Computational protocol: Dopaminergic neurons promote hippocampal reactivation and spatial memory persistence

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Protocol publication

[…] The distribution of transfected axons across brain areas was consistent with the known distribution of dopaminergic fibers. Specificity of virus transfection was quantified from both hemispheres of four bilaterally injected brains. Three consecutive sections centered on the injection coordinate (bregma −3.25) were selected. An exhaustive tiled grid of 20 μm thick (0.8 μm step) z-stacks to span the VTA was acquired from the top of each section using an epifluorescence microscope (Imager.M2 with filter set 38 and a Plan-Neofluar 40×/1.3 objective, Zeiss). Only grid tiles which were deemed to be within the VTA regions (A10 cell groups) were included in the counting. Greater than 900 cells were counted per brain. Three-dimensional reconstruction of transfected dCA1 axons was completed using the same microscope and Neurolucida software (MBF Bioscience). Published images were acquired using a confocal microscope (Imager.Z1 with LSM 710 scan head and Plan-Neofluar 5×/0.16, Plan-Apochromat 40×/1.3, 63×/1.4 or 100×/1.46 objective, Zeiss) in sequential scanning mode with the following excitation laser and emission filter wavelengths. DAPI: 405 nm, 409-499 nm; eYFP/DyLight488: 488 nm, 493-571 nm; Cy3: 543 nm, 566-729 nm. Acquisition and analysis were completed using ZEN 2008, v5.0 (Zeiss) and ImageJ (rsb.info.nih.gov) software. Z-stacks were flattened using maximum intensity projection. […]

Pipeline specifications

Software tools Neurolucida, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus