Laser capture microdissection

Fourteen sections of 14 μm thickness were sectioned from each frozen hippocampal sample, and sections were collected onto polyethylene terephthalate metal frame slides for laser capture microdissection (Leica, Milton Keynes, UK). Two additional sections were collected onto a microscopic slide (VWR International, UK) and stained briefly in 0.1% toluidine blue (pH 4.5) solution for 5 s to visualize the granule cell layer. laser capture microdissection (LCM 700; Leica, Milton Keynes, UK) was then carried out along the entire length of the GCL of multiple sections per case to capture basal and dispersed DGCs (Figure 2A). Basal samples included DGCs in the granule cell layer closest to CA4, while the dispersed samples included ectopic DGCs in the outer-granular layer and inner and outer molecular layers. A total tissue area of 9 ± 1 mm² was dissected for each case, and submitted for proteomic analysis.

The brains were isolated from decapitated animals, rapidly frozen using isopentane on dry ice, and stored at −80 °C until further stages. Afterwards, the brain Sects. (20 μm) containing the region of interest — the habenula — were cut using a Jung CM 3000 cryostat microtome (Leica, Wetzlar, Germany) according to Rat Brain Atlas [54 ]. The slices were then attached to PEN Membrane Glass Slides 2.0 µm (ThermoFisher Scientific, Waltham, MA, USA) and stained with Cresyl Violet from the LCM Staining Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Using laser capture microdissection (Leica, Wetzlar, Germany), the medial and lateral habenular nuclei were obtained.