Computational protocol: Esr1+ cells in the ventromedial hypothalamus control female aggression

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Protocol publication

[…] Tiled confocal images were acquired with 2 μm optical thickness using 20×, or 40× objectives (Zeiss LSM 510 or 700 microscope). The image acquisition settings were maintained for each experiment. Z-stacks were acquired for quantifying ChR2-EYFP and c-fos in catFISH experiments. Epifluorescent images were acquired for determining electrode location in (Zeiss Axio). Bright field images were acquired for CISH experiments in (Olympus AX70). ImageJ or Neurolucida (MBF Bioscience) were used to quantify the staining results by an observer blind to the experimental conditions. [...] Three group housed adult Vglut2-ires-cre × Ai6 female mice (10–12 weeks) were decapitated. The brains were flash frozen within 2 minutes and stored at −80 °C until sectioning. 20 μm thick coronal sections were obtained using a cryostat and mounted on slides that were dehydrated in 100% ETOH for 1 min and then dried by using a hair dryer. Slides were stored at −80 °C until use. Given that VMH is composed of largely glutamatergic cells that were surrounded by GABAergic cells and each subdivision of VMH has cell-poor boundary, the ZsGreen labeling in glutamatergic cells enabled recognition of the subdivisions. The anterior one third of the VMHvl was regarded as VMHavl while the remaining VMHvl was regarded as posterior VMHvl. VMHdm, VMHavl, VMHpvlm and VMHpvll was laser-dissected using LMD6000 (Leica; a fluorescent microscope combined with a movable laser for microdissection). Collected tissues were lysed in extraction buffer and total RNA was isolated using PicoPure RNA isolation kit (ThermoFisher). The RNASeq libraries were then prepared using Clontech SMARTer® Stranded Total RNA-Seq Kit (Cat # 635006) and purified using AMPure beads (Beckman coulter). The quantity of the library was examined using Qubit RNA HS assay Kit (Thermo Fisher Scientific) and qPCR. The quality of the library was determined using Bioanalyzer (Agilent). Once the libraries were deemed as high quality (free of adapter dimers and in concentration >2 nM), we pooled 8 samples equimolarly and sequenced them on Illumina HiSeq 2500 using high output mode to achieve greater depth of coverage. Read alignment was carried out using HISAT2 (https://github.com/infphilo/hisat2) against the mouse mm10 reference genome obtained from ENSEMBL database (http://useast.ensembl.org/). The alignment results were then exported to an open source software— Featurecounts to determine the number of reads for each gene. Then, the Bioconductor software package DESeq2 was used to identify genes that were differentially expressed across VMH subregions (adjusted p value < 0.05). Among those significantly differentially expressed genes, we selected a subset of vlm and vll specific genes with moderate to high level of expression (vlm specific: vlm/vll >1.2 and vlm normalized counts > 4 at log2 scale; vll specific: vll/vlm >1.2 and vll normalized counts > 4 at log2 scale) and conducted in situ hybridization of each gene to visualize its expression pattern in the VMH. […]

Pipeline specifications

Software tools ImageJ, Neurolucida
Application Microscopic phenotype analysis
Chemicals Estrogens, Progesterone