

of the interaction barrier is critical for revealing deficits in social interaction incentive in ASD models.

RESULTS

Shank3+/C and BTBR mouse models of ASD exhibit diminished social interaction incentive in a

modified elevated plus maze protocol.

We first confirmed the genotype of Shank3-deficient (Shank3+/C) mice (Figure 1A). WT mice

showed a PCR product with a size of 399 bp, whereas homozygous Shank3-deficient mice showed a 500

bp PCR product. Both PCR products (399 bp and 500 bp) were present in Shank3+/C mice, and a clear

decrease of Shank3 protein level was found (Figure. 1B, n = 4-5 per group, t(7) = 4.07, p = 0.005, t-test).

In order to evaluate whether the social deficits widely exhibited by the Shank3-deficient (Shank3+/C) and

BTBR mouse models of Autism spectrum disorder (ASD) are related to a reduction in the drive for social

engagement, we designed a modified protocol of the elevated plus maze (EPM). In the original EPM

testing format, a test mouse is placed into the center of the maze, and is allotted five minutes to explore

any of the four arms (Figure 2A, Baseline Trial). Since rodents express an innate preference for dark,

enclosed areas, the two open arms represent an aversive environment for mice. Thus, mice typically

demonstrate a strong preference for the enclosed arms, and the amount of time spent exploring the open

arms is thought to serve as a measure of anxiety-related behavior; specifically, reduced open-arm

exploration time is thought to represent elevated anxiety-like behavior[232]. Since the open arms of the

EPM represent an aversive environment, we wondered if we could gauge an animal’s drive to interact

with an object or social stimulus via their willingness to enter the open arm and endure exposure to an

innately aversive environment. Therefore, animals were tested in two modified formats of the EPM in

which one open arm contained either a novel object (wooden block) (Figure 2B, Object Incentive Trial)

or a social stimulus (age- and sex-matched WT mouse) (Figure 2C, Social Incentive Trial). The opposing

open arm was left empty for each trial, serving as an internal control for the animal’s baseline drive to

enter the open arm.

We first tested Shank3+/C and WT control mice in the three EPM trial types described. In the

Baseline Trial, Shank3+/C did not differ from WT mice in the total amount of time spent exploring the

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open arms (Figure 3A, left, t(23) =0.28, p = 0.78, t-test). Additionally, neither group demonstrated a

preference for either open arm (Figure 3A, right, n = 12-13 per group, F1,46 (interaction) = 0.47, p = 0.50, F1,46

(arm side) = 1.76, p = 0.19, F1,34 (genotype) = 0.11, p = 0.74, two-way ANOVA). To assess the incentive value of a

novel object, WT and Shank3+/C animals were tested in the Object Incentive Trial, in which a novel

object was placed in one open arm. WT and Shank3+/C mice did not differ in the amount of time spent

exploring either the object-containing arm or the empty arm (Figure 3B, n = 12 per group, F1,44 (interaction) =

0.83, p = 0.37, F1,44 (object v empty arm) = 3.06, p = 0.09, F1,34 (genotype) = 1.3, p = 0.26, two-way ANOVA). The

difference between the amount of time spent exploring the object-containing arm relative to the empty

arm (object arm time – empty arm time) also did not differ between WT and Shank3+/C mice (WT: 12.07

4.8 sec, Shank3+/C: 3.8 1.9 sec, t(22) = 1.61, p = 0.12). However, in the Social Incentive Trial, in

which a social stimulus was placed in one open arm of the EPM, WT mice spent significantly more time

exploring the social-stimulus-containing arm than the empty arm, whereas Shank3+/C mice did not exhibit

a preference for the arm containing the social stimulus, and spent significantly less time than WT animals

exploring the social arm (Figure 3C, n = 12 per group, F1,44 (interaction) = 8.30, p = 0.006, F1,44 (social v empty arm) =

5.48, p = 0.02, F1,44 (genotype) = 11.44, p = 0.0015, two-way ANOVA). In addition, the difference between the

amount of time spent exploring the social-stimulus-containing arm relative to the empty arm (social arm

time – empty arm time) was significantly greater for WT animals (WT: 20.62 4.7 sec, Shank3+/C: -2.14

3.5 sec, t(22) = 3.9, p = 0.0008), suggesting that WT animals have a significantly greater preference for

the social-stimulus-containing arm over the empty arm than Shank3+/C mice.

To quantify the animal’s level of motivation to explore the stimulus-containing-arm, we

calculated a “Stimulus Incentive Index” (SII) for each trial which represents the animal’s preference for

the stimulus-containing-arm versus the empty arm (calculated as: [time in stimulus-containing-arm – time

in empty arm] / total time in both open arms). In the Object Incentive Trial, WT and Shank3+/C mice

exhibited a similar and positive SII, indicating that both groups display a preference for the object-

containing-arm, and that the presence of the object enhances the drive for both WT and Shank3+/C mice

to enter that arm of the maze relative to an empty open arm (Figure 3D, n = 12 per group ). However in

the Social Incentive Trial, Shank3+/C mice demonstrated a negative SII that was significantly lower than

that of WT mice, suggesting that the incentive to engage in a social interaction is significantly reduced in

Shank3+/C mice (Figure 3D, n = 12 per group, F1,44 (interaction) = 15.15, p = 0.0003, F1,44 (trial type) = 3.86, p =

0.06, F1,44 (genotype) = 12.14, p = 0.0011, two-way ANOVA). Notably, Shank3+/C mice demonstrated a

significantly lower SII in the Social Incentive Trial than in the Object Incentive Trial, indicating that the

incentive to interact with a novel object exceeds the incentive to interact with a social stimulus in

Shank3+/C mice. Collectively, these data indicate that the aversive value presented by the open arm of the

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EPM is sufficient to deter Shank3+/C mice, but not WT mice, from pursuing a social interaction, and that

the incentive of social engagement is diminished in Shank3+/C mice.

To determine whether the modified EPM paradigm used here was sensitive to social incentive

deficits in other ASD models, we next attempted to replicate these findings in BTBR mice. Since ASD-

like deficits in BTBR mice are caused by an unclear genetic background and BTBR mice do not have a

distinct wild-type counterpart, experiments with BTBR mice were conducted without WT controls, and

all statistical comparisons were performed between open-arm side and trial type. BTBR mice did not

show any preference for either open arm of the EPM in the Baseline Trial (Figure 4A, n = 10 per group,

t(9) = 0.53, p = 0.61, paired t-test). In the Object Incentive Trial, BTBR mice demonstrated a significant

preference for the object-containing-arm (Figure 4B, n = 10 per group, t(9) = 2.94, p = 0.017, paired t-

test), however, BTBR mice did not show a preference for either arm in the Social Incentive Trial (Figure

4C, n = 10 per group, t(9) = 0.35, p = 0.73, paired t-test). Correspondingly, the SII was significantly lower

in the Social Incentive Trial than in the Object Incentive Trial (Figure 4D, n = 10 per group, t(9) = 3.95, p

= 0.003, paired t-test), indicating that the incentive to engage in a social interaction is reduced in BTBR

mice.

