Figure 5.

Histone acetylation levels in a cognitively impaired rat model. (a) Histological verification of the location and extent of cholinergic and entorhinal cortex (EC) lesions. A, C, E, and G are representative pictures of one sham-operated rat; B, D, F, and H are representative pictures of one rat subjected to the double lesions. A, B are typical examples of AChE-positive staining in the posterior hippocampus as observed at about −5.5 mm from Bregma (notice the marked decrease of dark staining accounting for cholinergic denervation in B); C, D are typical examples of ChAT-positive immunostaining in the medial septum at about +0.2 mm from Bregma (notice the dramatic reduction in D); E, F are examples of ChAT-positive immunostaining in the nucleus basalis magnocellularis and the substantia innominata (notice that the decrease is much less pronounced than in the septal region in F). G, H are examples of NeuN-positive staining in the posterior region of the cortex (coronal sections) illustrating the lesion extent in the medial and lateral entorhinal cortex at about Bregma 7.6 mm (notice the almost total loss of neuronal staining within the area delimited by the stippled line in H). All coordinates indicated in this caption are according to Paxinos and Watson (1998). Scale bar=1000 μm in A, B, G, and H, and scale bar=500 μm in C, D, E, and F. (b) Effects of double lesions on water maze performance. Upper panel: Acquisition data during the 3-day-training period are expressed as the mean (±SEM) of the distances to reach the platform in rats subjected to a lesion of septal cholinergic neurons and a fiber-sparing lesion of neurons in the EC (lesion, n=10) as compared with performance in the sham-operated controls (sham, n=11). Lower panel: Probe trial performance indicated as the mean time in the far quadrants (±SEM). Sham-operated control rats showed a performance in the target quadrant that was significantly above chance (ie, 15 s). In contrast, the double lesion affected spatial memory functions, as the lesioned rats did not focus their search in the appropriate quadrant. ^*p<0.05, significantly above chance. (c) Acetylated (Ac) and total (Tot) histone levels were measured by western blot analyses for H3 and H2B histones in total extracts obtained from the dorsal hippocampus of home cage rats (basal conditions; sham, n=6; lesion, n=8) or of trained rats (learning conditions: HPf group during 3 days for all rat groups; sham, n=7; lesion, n=7). Lysine acetylations measured are H3K9K14 and H2BK5K12K15K20. Typical western blots are represented in duplicates. Quantified results are represented as fold induction of the Ac/Tot ratio for each histone. The ratio obtained in the control condition is arbitrarily set at 100%. Sh, sham; Les, lesion. Student's t-test: ^**p<0.01. A significant decrease in acetylation of H2B is observed in the hippocampus of trained rats, but not in that of home cage rats. Acetylated-H3 levels tend to decrease as well only in learning conditions.