Computational protocol: Early Social Isolation Stress and Perinatal NMDA Receptor Antagonist Treatment Induce Changes in the Structure and Neurochemistry of Inhibitory Neurons of the Adult Amygdala and Prefrontal Cortex

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Protocol publication

[…] A volumetric analysis of the different nuclei of the amygdala (central, lateral, medial, basolateral, and basomedial) and regions of the PFC (infralimbic, prelimbic, dorsal cingulate, and ventral cingulate cortices) were performed by processing confocal images using the Volumest plugin of FIJI/ImageJ Software (NIH; ). To be able to differentiate the different regions of interest in the subsets of slices, 50-μm-thick sections from the GIN structure set of mice (n = 32) were counterstained with DAPI (1:10,000 in H2O, Sigma-Aldrich). Images of the regions-of-interest were acquired using a confocal microscope (Olympus FV-10). Volumes were estimated using Cavalieri's principle (). [...] All the structural parameters of the GAD-EGFP interneurons and THY1-YFP pyramidal neurons were studied using a laser scanning confocal microscope (Leica TCS SPE). In the case of pyramidal neurons, we focused our studies on the PFC, in particular, on the cingulated cortices because, due to the straight projection of the principal dendrite of pyramidal neurons to layer 1, it is only visible coronally in these two subregions of the PFC. To be consistent, interneurons were studied in the same subregions of the PFC. In addition, we studied the arborization and dendritic spine density of interneurons in the basolateral nucleus of the amygdala, which is known to project directly to the PFC and has an abundant population of fluorescent interneurons in GIN mice. Unfortunately, due to the overwhelming expression of the YFP in the amygdala, we could not to perform these structural analyses for pyramidal neurons.For the study of the dendritic arborization, six GAD-GFP expressing interneurons per animal and region were randomly selected (). Z-series of optical sections (0.8 μm step size; 40× objective) covering the dendritic tree of selected interneurons were obtained using the sequential scanning mode. To be suitable for analysis, these interneurons had to fulfill the following features: (1) the cell must not show any truncated dendrites, (2) the dendritic arbor of the cell must show at least a process with a length >150 μm, and (3) the soma must be located at least 30 μm deep from the surface of the tissue. The stacks obtained were then processed using FIJI (ImageJ, NIH) software to render 3D reconstructions. Neurons were traced using the “Simple neurite tracer” plugin, which also allowed us to analyze their Sholl profile in 3D (; ). The Sholl analysis consists on the measure of the number of intersections of the dendrites with spheres of increasing radius centered in the soma. The separation among the spheres of the analysis was set at 20 μm. For each animal, mean ± SEM was calculated and statistics were performed using the number of animals and the sample number (n; see below).For the analysis of dendritic spines, six GAD-GFP expressing interneurons and six THY1-YFP expressing pyramidal neurons per animal and region were randomly selected (). A 63× oil immersion objective and a 3.5× additional digital zoom were used to observe the first 150 µm of the dendrite in the case of interneurons and the first 200 µm of the dendrite in the case of pyramidal neurons in segments of 50 µm (Z-step size of 0.38 µm). Dendrites within EGFP- and YFP-positive interneurons were randomly selected, but they had to meet the following criteria to be included in the study: (1) their length should be at least 150 µm (for interneurons) or 200 µm (for pyramidal neurons), and (2) no other dendrites should be found crossing their trajectory. For interneurons, data were expressed as the total number of spines in the proximal (0–50 µm), medial (50–100 µm), and distal (100–150 µm) segments of the dendrite, depending on its distance from the soma. For pyramidal neurons, four segments were established: proximal (0–50 µm), medial (50–100 µm), medial-distal (100–150 µm), and distal (150–200 µm). The total number of spines in every dendrite (sum of the spines in the entire segment) was also analyzed. For each animal, mean ± SEM was calculated and statistics were performed using the number of animals and the sample number (n; see below). […]

Pipeline specifications

Software tools ImageJ, Simple Neurite Tracer, Sholl analysis
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals N-Methylaspartate, Dizocilpine Maleate