Diminished social interaction incentive in ASD models is not revealed under less aversive barrier

conditions.

We next questioned whether the diminished incentive for social interaction in Shank3+/C and

BTBR mice was selectively evoked by the aversive pressures of the modified EPM paradigm, or if this

deficit was pervasive and present under all conditions. To determine if the deficit was dependent upon the

severity of the aversive pressure imposed by the barrier to interaction (i.e. open space/bright

light/elevation in the open arm of the EPM), we designed a modified light-dark box (LDB) paradigm

which paralleled the modified EPM protocol. The LDB test measures anxiety-like behavior by gauging

the animal’s propensity to explore a brightly illuminated – and thus aversive - chamber relative to a dark

compartment[231]. In the modified LDB test used here, the animal’s intent to enter the aversive, brightly-

lit light chamber of the light-dark box was used to measure the drive to interact with an object or social

stimulus. Like the open arm of the EPM, the light chamber of the LDB is well-lit, however the light

chamber of the LDB is not elevated and is enclosed by transparent walls, both of which are in contrast to

the open arm of the EPM. Therefore, the light chamber lacks two key aversive features presented by the

open arm (elevation and exposure), and thus presents a less aversive barrier to interaction relative to the

EPM paradigm. In the three trial types used here, the light chamber contained either nothing (Figure 5A,

Baseline Trial), a wooden block under an inverted pencil cup (Figure 5B, Object Incentive Trial), or a

social stimulus under an inverted pencil cup (Figure 5C, Social Incentive Trial), and the amount of time

spent exploring the light and dark chambers was measured.

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We first compared WT and Shank3+/C mice in the modified LDB paradigm. In both the Object-

and Social Incentive Trials, WT and Shank3+/C mice spent significantly more time in the light box (and

less time in the dark box) than in the Baseline Trial, however WT and Shank3+/C mice did not differ in

the amount of time spent in the light box or dark box during any of the three trials (Figure 6A, n = 10-11

per group, F1,114 (genotype) = 0.0, p > 0.999, F2,114 (trial type) = 0.0, p > 0.999, F1,114 (box side) = 4.03, p = 0.047, three-

way ANOVA). Additionally, the “Stimulus Enhancement Index” (SEI) – which, due to the lack of an

internal control such as the empty arm in EPM, was calculated differently from the Stimulus Incentive

Index used prior (calculated as: [time in light-box during given trial type – time in light-box during

baseline trial] / time in light-box in baseline trial) did not differ between WT and Shank3+/C mice in either

the Object- or Social Incentive Trial (Figure 6B, n = 10-11 per group, F1,38 (genotype) = 0.53, p = 0.47, F1,38 (trial

type) = 1.86, p = 0.18, two-way ANOVA), indicating that the reduced incentive of social engagement in

Shank3+/C mice is masked under less severe aversive conditions.

We next tested BTBR mice in the modified LDB paradigm. BTBR mice spent significantly more

time exploring the light box in both the Object- and Social Incentive Trials than in the Baseline Trial

(Figure 7A, n = 10 per group, F2,18 (interaction) = 22.52, p < 0.0001, F2,18 (trial type) = 1.0, p = 0.39, F1,9 (box side) =

9.23, p = 0.01, two-way ANOVA). Additionally, the SEI for the Object- and Social Incentive Trials did

not differ in BTBR mice (Figure 7B, n = 10 per group, t(9) = 0.05, p = 0.96), indicating that BTBR mice

were similarly driven by both an object and a social stimulus to explore the light chamber of the LDB.

Collectively, these data indicate that the incentive to engage in a social interaction in Shank3+/C

and BTBR mice is selectively reduced in the modified EPM paradigm, but not in the modified LDB

paradigm, suggesting that the expression of this behavioral phenotype in these ASD models is specific to

the aversive pressures presented by the open arm of the EPM (bright light, open spaces, elevation) and

that the deterrent pressure imposed by the more mild aversive environment of the light chamber of the

LDB is insufficient to dissuade Shank3+/C or BTBR mice from exploring a compartment containing a

social stimulus.

DISCUSSION

Social deficits in Shank3+/C [102, 138, 227] and BTBR[228, 233, 234] mice have been reported

by our lab and other labs, indicated by a reduced preference for a social stimulus over a novel object in

the 3-chamber social preference test. In order to determine whether reduced incentive of social

engagement may contribute to the observed social deficits, we designed a new behavioral paradigm to

quantitate a Social Incentive Index. Through the use of this novel behavioral model, we determined that

both Shank3+/C and BTBR mice exhibit diminished motivational intent to interact with social stimuli,

which may be an underlying factor contributing to the widely observed social deficits.

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One way to evaluate the motivational value of a reward is to increase the effort required to obtain

the reinforcer[235, 236]. The progressive ratio operant task is a well-recognized behavioral paradigm

which has been widely used to evaluate motivation for reinforcers like sucrose, food, and addictive

drugs[235]. However, this paradigm requires long-term training and the success rate of learning the

paradigm for mice with social deficits (such as BTBR mice) is only 50%[237], which may be due to

learning impairments in the context of social reward[237]. Conditioned place preference (CPP) is another

widely-used method of measuring motivation. However, socially-deficient mice (BTBR[238]) are

likewise unable to develop CPP, which may also be due to an impaired learning capacity[239]. In our

paradigm, we used a simplified strategy in which the level of difficulty of obtaining a social reward was

increased. Since rodents express an innate preference for enclosed and dark spaces, they typically avoid

aversive environments such as the open arm of elevated plus maze and the light compartment of the light-

dark box. When a novel object or social stimulus is introduced to such a non-preferred environment, mice

must actively overcome the aversive pressures to engage in a play- or social interaction. In the baseline

EPM protocol, Shank3+/C mice showed no difference from WT mice, indicating that Shank3+/C mice do

not express any anxiety phenotype which could alter the Social Incentive Index. Furthermore, Shank3 +/C

and WT mice expressed comparable open-arm engagement time with the novel object in the Object

Incentive Trial, suggesting that Shank3+/C mice do not exhibit general incentivization deficits, but rather

these deficits are specific to social reward. Future studies should focus on whether Shank3 +/C mice also

express reward processing deficits for other rewards such as food, water and drugs of abuse.

