2.4.1. Habituation and baseline behavior

One week prior to surgical procedures animals were allowed to acclimate to the housing facility and underwent a habituation period that included handling, travel to the behavioral testing suite, administration of food treats (chocolate and fruit puffed cereal), and initiation of food restriction to encourage completion of behavioral tasks. For food restriction, animals were fed 5 g of food once-daily at completion of behavioral tasks, with daily weights to assure that animals did not lose ≥ 15% of their body weight. Baseline behavior included assessment of locomotor and anxiety behavior via 5 min exploration of a 50 gallon (63 cm diameter) open field arena (open field task). Animals were also habituated to the radial arm maze over a 3-day habituation period. On days one and two, animals were allowed free exploration of all eight food-baited arms for 15 min. On day three animals underwent a pre-injury working memory task, requiring visiting 7 of 8 un-baited arms in under 10 min with fewer than 4 arm revisits. All behavioral testing was video recorded and assessed using Ethovision XT 11.5 software (Noldus). Animals that could not complete this baseline task were excluded from the study. Animals that successfully completed the baseline behavior proceeded to surgical procedures. Two days post-injury, all animals were re-habituated to the radial arm maze in which they were allowed to explore the apparatus for 10 min without any treats present. Post-injury behavioral testing began 3 days post-injury (Fig. 1A). All animals underwent the full behavioral testing paradigm.

(A) Experimental design. Animals received baseline behavior prior to LFPI on Day 0 (D0). Three days after LFPI (D3) pattern separation task was started and continued daily for 10 days through D12. On D7 animals received a single intraperitoneal injection of bromodeoxyuridine (BrdU) after completion of behavioral testing. On D13 animals were tested on open field arena (OF) and then on novel object recognition (NOR). Tissue was harvested at the completion of behavioral testing. (B) Animals that received LFPI had significantly longer righting reflex time (RRT) as compared to sham animals. Error bars are standard error of the mean; ***p < 0.001. (C) In animals for immunoblotting, the hippocampus was isolated from fresh tissue and the dentate gyrus was dissected. Animals for immunohistochemistry were perfused with 4% paraformaldehyde and were cryosectioned at 20 µm. (D) Representative stained sections are shown from Sham (top row) and LFPI (bottom row) animals processed 2 weeks after injury. Cresyl violet staining illustrates lack of gross cortical or hippocampal damage. However, injury is evidenced by increased glial activity seen with GFAP staining in cortex and hippocampal dentate gyrus. Scale bar for GFAP is 250 µm.