Premium
Using an Optical Clearing Technique for Visualizing the Midbrain Cholinergic System in Mice
Author(s) -
De SantisSmith Alexandria,
Conciatori Tristan,
Allman Brian,
Rogers Kem,
Schmid Susanne
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.706.11
Subject(s) - cholinergic , midbrain , confocal , cholinergic neuron , confocal microscopy , coronal plane , neuroscience , slice preparation , anatomy , chemistry , microbiology and biotechnology , biology , biophysics , central nervous system , optics , physics
The 3D visualization of the midbrain cholinergic system is key for understanding its function and its relationship with other brain areas. In preliminary experiments, our lab used an optical clearing technique, CLARITY, along with confocal microscopy, to image cholinergic cells and projections in up to 4 mm thick brain slices. Brains from transgenic mice expressing YFP under the choline aceteyltransferase 1 (CHaT1) promotor were harvested and sliced into coronal sections of different thicknesses. The samples were then embedded into a hydrogel monomer solution for 3 hours and left to incubate at 37°C. Afterwards, the tissue samples were left in a clearing solution for lipid membrane removal and left to incubate at 37 °C between 19 to 44 days.When qualitatively studied, passive CLARITY was found to effectively clear coronal sections of 1, 2, 3 and 4 mm thick after 19, 26, 35 and 44 days of incubation, respectively. Confocal microscopy confirmed sufficient expression of YFP to visualize thin axons and single axon terminals of cholinergic neurons within the rostral regions of the mouse brain. Based on our preliminary results, our ultimate goal is to construct a 3D visualization of the entire midbrain cholinergic system and its projections in the mouse brain.