Our data also indicate diminished incentive for social interaction in BTBR mice. Unlike WT and

Shank3+/C mice, BTBR mice showed a notably strong preference for the novel object, indicating

increased novelty reward valence in BTBR mice which may correspond to the elevated interest in

restricted domains in human ASD patients[240]. Other studies have indicated decreased motivation for

other rewards such as food in BTBR mice[237], suggesting a generalized decrease of motivation in

BTBR mice. This discrepancy may be due to the complexity of test protocols and the impaired learning

ability of the tested mice.

One may speculate that the reduction of social interaction incentive in Shank3+/C and BTBR mice

in the modified EPM paradigm may be driven by social anxiety (or social aversion/avoidance). Following

this explanation, the observed avoidance of social cues should be unaffected by altering the level of

environmental aversiveness. In contrast, WT and Shank3+/C mice displayed a similar Social Enhancement

Index in the Social Incentive Trial of the LDB, suggesting that Shank3+/C mice do not exhibit avoidance

(or aversion) of social conspecifics under milder environmental conditions, and that the observed deficits

are dependent upon specific aversive pressures and not resulting from intrinsic social aversion. We also

hypothesize that in order to express the social incentive deficits, the aversive pressures imposed by the

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barrier to interaction must reach a certain threshold of severity which is sufficient to deter animals with

reduced incentive of social engagement. Relative to the open arm of the EPM, the light compartment of

the LDB represents a more mildly aversive environment, presenting light as the lone aversive stimulus, in

contrast to the EPM open arm which is well-lit, elevated and exposed (without walls). We thus speculate

that the aversive pressure imposed by light exposure alone in light-dark box is insufficient to suppress the

drive for social reward in Shank3+/C and BTBR mice. There is an apparent contradiction as Shank3+/C

mice display social withdrawal symptoms in the social approach test[138] where a social stimulus is

presented in the middle of open chamber similar to the light compartment of light-dark box, while this

deficit is not shown here. This may be due to the fact that, unlike the LDB test, during the habituation

phase of social approach test animals become very familiar with the open arena to avoid open space

induced anxiety. Additionally, the social approach test quantifies the amount of time the test mouse

spends directly interacting with the social cue, whereas here we quantified the amount of time spent in the

chamber with the social cue.

In the modified EPM protocols, Shank3+/C and BTBR mice displayed a significantly lower SII in

the Social Incentive Trial than the Object Incentive Trial, suggesting that in these ASD models, the

incentive to engage in an interaction with a novel object may exceed that of a social stimulus. However,

the SII for WT animals did not differ between the Object- and Social- Incentive Trials, which appears in

contrast to 3-chamber social preference findings in which WT animals routinely demonstrate a significant

preference for the social stimulus over a novel object[102, 138]. We theorize that the lack of difference

between the WT object- and social-stimulus incentive indexes is due to differences in testing parameters

between the traditional 3-chamber paradigm and the EPM models used here. Principally, the 3-chamber

paradigm exposes the test mouse to a social stimulus and a novel object simultaneously, forcing the test

animal to actively choose to interact with one stimulus over the other, which typically results in a time

distribution favoring the social stimulus over the object. Here, WT animals were exposed to novel- and

social-stimuli at different times, precluding the need for the animal to establish preference of one stimulus

over the other.

The biological mechanisms involved in social motivation rely on brain circuitry including the

amygdala, ventral striatum, orbital and ventromedial prefrontal cortex[241], along with mesolimbic

reward circuitry comprised of the nucleus accumbens and ventral tegmental area[242]. Synaptic

dysfunction has been reported in the PFC[102, 138] and VTA[243] of Shank3+/C mice, which may

compromise social reward processing. In BTBR mice, changes in the shape and localization of many

brain structures, including the hippocampus and amygdala[244] may also diminish the regulation of

social motivation.

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The molecular mechanisms underlying social motivation are closely related to the neuropipetide

oxytocin and its interaction with dopamine[245, 246]. Dopamine in the mesocorticolimbic system

influences the assignment of motivational salience by impacting the drive toward such rewards, without

affecting the pleasure derived from the reward itself[247, 248]. Oxytocin, which shares receptor

localization sites with the mesocorticolimbic dopamine system, acts with dopamine to specifically

increase the salience of social stimuli[245, 249, 250]. Oxytocin treatment has been shown in clinical

studies to improve social behavior in ASD patients[251, 252], and in preclinical studies to alleviate the

social deficits in Shank3+/C [253] and BTBR mice[254]. Therefore, the social motivation deficits

observed here in these mouse models may be related to oxytocin and the dopamine system. Future studies

could focus on examining this relationship.

In conclusion, we have used a simplified behavioral approach to assess the incentive of a social

interaction in ASD mouse models (Shank3+/C and BTBR), identifying diminished social interaction

incentive in both models, and providing a new strategy to facilitate the investigation of neurobiological

mechanisms for social reward and motivation deficits underlying neuropsychiatric disorders.

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Figure 1. Genotype confirmation for Shank3+/C mice. (A) Representative genotyping results (PCR) for wild-type (WT), homozygous Shank3-deficient (Shank3C/C) and heterozygous Shank3-deficient (Shank3+/C) mice. (B) Representative western blot showing Shank3 protein level in total protein lysate from prefrontal cortex of WT and Shank3+/C mice. (The data presented in this figure were generated by Dr. Zijun Wang).

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Figure 2. Graphic illustrating the three elevated plus maze (EPM) protocols used in the current study. (A) Baseline Trial, in which both open arms are left empty. (B) Object Incentive Trial, in which a novel object (wooden block) is placed under an inverted pencil cup in one open arm of the EPM. ( C) Social Incentive Trial, in which a social stimulus (age- and sex-matched WT mouse) is placed under an inverted pencil cup in one open arm of the EPM.

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Figure 3. Shank3+/C mice exhibit diminished social interaction incentive in a modified elevated plus maze (EPM) protocol. (A) Bar graph (mean SEM) showing the total amount of time wild type (WT) and Shank3 deficient (Shank3+/C) mice spent exploring both open arms of the EPM (left), and the amount of time spent exploring each individual arm (right) in the Baseline Trial. (B) Bar graph (mean SEM) showing the amount of time WT and Shank3+/C mice spent exploring the object-containing arm and the empty arm in the Object Incentive Trial. (C) Bar graph (mean SEM) showing the amount of time WT and Shank3+/C mice spent exploring the social-stimulus-containing arm and empty arm in the Social Incentive Trial. *p < 0.05, two-way ANOVA post hoc t-test. (D) Bar graph (mean SEM) showing the Stimulus Incentive Index for WT and Shank3+/C mice in the Object Incentive Trial (left) and the Social Incentive Trial (right). ***p < 0.0001, **p < 0.01, two-way ANOVA post hoc t-test.

