Computational protocol: Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities

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Protocol publication

[…] For morphological reconstructions, FSIs were subjected to whole-cell recordings with a pipette solution containing the following (in mM): 150 K gluconate, 4.6 MgCl2, 10 Hepes-K, 1 EGTA-K, 0.4 Na-GTP, 4 Na-ATP, 0.1 CaCl2, pH=7.2 and complemented with 0.4% biocytin. A period of 20 min after whole-cell break-in was respected and a high-resistance outside-out patch was obtained when the pipette was withdrawn. Slices were fixed by overnight immersion in 4% paraformaldehyde at 4 °C, rinsed in phosphate-buffered saline at 0.1 mM and, later, immersed for 2-h periods in a mixture of phosphate-buffered saline/Triton X-100 (0.1 mM and 0.1%, respectively). Biocytin was revealed in red with streptavidin-conjugated NL557 (R&D systems, Minneapolis, MN, USA), diluted 1:5000, always in phosphate-buffered saline/Triton X-100. After a final rinse in Tris-buffered saline (TBS), slices were mounted on coverslips with FlourSave reagent (EMD Millipore, Billerica, MA, USA) and secured with nail polish. Experiments were obtained from 8 to 18 different animals per genotype, which belonged to 5–9 different litters. A set of experiments with n=8 embraced at least five different animals per genotype. Confocal images were acquired using an Axiovert 200M-LSM 510 META microscope (Zeiss) equipped with a C-Apochromat × 40/1.2 and a 543-nm helium–neon laser. Band-pass emission filters were used for selective detection of the endogenous EGFP (500–550 nm) and red-biocytin labeling (565–615 nm). Single FSI images were acquired as 50–70-μm-thick Z-stacks composed of 2048 × 2048 pixel images (pixel size: 0.22 μm) with a Z-step of 0.62 μm. Reconstructions of neuronal projections were performed using the LSM Image Browser (Zeiss) and processed with a median filter (2 pixel radius) from Image J. Dendritic branching was quantified by Sholl analysis and their elongation measured with ‘moment calculator', an ImageJ plug-in (Francois Richard, University of Ottawa; http://imagej.nih.gov/ij/plugins/moments.html). Dendritic distribution was evaluated by tracing vectors from the soma (as the center) and the crossing dendrites within a 100-μm-radius circle. The angular variance of those vectors was calculated for each FSI according to Batschelet. […]

Pipeline specifications

Software tools Sholl analysis, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Genetic Diseases, Inborn