Analysis of the effects of unilateral LMAN lesioning on gene expression in birdsong neural circuitry

Analysis of the effects of unilateral LMAN lesioning on gene expression in birdsong neural circuitry Five birds received unilateral LMAN lesions, three with left-hemisphere lesions and two with right-hemisphere lesions. LMAN was electrolytically lesioned using a 100 kOhm platinum/iridium electrode. LMAN was stereotactically located at 4.7 mm AP, 1.7 mm ML, and 2.1 mm DV using a beak angle of 50 degrees. In one hemisphere, five penetrations were made, one at the given coordinates and four more +/- 300 microns from this center position. At each penetration, 100 μA of current at the anode was passed for 60 seconds. At experiment end, birds were euthanized as described in Euthanasia and brain preparation. To assess lesion completeness, 20 um coronal cryosections were collected at 100 um intervals across the anterior-posterior extent of LMAN onto SuperFrost Plus slides (FisherBrand), then Nissl stained as described in Standard Nissl stain, fresh-frozen cryosections. The volume of LMAN was estimated in ImageJ/FIJI by calculating the area of LMAN on the unlesioned side in each section that it was visible, interpolating a smooth curve in R across these measured areas and the known distance between cryosections, then calculating the area under the curve. This procedure was repeated for any residual LMAN visible on the lesioned side, and lesion percentage was calculated as 100 * (1 - [volume LMAN lesioned] / [volume LMAN unlesioned]). LMAN was considered lesioned if more than 25% of volume was spanned by the lesion.