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Figure 4. BTBR mice exhibit diminished social interaction incentive in a modified elevated plus maze (EPM) protocol. (A) Bar graph (mean SEM) showing the total amount of time BTBR mice spent exploring both open arms of the EPM (left), and the amount of time spent exploring each individual arm (right) in the Baseline Trial. (B) Bar graph (mean SEM) showing the amount of time BTBR mice spent exploring the object-containing arm and the empty arm in the Object Incentive Trial. *p < 0.05, t-test. (C) Bar graph (mean SEM) showing the amount of time BTBR mice spent exploring the social-stimulus-containing arm and empty arm in the Social Incentive Trial. (D) Bar graph (mean SEM) showing the Stimulus Incentive Index for BTBR mice in the Object Incentive Trial (left) and the Social Incentive Trial (right). **p < 0.01, t-test.

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Figure 5. Graphic illustrating the three light-dark box protocols used in the current study. (A) Baseline Trial, in which the light chamber is left empty. (B) Object Incentive Trial, in which a novel object (wooden block) is placed under an inverted pencil cup in the light chamber. (C) Social Incentive Trial, in which a social stimulus (age- and sex-matched WT mouse) is placed under an inverted pencil cup in the light chamber.

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Figure 6. Shank3+/C mice do not exhibit reduced social interaction incentive when presented with a less aversive barrier to interaction. (A) Bar graph (mean SEM) showing the amount of time WT and Shank3+/C mice spent exploring the light and dark compartments of the light-dark box in the Baseline Trial (left), in the Object Incentive Trial (middle), and in the Social Incentive Trial (right). *: indicates statistical difference from time in light-box during the baseline trial for each respective group. #: indicates statistical difference from time in dark-box during the baseline trial for each respective group. ***p < 0.0001, ###p < 0.0001, three-way ANOVA post hoc t-tests. (B) Bar graph (mean SEM) showing the Stimulus Enhancement Index for WT and Shank3+/C mice in the Object Incentive Trial (left) and Social Incentive Trial (right).

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Figure 7. BTBR mice do not exhibit reduced social interaction incentive when presented with a less aversive barrier to interaction. (A) Bar graph (mean SEM) showing the amount of time BTBR mice spent exploring the light and dark chambers of the light-dark box in the Baseline Trial (left), in the Object Incentive Trial (middle), and in the Social Incentive Trial (right). *: indicates statistical difference from time in light-box during the baseline trial. #: indicates statistical difference from time in dark-box during the baseline trial. **p < 0.001, ##p < 0.001, two-way ANOVA post hoc t-tests. (C) Bar graph (mean SEM) showing the Stimulus Enhancement Index for BTBR mice in the Object Incentive Trial (left) and Social Incentive Trial (right).

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Chapter VI:

Discussion and Conclusions

Copy number variations (CNVs) of the 16p11.2 gene locus are associated with a range of clinical

presentations and developmental trajectories, with differing symptomatologies and severities across those

afflicted. The diverse outcomes observed in human patients with 16p11.2 CNVs may represent an

intricate molecular profile driven by the tandem deletion or duplication of 22-27 genes. Due to the

complexity of these downstream transcriptional and cellular consequences, it remains challenging to

comprehensively evaluate the systems affected and their relative contributions in producing the observed

symptoms. There is thus great value in the identification of common-ground phenotypes and molecular

pathologies that are consistent across patients with either 16p11.2 deletions or duplications, especially

those that may be therapeutically targetable. The work presented in this thesis has effectively identified

core synaptic dysfunctions in mouse models of both 16p11.2 deletions and duplications which, when

corrected, are capable of reversing the observed impairments in sociability and cognition. This has

produced putative therapeutic targets for the social and cognitive deficits in both 16p11.2 deletions and

duplications, and thus may offer translational value for the development of drug-based therapies for

16p11.2 CNVs.

In Part I of this thesis, we identified behavioral abnormalities in 16p11.2 deletion and

duplication mice that correspond with the human clinical features of these copy number variations.

Specifically, 16p11.2 deletion mice display social and cognitive deficits, while duplication mice display

social and cognitive deficits, hypolocomotion, and enhanced self-grooming. Interestingly, these

phenotypes appear to be relatively consistent across each mouse, though some variability is certainly

observed. This is in contrast to the scattered symptomatologies reported in human 16p11.2 CNV carriers.

The reasoning behind this is unclear, though it is possible that the mouse models express a genetically

well-controlled and consistent variant of the 16p11.2 locus, whereas human patients may express a partial

deletion or duplication of the 16p11.2 region which only affects a portion of the 29 total genes.

We also report synaptic pathologies in the prefrontal cortex (PFC) of 16p11.2 deletion and

duplication mouse models, which can be targeted to effectively restore social and cognitive function.

Interestingly, restoring these synaptic functions exclusively in the PFC is sufficient to reverse the

behavioral deficits in both models, suggesting that PFC plays a critical role in 16p11.2 pathology. While

the PFC is highly important in social cognition and higher-level executive functions [109, 255], it is

unlikely that local PFC dysfunction alone is responsible for the observed behavioral phenotypes. Rather,

it is proposed that abnormal excitability in medial PFC disrupts network activity affecting other brain

regions, including other cortical regions and limbic structures. In support of this, an external study of the

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16p11.2 duplication model reports disrupted hippocampal-orbitofrontal-amygdala connectivity [73].

Future studies should focus on characterizing the electrophysiological profiles of other brain regions in

16p11.2 deletions and duplications, to inform a more clear and comprehensive understanding of the brain-

wide synaptic consequences.

The direct link between the genes in the 16p11.2 locus and the observed synaptic pathologies

remains unclear in both models. It is very interesting that NMDAR-mediated synaptic transmission is

impaired in 16p11.2 deletion PFC, but intact in the duplication model. This suggests that deletion - but

not duplication - of the 16p11.2 region selectively impairs a cellular process resulting in NMDAR

dysfunction. Conversely, GABAergic deficits are selectively observed in 16p11.2 duplication mice. It is

unclear which of the genes in the 16p11.2 region may be responsible for these downstream effects,

though there are several suspected genes: Taok2 has been shown to mediate dendrite formation and

maturation, as well as PSD95 stability [91, 256], so its downregulation may result in the de-stabilization

of glutamatergic synapses in 16p11.2 deletion mice. Mapk3, which plays a major role in neuronal

proliferation and synapse formation, has been implicated as a network hub in 16p11.2 deletion neurons

[81]. Presynaptic dysfunction could be driven by altered expression of the activity-dependent presynaptic

vesicle proteins Doc2a [257, 258], or Prrt2 [259]. The 16p11.2 region also contains the transcription

factors Tbx6 and Maz, which could evoke downstream transcriptional effects leading to synaptic

dysfunction. The exact role of these genes in producing the observed phenotypes remains to be

investigated.

