Visual Deprivation Decreases Somatic GAD65 Puncta Number on Layer 2/3 Pyramidal Neurons in Mouse Visual Cortex
Figure 1
Representative examples of GAD65 immunohistochemistry in layer 2/3 of visual cortex obtained from YFP expressing mice that are reared under different experimental conditions. Confocal images obtained from mice that were normal reared for 5 weeks (A1–A4), dark reared from birth until 5 weeks old (B1–B4), and dark reared from birth for 5 weeks that were subsequently exposed to 3 days of light (C1–C4) are shown. Single section images of staining in layer 2/3 of visual cortex are shown in A1, B1, and C1. GAD65 immunoreactivity (Alexa633 conjugated secondary antibody) is shown in red, and YFP expression in a subset of pyramidal neurons is shown in green. A2, B2, and C2 show the plane of each image section. Note GAD65 staining throughout the thickness of the sections. The total thickness of each section was 20m. White arrows point to a neuron in each image field that was used for 3 dimensional (3D) reconstructions as shown in A4, B4, and C4. Single plane cropped images of the soma of selected neurons are shown in A3, B3, and C3. A stack of 20 single plane cropped sections were reconstructed to generate a 3D image using the image analysis program Volocity (Improvision). A constant threshold was applied to all images to get rid of the background in both GAD65 and YFP channels. GAD65 puncta not contacting the soma of YFP neuron were discarded using the image analysis software Volocity (Improvision) to obtain the final 3D images as shown in A4, B4, and C4. These final images were used for quantification.
