Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice

Denervation of the dentate gyrus by entorhinal cortex lesion has been widely used to study the reorganization of neuronal circuits following central nervous system lesion. Expansion of the non-denervated inner molecular layer (commissural/associational zone) of the dentate gyrus and increased acetylcholinesterase-positive fibre density in the denervated outer molecular layer have commonly been regarded as markers for sprouting following entorhinal cortex lesion. However, because this lesion extensively denervates the outer molecular layer and causes tissue shrinkage, stereological analysis is required for an accurate evaluation of sprouting. To this end we have performed unilateral entorhinal cortex lesions in adult C57BL/6J mice and have assessed atrophy and sprouting in the dentate gyrus using modern unbiased stereological techniques. Results revealed the expected increases in commissural/associational zone width and density of acetylcholinesterase-positive fibres on single brain sections. Yet, stereological analysis failed to demonstrate concomitant increases in layer volume or total acetylcholinesterase-positive fibre length. Interestingly, calretinin-positive fibres did grow beyond the border of the commissural/associational zone into the denervated layer and were regarded as sprouting axons. Thus, our data suggest that in C57BL/6J mice shrinkage of the hippocampus rather than growth of fibres underlies the two morphological phenomena most often cited as evidence of regenerative sprouting following entorhinal cortex lesion. Moreover, our data suggest that regenerative axonal sprouting in the mouse dentate gyrus following entorhinal cortex lesion may be best assessed at the single-fibre level.