Figure 4: TBS-induced ERK activation is differentially affected by pre- or post-TBS CI-III treatment.

Hippocampal slices were prepared from adult rats and subjected to various experimental conditions. At the indicated times, slices were fixed, re-sectioned and processed for immunohistochemistry for p-ERK. Images of stimulated and unstimulated regions from two most representative sections of each slice were chosen for analysis. (a) Representative images. Top: p-ERK immunoreactivity at 5 min after TBS (10 bursts of 4 pulses (100 Hz) delivered at 5 Hz) in slices treated with vehicle or CI-III (10 μM) for 10 min before and continued for 5 min after TBS. Bottom: p-ERK immunoreactivity at 50 min after TBS in slices treated with vehicle or CI-III (10 μM) for 30 min starting 10 min after TBS. Scale bar, 20 μm. (b) High-magnification images of dendrites in stimulated region double-stained with p-ERK and PSD-95 antibodies, showing the co-localization of p-ERK and PSD-95 (arrows). Scale bar, 2 μm. (c) Quantitative analysis of p-ERK immunostaining. The average p-ERK intensity was determined in a 120 × 60 μm² area in the dendritic field of CA1. Image intensities from stimulated regions were subtracted from those of unstimulated regions of the same slice to correct for slice-to-slice variation in staining intensity, as described inref. ³⁸. Shown are means±s.e.m. of n=8. *P<0.01, one-way analysis of variance followed by Bonferroni test.