Abstracts

Rationale: Temporal lobe epilepsy is associated with a variety of structural abnormalities of the hippocampus. The organization of the hippocampus, with distinct lamina, compact cell layers, and well-defined fiber pathways, has made identifying and characterizing structural changes in this region a relatively easy task for the neuroanatomist. One aspect of hippocampal anatomy that has continued to be less tractable, however, has been assessing the extensive longitudinal fiber projections of some important cell types, such as mossy cells. Traditional methods for examining hippocampal structure require sectioning the hippocampus in a plane perpendicular to the longitudinal axis. While this is ideal for revealing the hippocampal principle cell layers, it unavoidably severs the longitudinal fibers. Here we describe a protocol which allows researchers to image deep within the intact hippocampus at micron-level resolution, thereby allowing for analysis of fibers traversing all hippocampal axes. Methods: C57/Blk6 transgenic mice expressing Thy1-GFP were treated with pilocarpine to induce status epilepticus. At various time points after status epilepticus treatment, animals were sacrificed and whole brains were excised, paraformaldehyde fixed and placed in the newly developed SCALE tissue clearing reagent (Nature Neuroscience 2011, 14(11): 1481-88). Hippocampi were dissected to improve clearing efficiency. Two-six weeks later, hippocampi were imaged using a Nikon A1R MP upright confocal microscope in both single and multi-photon modes. Imaris Imaging software was used for data analysis and quantification. Results: Within two-weeks, samples were sufficiently cleared for deep imaging. Endogenous GFP expression was not adversely affected. This methodology allowed us to image all the way through the hippocampus, from granule cells at the interior surface of the hippocampus, to CA1 stratum oriens at the exterior surface, a depth of greater than 1.0 mm. From this data, neurons from all hippocampal axes may be reconstructed with micron-level precision allowing for quantification of cell density, spine number, mossy fiber sprouting, and other potentially important structural changes that have been observed during epileptogenesis. Conclusions: While the numerous anatomical changes already described in the epileptic hippocampus are undeniably important, there may be significant - yet to be discovered - changes among longitudinal and laminar circuits that have resisted identification with traditional methods. With sufficient computing power, it should be possible to reconstruct the entire hippocampus with micron-level resolution. This approach should greatly facilitate assessing long-projection circuit changes in temporal lobe epilepsy.