In Part II of this thesis, we evaluated and improved the efficacy of an existing behavioral assay

for measuring sociability in mice, and produced an entirely novel approach. The three-chamber social

preference test, originally proposed by Dr. Jacqueline Crawley [184], assesses the mouse’s preference for

a social stimulus versus a non-social stimulus. This test has been used for many years and by many

researchers, though over time it has been subjected to numerous modifications which have resulted in an

array of differing protocols used across the literature with inconsistent findings. Additionally, this

protocol contains several conceptual imperfections which we believed may impact the efficacy of the test.

In order to address these issues, we designed a standardized protocol with several modifications that

improve the accuracy of the testing measures. We demonstrate that our protocol displays heightened

sensitivity to social deficits in several transgenic mouse models of ASD relative to the original protocol,

while remaining sensitive to sex-specific changes in sociability. We hope that this protocol will enable the

autism research field to produce more consistent and comparable findings across laboratories.

Aside from the three-chamber social preference test, there are several other methods of assessing

sociability in mice, though none of these behavioral assays evaluate the influence and contribution of

social motivation. Intuitively, there is a motivational component underlying the desire for social

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engagement, as social interactions can be rewarding. We hypothesized that this social motivation may be

impaired in mouse models of ASD, and sought to design a paradigm to assess it. In order to accomplish

this, we combined components of the elevated plus maze (EPM) – a behavioral test typically used to

assess anxiety [260] – and social approach tests. The EPM is a plus-sign-shaped maze that contains two

open arms, and two arms enclosed by walls. The open arms represent an aversive environment, as mice

prefer enclosed and dark areas. Thus, in order to test the mouse’s motivation to engage in a social

interaction, we placed an unfamiliar wild-type (WT) mouse under a cup in one of the empty arms, to

determine whether the mouse would be willing to enter an aversive environment in order to engage in a

social interaction. Indeed, we found that WT mice spent significantly more time in the open arm when it

contained either a social stimulus or an object. Interestingly, we observed that two mouse models of ASD

similarly spent more time in the open arm when it contained an object, but not when it contained a social

stimulus. This suggests that the incentive value of a social interaction may be reduced in transgenic ASD

models, while the incentive value of interacting with a play object is not.

This behavioral paradigm may be similarly applicable for the evaluation of anhedonia or social

withdrawal in mouse models of schizophrenia, or in other models of affective disorders. We hope that this

assay will meaningfully expand the repertoire of behavioral tests available to the field, and enable more

in-depth assessments of social behavior by stratifying this broad topic into multiple components,

including social preference, social motivation, and others.

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Chapter VII:

Materials and Methods

Animals

All animals were maintained on a 12-hour light (6:00 am – 6:00 pm)/dark (6:00 pm – 6:00 am)

cycle. All animals were group-housed with 1-3 gender-matched conspecifics and provided standard

enrichment. Researchers were blind to genotypes during all behavioral experiments. All animal studies

were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the

State University of New York at Buffalo.

Mice expressing C-terminal (exon 21)-deleted Shank3 (Shank3 tm1.1Pfw/J) with significant loss of

full-length Shank3 expression were purchased from the Jackson Laboratory (Bar Harbor, ME, USA,

Stock #018398). These mice were backcrossed at least 5 generations to C57BL/6J mice at the Jackson

Laboratory. Upon arrival at our laboratory, these mice were backcrossed 3 more generations to C57BL/6J

mice (Jackson Laboratory, Stock #: 000664) before any experimental use. All subsequent breeding,

genotyping, and colony maintenance was performed in-house as previously described[110]. Heterozygous

Shank3+/C mice were crossed to produce litters containing WT, heterozygous Shank3+/C, and

homozygous Shank3+/C offspring, which were identified via in-house genotyping. Genotyping of these

mice was determined by Polymerase Chain Reaction (PCR) on their tail genomic DNA. The primers used

in genotyping were the wild-type reverse primer (5’- ATG TCC TGT TGC AGG TAG GG -3’), the

common forward primer (5’- GTG TCC CCT CAT TGA TGT TG -3’), and the mutant reverse primer

(5’- CTC TGC CAC CTT CTG CCT AC-3’). Only heterozygous Shank3+/C mice (6-8 weeks old, male)

and age-matched WT littermates (male) were used in this study. Female Shank3+/C mice lack autism-like

social deficits, so they (along with female WT animals) were not used. BTBR T+Itpr3tf/J (BTBR) mice

were obtained from The Jackson Laboratory (Stock #: 002282) and bred in house. Both male and female

BTBR mice (6-8 weeks old) were used for all experiments described. Mice of all genotypes were group-

housed (2-4 per cage) with littermates of the same gender. Shank3+/C mice were housed with littermates

of any genotype (WT or Shank3+/C); BTBR mice were housed only with BTBR littermates.

Both male and female WT (C57BL/6J) and dopamine D4 receptor knockout mice (D4KO) were

used in the current study. All behavioral testing was performed at 6-8 weeks old. WT littermates were

used as controls.

16p11.2dp/+ mice carrying a heterozygous duplication of the 7F3 chromosomal region homologous

to human 16p11.2 were initially generated on a hybrid C57BL/6N:129Sv background before being

backcrossed 10 generations onto the C57BL/6 inbred strain and subsequently maintained on the C57BL/6

genetic background. Thus, the WT and 16p11.2dp/+ animals used in the current study represent a pure

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C57BL/6 background. For all experiments, wild-type (WT) littermates were used as controls. 7-9-week-

old (both male and female) WT and 16p11.2dp/+ mice were used for all experiments. Similar results were

obtained from 16p11.2dp/+ mice of either gender, so results were pooled together.

16p11.2 deletion mice (Jackson Labs, Bar Harbor, ME) with one copy [heterozygous for a

deletion or deficiency allele] of the chromosomal region corresponding to 16p11.2 were generated using

chromosome engineering. Wild-type littermates (C57BL/6N129Sv) were used as controls. Both male and

female 16p11.2 deletion/WT mice (7-8 weeks old) were used.

Cul3flox/flox mice from Jackson Laboratory (stock #: 028349) were bred with Emx1-IRES-Cre mice

(stock #: 005628) to generate forebrain-specific Cul3 knockout mice (Cul3f/-). Both male and female

Cul3f/- and Cul3f/f mice were used in this study. All behavioral testing was performed at 6-8 weeks old.

Behavioral Techniques

For all behavioral testing, the animal was habituated to the testing room for at least 30 minutes

prior to testing. All behavioral testing was performed on at least 2-3 independent litters. If multiple

behavioral tests were performed, the sequence in which the animals were exposed to each test was

randomized between litters, and tests were performed 24 hours apart, with one test performed per day.

Sample sizes were determined based on power analyses. All behavioral testing apparatuses were wiped

down with 75% ethanol between animals and trials. All scoring was performed manually by a researcher

blind to both genotype and treatment. All testing (unless otherwise indicated) was performed in a dimly lit

room. All animals were tested at the same time of the day. The Anymaze behavior tracking software

(Stoelting, Wood Dale, IL) was used to record footage of behavior tests and generate heat maps.

Barnes Maze

The test animal is first habituated to the platform with no escape box attached for 5 minutes, then

returned to its home cage for 3 minutes, and the platform is cleaned. The mouse is then returned to the

platform for a 3-minute trial (T1), where it is allowed to locate and enter the escape box. If the mouse

does not find the box within 3 minutes, it should be gently guided toward the box. When the mouse

locates and enters the box, a cardboard cover is placed over it, and the animal is allowed to rest in the

dark for about one minute before being removed from the box. The mouse is then returned to the home

cage for another 3 minutes. This is followed by an identical trial (3 minutes – T2). For both T1 and T2, be

sure to use the same box position. Return the animal to the home cage for a 15-minute break. After the

15-minute break, return the mouse to the platform for a 5-minute trial with no escape box, and record the

amount of time spent exploring the correct hole, and the incorrect holes. Then calculate the Spatial

Memory Index as (time exploring correct hole – time exploring incorrect holes)/(total time exploring).

Self-Grooming

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Place the test mouse into a clean, lidded cage (empty besides from corn bedding) for 30 minutes.

During the first 20 minutes, the animal is allowed to habituate to the environment. After 20 minutes,

begin recording video of the animal for 10 minutes. Footage is best taken from the side, where the animal

is clearly visible. Manually score the amount of time spent grooming. Be sure to use a new cage for each

animal. For scoring purposes, self-grooming behavior was defined as any repetitive grooming motion (i.e.

licking, brushing) directed at the front paws, head/ears, or abdomen.

Novel Object Recognition

The test mouse is placed on a circular open white platform (2-foot diameter) for 5 minutes for

habituation, then returned to its home cage. The mouse is then placed on the platform with two identical

objects for 5 minutes, then returned to its home cage again. The mouse is then allowed to explore the

platform containing one of the original objects (familiar) and a new object (novel) for 5-min, during

which the amount of time spent interacting with each object is manually scored. Discrimination ratio is

calculated as: (novel-object time – familiar-object time) / (novel-object time + familiar-object time).

Forced Swim Test

The test mouse is placed into a 5L beaker containing 3L of water at about 23-25 degrees Celsius.

The animal is placed in the water slowly, by its tail. The test mouse is then left in the beaker for 6

minutes. The first two minutes are not measured, as the animal is likely to swim for the full duration.

During the final four minutes, the amount of time spent mobile is measured (this should be easier to

record and more consistent than immobile time). The amount of time spent immobile is reported, as this

variable correlates directly with perceived level of depression.

Startle Response/Pre-Pulse Inhibition (PPI)

The acoustic startle response test was performed in an SR-LAB startle chamber (San Diego

Instruments, San Diego, CA). The test consisted of three trial types: null trials with only a white noise

background (66 dB), pulse-only trials with 40 ms white noise stimulus (90, 100, 110, and 120 dB), and

pre-pulse trials, where the 120 dB startle stimulus was preceded 100 ms earlier by a 20 ms pre-pulse

stimulus that was either 70, 76, or 85 dB. Mice were placed into the plexiglass holder and habituated to

the apparatus for 5 minutes before testing. Each test consisted of 74 trials with 30 pulse-only trials, 11

null trials, and 33 pre-pulse trials. Trial order was the same for all animals, with 10 pulse-only trials

followed by combinations of the pre-pulse and null trials, then terminating with 10 pulse-only trials. The

maximum startle response was measured for each trial. Pre-pulse inhibition (PPI, percentage) = (1 – pre-

pulse trial response / pulse-only trial response) x 100.

Elevated Plus Maze

The test mouse was placed into the center (facing into a closed arm) of a plus-shaped apparatus

with four arms (3” wide x 15.5” long), two of which were enclosed with 11” tall opaque walls, while the

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two open arms did not have walls. Testing was conducted in a brightly lit room, with the open arms

illuminated by overhead lighting (200 lux) and the closed arms/center shielded from light by upright walls

(3 lux), consistent with suggested lighting levels for the elevated plus maze[232, 260].

For the modified protocols included in this study (Object Incentive Trial/Social Incentive Trial),

either a novel object (wooden block) or a social stimulus (randomly selected, unfamiliar age- and sex-

matched WT mouse) was placed in one open arm of the maze under an inverted pencil cup with a water

bottle placed on top to prevent the test mouse from climbing the cup. The other arm was kept empty for

each trial to serve as an internal control sensitive to changes in the animal’s baseline open arm entry. The

open arm in which the stimulus was placed was alternated between tests to control for the potential

preference for either open arm. For each trial type, the test animal was allowed to explore for 5 minutes,

and the amount of time spent in each open arm was manually quantified by a genotype-blind researcher.

For scoring purposes, animals were considered to be in the open arm when all four paws were touching

the floor of that arm. Each test animal was exposed to all three trial types described (Baseline, Object-,

and Social Incentive Trials) on three separate days, with a 24-hour interval. Animals were first tested in

the Baseline Trial, and the order of the Object and Social Incentive trials was counterbalanced over the

subsequent two days in order to avoid practice effects.

Temporal Order Recognition

The test mouse was first placed into a rectangular enclosure (L: 48.2 cm, W: 38.1 cm, H: 25.4

cm) with opaque walls for 3 min for habituation, then returned to its home cage for 5 min. The mouse was

then returned and allowed to interact with two copies of the same object (object 1) placed in opposing

corners of the enclosure for 4 min. After a 1-hour break, the mouse was exposed to a second pair of

identical objects (object 2) for 4 min. After a three-hour break, mice were then exposed to one copy of

each object for 5 min. The location and identity of the objects used were counterbalanced across animals

to avoid object-driven effects. The amount of time spent investigating each object was recorded and used

to calculate a discrimination ratio: [novel object time (object 1) – familiar object time (object 2)/total time

investigating both objects].

