Excitatory Synaptic Dysfunction In The Anterior Cingulate Cortex Underlies Social Deficits In A Shank3 Mutant Mouse Model Of Autism Spectrum Disorder

BackgroundAutism spectrum disorder（ASDs）is a group of neurodevelopmental syndromes.Patients display behavioral abnormalities in infants and young children stage,and the behavioral deficits last for life,requiring long-term rehabilitation treatment and life-long support services,which causes huge economic burdens to the family and society.Social behavior deficit is one of the core symptoms of patients with autism.The social deficits of different patients are highly heterogeneous,making the treatment and research of social disorder extremely difficult.One of the reasons is that the neural circuit of social behaviors has not yet been fully understood.Clinical studies have shown that the anterior cingulate cortex（ACC）changes in patients when they performed social-related behaviors,while the ACC of patients with social disorders shows abnormal metabolic and functional connections.Furthermore,the ACC is connected with multiple neural networks that regulate different stages of social behavior,such as observing learning,social sentiment,and value processing networks.However,whether the ACC is involved in ASDs related social deficits and how it is involved in it has not yet been studied.The Shank family（Shank1-3）encodes excitatory postsynaptic scaffold proteins,connecting with PSD-95,Homer and other postsynaptic molecules,interacting with glutamate receptors,and cytoskeletal proteins to promote the formation of synapses,the development of dendritic spines and the stability of synaptic function.Genetic studies have found that SHANK3 was monogenic cause of ASD,and Shank3 knockout mice have typical ASDs-like manifestations such as social behavior disorder and stereotyped behavior.Therefore,Shank3 knockout mice are one of the desirable animal models for studying ASDs.In the current study,we found that Shank3 deficiency leads to structural and functional abnormalities in ACC pyramidal neurons.The resulting hypoactivity of the pyramidal neurons in the ACC play a causal role in social interaction deficits.Optogenetic specific activation and inhibition of ACC pyramidal neurons could improve the social behavior disorder of Shank3 knockout mice or mimic ASDs-like social behavior abnormalities in wild-type mice.By locally specifically knocking out or re-expressing Shank3 in the ACC,ACC synaptic function and animal social behaviors can be adjusted in both directions.CX546,a positive allosteric modulator of AMPA receptors administered systemically or locally in ACC can enhance the excitatory synaptic function of ACC and improve the social behavior disorder of Shank3 knockout mice.This study reveals the novel mechanisms of the ACC dysfunction in social behavior disorder in ASDs,and builds the causal link between ACC excitatory synaptic dysfunction and ASD-like social deficits in the Shank3 mutant mouse model of ASDs,and emphasize that the ACC is a potential therapeutic target for developing interventions for social deficits.Methods1)Observing excitatory synapse morphology:We used adeno-associated virus（AAV） and Semliki forest virus（SFV）to sparsely label ACC pyramidal neurons,and analyzed the dendritic branches and dendritic spines’morphology of reconstructed neurons through Imaris3D image analysis.Electron microscope was used to observe the excitatory synapses of the ACC,and the length and thickness of the postsynaptic densities were analyzed.2)Testing excitatory synaptic function:We performed whole-cell patch clamp to record ACC pyramidal neurons on brain slices,analyzed miniature excitatory postsynaptic currents（m EPSCs）,paired pulse stimulation（PPR）,and glutamate receptors-mediated excitatory currents,long-term potentiation（LTP）and other synaptic function related parameters.The expression levels of different glutamate receptor subtypes were detected by Western Blot.3)Monitoring of ACC neuron activities in vivo:We adopted in vivo calcium signal imaging method in freely moving mice.When the mice are in social interaction,the calcium signal activity is recorded by optical fiber simultaneously.We also performed multi-channel electrophysiological recordings combined with optogenetic stimulation methods,testing the influence of different optogenetic activation protocols on ACC neuronal activity.4)Manipulating ACC neuronal activities in vivo:We injected AAV-Ca MKIIa-Ch R2-EYFP/AAV-Ca MKIIa-Np HR-EYFP virus vector in the ACC,and used an implanted fiber to deliver blue or yellow laser,activating or silencing ACC neurons in vivo.The DREADDs virus vector was injected to express h M3Dq locally in ACC,and CNO was administrated intraperitoneally for in vivo chemo-genetic regulation.5)Knockout Shank3 in the ACC:The AAV-CMV-Sa Cas9-sg RNA virus vector was applied to specifically knock out Shank3 in ACC neurons.6)Restoring Shank3 in ACC pyramidal neurons:AAV-Ca MKIIa-cre-GFP was injected locally in Shank3^fx/fx transgenic animals to specifically re-express Shank3 in ACC pyramidal neurons.7)Pharmacological enhancement of AMPA receptors:Intraperitoneal injections of different doses of CX546,or local infusion after ACC implantation of bilateral cannulas were used to enhance the activity of AMPA receptors in vivo.8)Behavior testing:Three-box,social interaction in home cage experiments were performed to test the sociability of mice;continuous recording combined with video analysis was used to detect mouse stereotyped grooming behavior;open field experiment,elevated plus maze experiment,marbles burying experiments as well as novel object recognition experiments were performed to detect anxiety-like behaviors in mice.Results1)The dendritic complexity and the densities of total spines and mushroom-shaped spines were significantly lower in the pyramidal cells of Shank3 knockout mice than in those of WT mice.And the length and thickness of postsynaptic densities were significantly lower in Shank3 knockout mice than in WT mice.2)The frequency and amplitude of m EPSCs were significantly lower in Shank3 knockout mice than those in WT mice.The AMPAR-mediated EPSC was impaired compared with WT mice.Glu R1 expression was reduced,and paired training paradigm failed to induce LTP in Shank knockout mice.3)Shank3 deficiency leads to hypoactivity in pyramidal neurons,with no significant changes in intrinsic electrophysiological properties.ACC neuronal activity was significantly reduced in the Shank3 knockout mice when they approached unfamiliar mice.4)Optogenetics and chemogenetics activation of ACC pyramidal neurons can improve the social behavior in Shank3 knockout mice,and optogenetics inhibition of ACC pyramidal neurons can lead to ASDs-like social deficits in wild-type mice.5)Selective deletion of Shank3 in the ACC sufficed to induce similar synaptic impairments and social behavior abnormalities,but not other behavioral changes;ACC local restoration of Shank3 can partially rescue synaptic functions and social behaviors in Shank3 knockout mice.6)Systemic or local administration of AMPA receptor positive allosteric modulator CX546 can improve the function of excitatory synapses and social deficits in Shank3 knockout mice.ConclusionIn the current study,we found that Shank3 deficiency leads to dendritic spine loss and impaired synaptic transmission in ACC pyramidal neurons.The resulting hypoactivity of pyramidal neurons in the ACC plays a causal role in social deficits.Selective knockout Shank3 in the ACC sufficed to induce both synaptic dysfunction and social behavioral deficits that were comparable to those observed in global Shank3 knockout mice.Selective activation ACC pyramidal neurons,restoring SHANK3 expression in ACC pyramidal neurons or systemic administration of AMPA receptor PAM could boost synaptic function and improve social behavior.Our findings provide direct evidence supporting a causal link between ACC dysfunction and social deficits in Shank3 mutant mouse model,and emphasize that the ACC is a potential therapeutic target for developing interventions for social deficits.