Computational protocol: Ventromedial prefrontal cortex mediates sex differences in persistent cognitive drive for food

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Protocol publication

[…] The acquisition of images was conducted with a Zeiss Axio Image Z2 fluorescence microscope (Carl Zeiss Microscopy GmbH; Jena, Germany) and attached Hamamatsu ORCA-R2 camera (Bridgewater, NJ, USA). To determine the location and extent of the AAV expression, images of the areas with viral expression and the adjacent thionin-stained tissue were acquired at 10x. Neuroanatomical borders were drawn onto the thionin-stained image and then transposed to the adjacent fluorescent image using ImageJ software (NIH). The spread of AAV expression were then drawn on computerized versions of the standard rat brain atlas templates using illustration software (Adobe Illustrator CS5.5). Based on this analysis, well-defined and localized AAVs expressions were identified and included for data analysis (at least 50% in the prelimbic and infralimbic regions at bregma AP +3.60, +3.20 or +2.80; with less than 25% of the injection spread outside this target region). Only these subjects were included in behavioral data analysis. For Fos analysis, 2 × 2 tiled images were taken at 20× (tissue area approximately 820 × 630 μm) in the prelimbic area of the vmPFC (+3.20 mm AP from bregma). Three brains were excluded from Fos analysis (one male Control Virus + CNO from Experiment 2, one female DREADD + CNO and one female Control Virus + CNO from Experiment 3) due to inadequate viral expression in the prelimbic area at bregma +3.20 mm AP. Images were pseudocolored with green for DREADDs, red for Fos and blue for DAPI (nuclear counterstain). Single AAV, single Fos and double-labeled (AAV and Fos) cells were determined based on characteristic nuclear (Fos) or cytoplasmic (AAV) labeling by an investigator ‘blind’ to experimental condition. Single (AAV or Fos) and double-labeled (AAV and Fos) cells were counted. Double-labeled cells were expressed as a percentage of total AAV cells and total Fos cells. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens