Six hours after LPS or saline injection, animals belonging to cohort 2 were sacrificed, trunk blood was collected, brains were removed, and ventral and dorsal hippocampi were dissected and immediately stored at −80 °C until RNA extraction. This time point was selected based on published and preliminary results suggesting that changes in the expression levels of inflammatory markers at this time are indicative of the early immune response and may underlie late induced effects on behavior (Terrando et al., 2010; Biesmans et al., 2013).

Trunk blood was collected after decapitation for the determination of glucocorticoid levels as an indication of HPA activation. Experiments were performed from 9:00 am to 4:00 pm during the light period. To improve serum separation from whole blood, samples were let to clot 15 min at room temperature and 1 h on ice before centrifugation (1,000 × g for 15 min). Serum was transferred into clean tubes and stored at −80 °C until the assay.

Assessment of serum corticosterone levels was done by means of enzyme immunoassay (EIA) using a commercially available kit (Arbor Assays, Ann Arbor, MI, United States), which utilizes a microplate reader set at 450 nm, following the manufacturer’s instructions. Serum samples were diluted 1:150 in appropriate assay buffer and assayed in duplicate. The detection limit of the assay was 16.9 pg/ml; intra- and interassay coefficients of variations were 8.15% and 17.93%, respectively.

RNA extraction and DNAse treatment were performed as previously described (Alboni et al., 2013a) using GenElute™ Mammalian Total RNA Miniprep Kit and DNase70-On-Column DNAse I Digestion Set (Sigma-Aldrich®, Milan, Italy). Two µg of total RNA was reverse-transcribed with High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States) and Real-Time PCR was performed, as previously described (Benatti et al., 2011), in ABI PRISM 7900 HT (Thermo Fisher Scientific, Waltham, MA, United States) using Power SYBR Green mix (Thermo Fisher Scientific, Waltham, MA, United States) and specific forward and reverse primers at a final concentration of 150 nM (see Supplementary Table S1 for primer sequences). Ct (cycle threshold) value was determined by the SDS software 2.2.2 (Thermo Fisher Scientific, Waltham, MA, United States); mRNA expression was calculated with the ΔΔCt method with cyclophilin A (CypA) as endogenous control.

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