Proteomic and transcriptional profiling of rat amygdala following social play. Proteomic and transcriptional profiling of rat amygdala following social play

Social play is a frequently studied behavior and it is the most characteristic form of social interaction observed in adolescent rats. Social play is necessary for adolescents to develop proper cognitive, emotional, and social competency. Deficits in social play have been observed in several neurodegenerative disorders such as autism, schizophrenia, and attention deficit hyperactivity disorder. However, the information available on neural substrates and the mechanism involved in social play is still limited. This study characterized social play by proteomic and transcriptional profiling studies. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioral testing. The proteomic analysis led to the identification of 170 differentially expressed proteins (p≤0.05) with 67 upregulated and 103 downregulated proteins. The transcriptomic analysis led to the identification of 188 genes (adjusted p≤0.05) with 55 upregulated and 133 downregulated genes. Based on both protein and gene expression data, DAVID analysis revealed that social play altered neurotransmitter signaling including GABAergic and glutamatergic signaling and G-protein coupled receptor (GPCR) signaling. These data suggest that the synaptic levels of GABA and glutamate increased during play. Ingenuity Pathway Analysis (IPA) confirmed these alterations. IPA also revealed that differentially expressed genes/proteins in our data had significant over representation of additional neurotransmitter signaling systems, including the opioid, serotonin, and dopamine systems, suggesting that play alters the systems involved in the regulation of reward. In addition, corticotropin-releasing hormone signaling was altered indicating that an increased level of stress occurs during play. Our data suggest that increased inhibitory GPCR signaling in these neurotransmitter pathways occurs following social play as a physiological response to regulate the induced level of reward and stress and to maintain the excitatory-inhibitory balance in the neurotransmitter systems. Overall design: Social play was performed on male Sprague Dawley rats on postnatal day 38 and gene expression in the amygdala was determined following behavioral testing. Total RNA was isolated from amygdala using a Qiagen miRNA easy micro kit (Germantown, MD) which is specialized for isolation of RNA from lipid rich tissue. The RNA quality was verified by NanoDrop. Each RNA sample containing 1 µg of RNA was used for poly A selected RNA-seq library preparation using the NEB Ultra-Directional RNA Library Prep Kit for Illumina, Cat# E7420. Each library was barcoded using NEBNext Multiplex Oligos for Illumina (Cat# E6609) based on the manufacturer's protocols. From total of 24 RNA-Seq libraries, 8 individual RNA-Seq libraries were equimolar pooling into one pooled library sample and sequenced with 1 lane of paired-end 100bp (2x100) using Illumina HiSeq4000 sequencer. Total of 3 lanes were subjected to sequencer.

社交玩耍（Social play）是一项被广泛研究的行为，也是青春期大鼠最具特征性的社交互动形式。社交玩耍对于青少年大鼠发育正常的认知、情感与社交能力至关重要。社交玩耍的缺陷已在自闭症、精神分裂症、注意缺陷多动障碍等多种神经退行性疾病中被观测到。然而，目前关于社交玩耍的神经底物及相关机制的研究信息仍然有限。

本研究通过蛋白质组学与转录组学分析对社交玩耍进行了表征。实验于出生后第38天对雄性斯普拉格-道利大鼠（Sprague Dawley rats）开展社交玩耍行为测试，行为测试结束后检测其杏仁核（amygdala）中的蛋白质与基因表达水平。蛋白质组学分析共鉴定出170种差异表达蛋白（differentially expressed proteins，p≤0.05），其中67种表达上调，103种表达下调。转录组学分析共鉴定出188个基因（校正后p≤0.05），其中55个基因表达上调，133个基因表达下调。

基于蛋白质与基因表达数据，DAVID分析显示社交玩耍改变了神经递质信号通路，包括γ-氨基丁酸能（GABAergic）、谷氨酸能（glutamatergic）信号通路以及G蛋白偶联受体（G-protein coupled receptor, GPCR）信号通路。上述结果提示，玩耍过程中杏仁核内γ-氨基丁酸与谷氨酸的突触水平有所升高。Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）验证了这一变化。此外，IPA分析还发现，本研究数据中的差异表达基因/蛋白显著富集于其他神经递质信号系统，包括阿片样物质、5-羟色胺与多巴胺系统，提示社交玩耍可改变参与奖赏调控的信号系统。同时，促肾上腺皮质激素释放激素（corticotropin-releasing hormone）信号通路发生改变，表明玩耍过程中应激水平有所升高。本研究数据表明，社交玩耍后这些神经递质通路中的抑制性GPCR信号通路会增强，以此作为生理应答调控诱导产生的奖赏与应激水平，并维持神经递质系统的兴奋-抑制平衡。

整体实验设计：本研究于出生后第38天对雄性斯普拉格-道利大鼠开展社交玩耍行为测试，行为测试结束后检测其杏仁核中的基因表达水平。使用Qiagen miRNAeasy微型试剂盒（Qiagen miRNA easy micro kit，Germantown, MD）从杏仁核中提取总RNA，该试剂盒专为从富脂组织中分离RNA而设计。通过NanoDrop检测RNA质量。取每份含1μg RNA的样品，使用适配Illumina平台的NEB超定向RNA文库制备试剂盒（NEB Ultra-Directional RNA Library Prep Kit for Illumina, Cat# E7420）进行聚腺苷酸选择RNA测序文库构建。根据制造商方案，使用NEBNext多重寡核苷酸（NEBNext Multiplex Oligos for Illumina, Cat# E6609）对每个文库进行条形码标记。将24个RNA测序文库中的8个独立文库按等摩尔浓度混合为一个混合文库样品，使用Illumina HiSeq4000测序仪以双端100bp（2×100）模式进行测序，共完成3个测序泳道的测序。