Proteomic and transcriptional profiling of rat amygdala following social play

Abstract

Social play is the most characteristic form of social interaction which is necessary for adolescents to develop proper cognitive, emotional, and social competency. The information available on neural substrates and the mechanism involved in social play is 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 (FDR ≤ 0.05) with 55 upregulated and 133 downregulated genes. DAVID analysis of gene/protein expression data revealed that social play altered GABAergic signaling, 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 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. Overall, 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.

Keywords: Amygdala; Behavior; Gene expression; Protein expression; Reward; Social play.

Figure 1

Cellular location (A) and biological processes (B) represented by differentially expressed proteins identified by DAVID. The x-axis indicates the number of proteins that are associated with each cellular component (a) or biological processes (B). P-value of ≤ 0.05 was considered while selecting enriched GO terms.

Figure 2

Cellular location (A) and biological processes (B) represented by differentially expressed genes identified by DAVID. The x-axis indicates the number of genes that are associated with each cellular component (a) or biological processes (B). P-value of ≤ 0.05 was considered while selecting enriched GO terms.

Figure 3

Ingenuity Pathway Analysis (IPA) identified canonical pathways represented by altered protein (A) and gene (B) expression during social play. The blue color indicates the inhibition of pathway, orange color indicates the activation of pathway, and white color indicates that there is no activation/ inhibition of the pathway. Gray color means IPA cannot predict about the activation state of that pathway. The color coding was given based on Z-score. Threshold (dot line) line indicates the p-value of 0.05 or −log (P-value) of 1.3. Ratio which is represented in orange solid line refers to the number of molecules from the dataset that map to the pathway listed divided by the total number of molecules that define the canonical pathway from within the IPA knowledgebase.

Figure 4

IPA identified molecular and cellular functions represented by altered protein (A) and gene (B) expression during social play. Threshold line indicates the p-value of 0.05 or −log (P-value) of 1.3.

Figure 5

IPA identified physiological functions represented by protein (A) and gene (B) expression during social play. Threshold line indicates the p-value of 0.05 or −log (P-value) of 1.3.

Figure 6

One of the top networks identified from differentially expressed proteins in social play is associated with different canonical pathways such as the dopamine receptor signaling, opioid signaling, GABA receptor signaling, G-protein coupled receptor signaling, serotonin receptor signaling, and glutamate receptor signaling. Differential expression of proteins is indicated by color: red color indicates the upregulation, green color indicates the down regulation, and white indicates that those proteins were not in the dataset. The intensity of color indicates the level of regulation i.e., fold change.

Figure 7

The western blot analysis of protein expression of regulatory G-protein signaling 7 (RGS7) (A), G alpha o (Gαo) (B), Monoamine oxidase A (MAOA) (C), and GABA type A receptor (D) in adolescent rats who were either behaviorally naive or had completed a ten-minute interaction session with a play partner. The behavioral rats were sacrificed three hours following social play and protein expression in the amygdala was measured. The proteins levels were normalized to the β-actin levels. Values are expressed as mean ± SEM.