This data collection contains spatially resolved single-cell transcriptomics datasets acquired using MERFISH on the mouse hypothalamus collected by the Xiaowei Zhuang and Catherine Dulac Laboratories at Harvard University and Howard Hughes Medical Institute. * The dataset contains MERFISH images of 42 experiments, which include 403 coronal slices of the mouse hypothalamus region (10 um thick slices, every 100 um interval) collected from 12 animals exposed to different behavioral conditions including parenting (par), aggression (agg), infanticidal (inf), and sickness induced by lipopolysaccharide (LPS). For each mouse, 3-4 experiments were performed to span the right half of the hypothalamus (from anterior to posterior), and each experiment was named by animal id (Mouse_male for the virgin males and Mouse_mo for the mothers) plus the behavior id and sample id, e.g. mouse_male_agg1_sample1, mouse_mo_par1_sample3 (see details in the section below). For each experiment, multiple coronal slices (7 - 12 slices) were included on the same coverslip and were imaged together. Mouse_male_agg1 – corresponds to a male mouse (replicate 1) exposed to a castrated male intruder who has been painted with adult-male urine over the anogenital area and the head-nose area, and showed aggression towards the intruder. Mouse_male_agg2 – corresponds to a male mouse (replicate 2) exposed to a castrated male intruder who has been painted with adult-male urine over the anogenital area and the head-nose area, showed aggression towards the intruder. Mouse_mo_agg1 – corresponds to a mother mouse (replicate 1) exposed to a juvenile intruder of a different strain and showed aggression towards the intruder. Mouse_ mo_agg2 – corresponds to a mother mouse (replicate 2) exposed to a juvenile intruder of a different strain and showed aggression towards the intruder. Mouse_male_inf1 – corresponds to a male mouse (replicate 1) who showed lethal aggression to three pups introduced to his home cage. Mouse_male_inf2 – corresponds to a male mouse (replicate 2) who showed lethal aggression to three pups introduced to his home cage. Mouse_mo_par1 – corresponds to a mother mouse (replicate 1) exposed to three pups introduced to her home cage and showed parenting behavior. Mouse_mo_par2 – corresponds to a mother mouse (replicate 2) exposed to three pups introduced to her home cage and showed parenting behavior. Mouse_male_LPS1 – corresponds to a male mouse (replicate 1) exposed to intraperitoneal injection of lipopolysaccharide. Mouse_male_LPS2 – corresponds to a male mouse (replicate 2) exposed to intraperitoneal injection of lipopolysaccharide. Mouse_male_PBS1 – corresponds to a male mouse (replicate 1) exposed to intraperitoneal injection of phosphate buffered saline. Mouse_male_PBS2 – corresponds to a male mouse (replicate 2) exposed to intraperitoneal injection of phosphate buffered saline. * In this dataset, a total of 360 genes were imaged. Among the 360 genes, 350 genes were imaged using MERFISH, which encodes individual genes with error-robust barcodes (22-bit, Hamming distance 4, Hamming weight 4, binary code), imprints the barcodes onto the RNAs using combinatorial labeling with encoding probes, and measures the barcodes bit-by-bit using sequential hybridization of readout probes. The 22 bits are imaged in 11 hybridization rounds with two-color imaging each round. The remaining 10 genes were imaged by 5 sequential rounds of two-color FISH and an additional such round was acquired using no fluorescent probes to estimate local background. In addition to gene imaging, additional imaging rounds were included to image markers (polyT and DAPI) for cell segmentation. All bits used for MERFISH imaging of the 360 genes and all segmentation markers were imaged in 2 z-planes spaced apart 1.5 um. * Each of the subdirectory folders named by animal id contains the raw images of one experiment. Each experiment contains many fields of view (FOVs) and each dax (or tif) file in the folder corresponds to the images of one FOV. Raw image files named Epi-545s1-473s2-408s2_{FOV}.dax (or Epi-545s1-473s2-408s2_{FOV}.tif) where {FOV} is the number of the FOV contain 1 z-plane of the fiducial beads and 2 z-planes of polyT and DAPI images. Raw image files named Epi-750s2-635s2-545s1_{FOV}_{round}.dax (or Epi-750s2-635s2-545s1_{FOV}_{round}.tif) where {round} is the number of imaging round with two channels contain 2 z-planes of 750 channel and 650 channel images, and 1 z-plane of the fiducial beads. Inf files contain the data type, frame dimensions and number of frames information corresponding to the FOV, xml files contain filming and stage positions, and off files contain the z-plane positions corresponding to the FOV. See data_organization.csv files for detailed channel information. * The processed_data folder contains the subdirectories name by animal id, each of which contains the following files: • {animal_id}_{behavior_id}_{sample_id}_cellSegmentation.csv: Segmented cell boundary coordinates (x,y,z) in the unit of microns for each cell. The first column (cellID) is a unique cell identifier, the second column (z_height) is height of the 2 z-planes relative to the coverslip. The third and fourth columns (x-boundary, y_boundary) are the x and y boundary coordinates, respectively, arranged as a 3-tiered list (list of lists of lists), wherein the top tier corresponds to the z-heights (as ordered in the second column), the second tier corresponds to the number of discrete areas of the segmentation for each z plane (note that in most cases, this is a single area, but in some instances the cell boundary was split into more than one discrete area), and the third tier (lowest) contains a list of the boundary coordinates. Finally, the fifth column (slice_id) gives the slice number to which each cell belongs. Note that the same coordinate system is used for the cell boundaries and decoded spots (below). • spots_{animal_id}_{behavior_id}_{sample_id}.csv: Decoded spot location (x,y,z) in the unit of microns and their target gene identity for each experiment. The same coordinate system was used in cellSegmentation.csv and spots_{sample_id}.csv for each experiment, and hence the spots can be parsed into the segmented cells by comparison of their coordinates with the cell boundary locations. • counts.h5ad: Cell by gene matrix of the whole dataset. We also provide these files that are associated to this dataset: • genes_combinatorial.txt: List of target genes that are imaged in combinatorial imaging rounds. • genes_sequential.txt: List of target genes that are imaged sequentially after the combinatorial imaging rounds. • probes.fasta: Provides sequences all encoding probes used for hybridization • codebook.csv: Provides the barcodes that encode individual genes measured in the combinatorial imaging rounds. • dataset_group.csv: Provides group_id for datasets. Each group takes its specific data_organization_{group_id}.csv and microscope_{ group_id}.json (below) • data_organization_{group_id }.csv: Provides information on how individual channels and z-planes are ordered in the multi-frame dax(or tif) file for each field of view of raw images specific to the datasets in {group_id}. • microscope_{group_id }.json: Provides parameters of the microscope that were used with pixel size and orientation of images specific to the datasets in {group_id}. • slice_FOV folder: contains csv files, named by {animal_id_{behavior_id}_{sample_id}. Each file provides the set of the FOVs that corresponds to each slice according to its slice_id. • fov_coordinates folder: contains csv files, named by {animal_id}_{behavior_id}_{sample_id}. Each file contains two columns, the x and y coordinates of the FOV in µm. These coordinates can be used to arrange the FOVs of an experiment as they were arranged on the coverslip.