Fig. 1. Experimental Design and Timeline.

10-month-old C57BL/6JNia male mice were randomly assigned to one of two treatment conditions: Vehicle (Saline) or Intermittent LPS. To reliably induce a moderate sickness response from which subjects made a full recovery, mice received one injection every 15 days whereby the dosage of LPS was progressively increased with each subsequent injection such that: Injection 1 = 0.4 mg/kg, Injection 2 = 0.8 mg/kg, Injection 3 = 1.6 mg/kg, Injection 4 = 3.2 mg/kg, Injection 5 = 6.4 mg/kg. Health and sickness behavior was measured at baseline (one day prior to the first injection) to verify all mice displayed equivalent health without signs of sickness, at 4 h post-injection (Day 1) to verify induction of moderate sickness among LPS-treated mice, and at 14 days post-injection (one day prior to the subsequent injection) to verify a lack of sickness. Following the final injection (Injection 5), mice were randomly assigned to one of two cohorts (Behavior Naïve or Behavior Tested). Tissue was collected from the Behavior Naïve cohort approximately 2–3 weeks following the final injection, while tissue collection for the Behavior Tested cohort took place approximately 3–4 weeks later (approximately 5–6 weeks following the final injection).