Repeated Social Defeat
Objective: To determine if repeated social defeat induces anxiety-like behavior and microglial reactivity through β-adrenergic receptor signaling and IL-1 receptor activation
Gather these items before starting the experiment. Check off items as you prepare.
Equipment1
Not specified in provided text • Not specified • Not specified • Not mentioned
Materials2
Software1
Not specified in provided text • Not mentioned
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Protocol Steps
Repeated Social Defeat Exposure
Mice were exposed to repeated social defeat stress paradigm to induce anxiety-like behavior and immune alterations
Note: This is a psychosocial stress paradigm where mice experience repeated social defeat
View evidence from paper
“Repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior”
Behavioral Testing for Anxiety
Anxiety-like behavior was assessed following repeated social defeat exposure
Note: β-adrenergic receptor-dependent anxiety-like behavior was measured
View evidence from paper
“Repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner”
Brain Tissue Collection and c-Fos Staining
Brain tissue was collected and analyzed for c-Fos staining in regions associated with fear and threat appraisal
Note: c-Fos staining was increased in brain regions associated with fear and threat appraisal
View evidence from paper
“Repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal”
Flow Cytometry Analysis of Immune Cells
Analysis of CD11b+/CD45high/Ly6Chigh macrophages that trafficked to the brain and inflammatory markers on microglia and macrophages
Note: Markers analyzed included CD14, CD86, and TLR4 on microglia and macrophages
View evidence from paper
“Repeated social defeat also significantly increased the number of CD11b+/CD45high/Ly6Chigh macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86)”
Microglial Morphology Assessment
Assessment of microglial morphology in medial amygdala, prefrontal cortex, and hippocampus to identify deramified microglia
Note: Deramified microglia presence was increased in these brain regions
View evidence from paper
“Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus”
mRNA Analysis of Microglia
mRNA analysis of microglia to measure interleukin (IL)-1β and glucocorticoid responsive genes (GILZ and FKBP51)
Note: Repeated social defeat increased IL-1β levels and reduced GILZ and FKBP51 levels
View evidence from paper
“mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]”
Propranolol Treatment
Administration of propranolol (β-adrenergic receptor antagonist) to prevent stress-dependent changes in microglia and macrophages
Note: Propranolol prevented the stress-dependent changes in microglia and macrophages
View evidence from paper
“The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist”
Ex Vivo Microglia Culture and Stimulation
Microglia were isolated from socially defeated and control mice, cultured ex vivo, and stimulated with lipopolysaccharide
Note: Microglia from defeated mice produced higher levels of IL-6, TNF-α, and MCP-1
View evidence from paper
“Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide”
IL-1 Receptor Type-1 Deficient Mouse Studies
Repeated social defeat was tested in IL-1 receptor type-1-deficient mice to assess IL-1 receptor dependence
Note: Social defeat increased c-Fos activation but did not promote anxiety-like behavior or microglia activation in IL-1R1-deficient mice
View evidence from paper
“Repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1”