3-Chamber Social Preference Test

The test mouse was first placed into a Plexiglass arena (L: 101.6 cm, W: 50.8 cm, H: 50.8cm)

containing two empty inverted pencil cups for a 10-minute habituation period. On the following day, the

mouse was reintroduced to the apparatus for a 10-minute trial in which the pencil cups contained two

identical objects. The animal was then returned to its home cage for five minutes. The animal was then

placed into the apparatus for a 10-minute trial (social preference test), in which one cup contained a novel

object (non-social stimulus) while the other contained an age- and gender-matched WT mouse (social

stimulus). The amount of time spent interacting with each stimulus was recorded. The preference index

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was calculated as (social time – non-social time) / (social time + non-social time). Five minutes later, the

animal was tested in a final 10-minute trial, in which the non-social stimulus was replaced by a novel age-

and gender-matched WT mouse (novel social stimulus), while the other mouse from the previous trial

remained (old social stimulus). The amount of time spent interacting with each stimulus was recorded.

The preference index was calculated as (novel-social time – old-social time) / (novel-social time + old-

social time).

MK-801 Induced Hyperlocomotion

Locomotor activity was measured using a transparent plastic cage (40 x 40 x 30 cm) containing a

thin layer of corncob bedding and equipped with movement-detecting photocell beams. The test mouse

was placed into the cage for 30 minutes to measure baseline locomotive activity. After 30 minutes, the

animal was removed from the cage and given a saline injection (i.p.), to serve as a control for any

behavioral response invoked by the injection stimulus. The mouse was promptly returned to the cage and

locomotion was recorded for another 30 minutes. The test mouse was then removed again and

administered MK-801 (2 mg/kg, i.p.), and promptly returned to the cage for another 90-minute test

session. The total distance travelled was measured in 5-minute intervals by the Versa Max animal activity

monitoring software. Data collection was stopped during injection administration, and resumed upon the

mouse’s return to the cage.

Marble Burying Test

Twenty glass marbles were evenly spaced in five rows of four on a sawdust bedding in an

otherwise empty animal housing cage. The test mouse was placed into the cage for 30 minutes, and the

number of marbles buried (to 2/3 depth) was measured.

Social Withdrawal Test

The test animal was habituated in a rectangular apparatus (L: 67.7 cm, W: 50.8 cm, H: 50.8 cm)

containing a capsule (inverted pencil cup, placed in the center) for 10 minutes, then returned to its home

cage. A social stimulus (an age- and sex-matched mouse) was then placed inside the capsule. The test

animal was placed into the apparatus to explore for 10 minutes. The amount of time the test animal spent

interacting with the social stimulus was measured.

Open Field Test

The test mouse was placed into an empty Plexiglass arena (L: 101.6 cm, W: 50.8 cm, H: 50.8cm)

for 30 minutes. Anymaze behavior tracking software was used to measure total distance travelled and

time in center. The number of fecal boli produced by the animal during the testing period was manually

counted by the researcher.

Rota-Rod

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The test mouse was placed onto an accelerating rotarod (SD Instruments, San Diego, CA), which

slowly accelerated from 4 to 40 revolutions per minute over a 5-min test session. The mouse was required

to walk forward in order to remain on top of the rotating cylinder rod. After a practice trial in which the

mouse was allowed to familiarize with the apparatus, two test trials were conducted. For each trial, the

amount of time that the mouse remained on the rotarod before falling off was recorded (latency to fall).

Values presented for each mouse represent the average latency to fall between the two test trials.

Light/Dark Box

The light-dark box used in the current study is comprised of two 13.75”x10.375”x13.5”

compartments joined by a 3”x5” doorway. The walls of one compartment (the light box) are made of

transparent glass to allow the area to be illuminated by overhead lighting (200 lux), while the walls of the

other compartment (dark box) are opaque black to prevent the transmission of light and ensure darkness

within the compartment (3 lux). The lighting levels used here are consistent with reported experimental

guidelines[261]. The test animal was placed into the dark box and allowed 5 minutes to explore the

apparatus. The amount of time spent exploring the light box (defined as all four paws inside the light box)

was manually quantified by a genotype-blind researcher. In the modified Object Incentive and Social

Incentive Trials, either a novel object (wooden block) or a social stimulus (randomly selected, unfamiliar

age- and sex-matched WT mouse), respectively, was placed in the center of the light box, under an

inverted pencil cup with a water bottle placed on top to prevent the test mouse from climbing the cup.

Electrophysiological Recordings

Whole-cell voltage-clamp recording was used to measure synaptic currents in layer V mPFC

(prelimbic and infralimbic) pyramidal neurons as previously described. Mouse brain slices (300 µm)

were positioned in a perfusion chamber attached to the fixed stage of an upright microscope

(Olympus) and submerged in continuously flowing oxygenated ACSF (in mM: 130 NaCl, 26

NaHCO3, 1 CaCl2, 5 MgCl2, 3 KCl, 1.25 NaH2PO4, 10 glucose, pH 7.4, 300 mOsm). For EPSC

recordings, the pipette contained the following solution (in mM: 130 cesium-methanesulfonate, 10

CsCl, 4 NaCl, 10 HEPES, 1 MgCl2, 5 EGTA, 2 QX-314, 12 phosphocreatine, 5 MgATP, 0.2 Na3GTP,

0.1 leupeptin, pH 7.2-7.3, 265-270 mOsm). For NMDAR-EPSC, Bicuculline (10 μM) and CNQX (25

μM) were added into the ACSF, and the cell (clamped at -70 mV) was depolarized to +40 mV for 3s

before stimulation to fully relieve the voltage-dependent Mg2+ block. Membrane potential was held at -

70 mV for AMPAR-EPSC recordings (10 μM bicuculline and 25 μM D-APV were added). For

GABAAR-IPSC recording (holding at -70 mV), 25 μM CNQX and D-APV were added into the ACSF.

The recording pipette contained the following internal solution (in mM: 100 CsCl, 30 N-methyl-D-

106

glucamine, 10 HEPES, 4 NaCl, 1 MgCl2, 5 EGTA, 2 QX-314, 12 phosphocreative, 5 MgATP, 0.5

Na2GTP, pH 7.2-7.3, 265-270 mOsm).

