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. 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.