Evoked synaptic currents were generated with a pulse from a stimulation isolation unit

controlled by an S48 pulse generator (Grass Technologies, West Warwick, RI). A bipolar stimulating

electrode (FHC, Bowdoinham, ME) was placed ~100 μm from the neuron under recording. Input-

output responses of synaptic currents were elicited by a series of pulses with varying stimulation

intensities (20–130 µA) delivered at 0.05 Hz. To record the evoked action potential, slices were bathed

in a modified ACSF (in mM: 130 NaCl, 26 NaHCO3, 1 CaCl2, 0.5 MgCl2, 3.5 KCl, 10 glucose, 1.25

NaH2PO4) to slightly elevate basal neuronal activity [62]. Whole-cell current clamp techniques were used

to measure action potential firing with the internal solution containing (in mM: 20 KCl, 100 K-gluconate,

10 HEPES, 4 ATP, 0.5 GTP, and 10 phosphocreatine) [62, 215] (Tan et al., 2018; Wang et al., 2018). A

small depolarizing current was applied to adjust the membrane potential to -70 mV. And steps of

depolarizing currents (-30 pA – 300 pA) were injected. Data analyses were performed with the Clampfit

10.0.7 software (Molecular Devices, Sunnyvale, CA, USA).

Viral Vectors and Animal Surgeries

Npas4 CRISPR-lentiviral activation particles were obtained from Santa Cruz Biotechnology (sc-

432569-LAC). Stereotaxic injection of the virus (1.5 l total volume, bilateral) to the medial PFC

(prelimbic infralimbic regions; 0.25mm ML/2.0mm AP/2.0mm DV) was performed as described

previously [110]. In brief, mice were anesthetized and placed on a stereotaxic apparatus (David Kopf

Instruments). The injection was performed with a Hamilton syringe (gauge 31) at a speed of 0.2 l/min,

and the needle was kept in place for an additional 5 min. The virus was delivered bilaterally to the target

area using the following coordinates: 2.0 mm anterior to bregma; 0.25 mm lateral; and 2.0 mm dorsal to

ventral. Mice of either genotype were randomly assigned to treatment groups. All behavioral and

electrophysiological tests were conducted 10 days after surgery. Fluorescence images were taken on a

Leica DMi8 inverted fluorescent microscope.

Biochemical Techniques

Immunohistochemistry

Mice were anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde

(PFA) before brain removal. Brains were post-fixed in 4% PFA for 2 days and cut into 30-m slices.

Slices were cut coronally and washed and blocked for 1 hour in PBS containing 5% donkey serum and

0.3% Triton for permeabilization. After washing, slices were incubated with the primary antibody for 48

hours at 4°C. After washing three times (30 minutes with gentle shaking) in PBS, slices were

107

incubated with secondary antibody for 1 hour at room temperature, followed by three washes with

PBS. Slices were mounted on slides with Vectashield hardset mounting media with DAPI (Vector

Laboratories). Images were acquired using a Leica DMi8 fluorescence microscope. All specimens

were imaged under identical conditions and analyzed with identical parameters using ImageJ software.

RNA-Sequencing and Analysis

PFC samples were obtained from three WT mice and three 16p11.2dp/+ mice. We generated strand-

specific RNA libraries from 1 μg purified RNA using TruSeq stranded total RNA plus Ribo-zero kits

(Illumina). The sequencing was performed at the Genomics and Bioinformatics Core of State University

of New York at Buffalo. Single end reads per sample were obtained using the HiSeq 2500 platform from

Illumina. Reads were first trimmed using Cutadapt to remove the 3’ end adapters and trailing sequences,

followed by aligning to mouse RefSeq mRNAs using TopHat2. Transcript counts were estimated using

HTSeq. Differences in gene expression levels between samples were assessed with edgeR and calculated

as log2 fold change. Functional protein classification analyses were undertaken using Panther, and genes

lacking classifications (defined as “other”) were manually assigned appropriate categories. For

comparisons to ASD-susceptibility genes, we used the list on the SFARI database.

Quantitative Real-time RT-PCR

Total RNA was isolated from mouse PFC punches using Trizol reagent (Invitrogen) and treated

with DNase I (Invitrogen) to remove genomic DNA. SuperScript III first-strand synthesis system for RT-

PCR (Invitrogen) was used to reverse-transcribe mRNA into cDNA, followed by treatment with RNase H

(2 U/I) for 20 minutes at 37°C. Quantitative real-time RT-PCR was performed using the iCycler iQ™

Real-Time PCR Detection System and iQ™ Supermix (Bio-Rad) according to the manufacturer’s

instructions. GAPDH was used as the housekeeping gene for quantitation of the expression of target

genes in samples from WT vs. 16p11.2dp/+ mice. Fold changes in the target genes were determined by:

Fold change = 2-Δ(ΔCT), where ΔCT = CT(target) – CT(GAPDH), and Δ(ΔCT) = ΔCT(treated group) -

ΔCT(WT). CT (threshold cycle) is defined as the fractional cycle number at which the fluorescence

reaches 10x the standard deviation of the baseline. A total reaction mixture of 20 L was amplified in a

96-well thin-wall PCR plate (Bio-Rad) using the following PCR cycling parameters: 95°C for 5 min

followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec. Primers for all target

genes are listed in Table S3.

Western Blotting of Nuclear/Synaptosomal Proteins

Nuclear extracts from mouse brains were prepared as previously described [102] with

modifications. Eight prefrontal cortex punches (diameter: 2mm) from fresh mouse brain slices (300 μm)

per animal were collected and homogenized with 500 μL homogenization buffer (20 mM Tris-HCl, pH

7.4, 10 mM NaCl, 3 mM MgCl, 0.5% NP-40, 1 mM PMSF, with cocktail protease inhibitor). The

108

homogenate was incubated on ice for 15 min and followed by centrifugation at 3,000 g, 4°C for 10

minutes. The nuclear pellet was resuspended in 50 μL nuclear extract buffer (100 mM Tris-HCl, pH 7.4,

100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 10% glycerol, 1 mM PMSF, with cocktail

protease inhibitor) and incubated on ice for 30 min with periodic vortexing to resuspend the pellet. After

centrifugation, the supernatant for nuclear fractions was collected, boiled in 2 × SDS loading buffer for 5

min and then separated on 10% SDS-polyacrylamide gels.

Subcellular fractions were prepared as follows. punches of frontal cortex were cut out, weighed,

and homogenized in ice‐cold lysis buffer (10 ml/g, 15 mM Tris, pH 7.6, 0.25 M sucrose, 1 mM PMSF, 2

mM EDTA, 1 mM EGTA, 10 mM sodium pyrophosphate, and protease inhibitor tablet). After

centrifugation at 800 × g for 5 min to remove nuclei and large debris, the remaining supernatant was

subjected to 10,000 × g centrifugation for 10 min. The crude synaptosome fraction (pellet) was suspended

in lysis buffer containing 1% Triton X‐100 and 300 mM NaCl, homogenized again, and centrifuged at

16,000 × g for 15 min. Triton insoluble fraction, which mainly includes membrane‐associated proteins

from synaptosomes, was dissolved in 1% SDS. Samples were boiled in 2 × SDS loading buffer for 5 min

and separated on 7.5% SDS‐PAGE.

109

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