Whole brain Fos+ cell distribution after a 1h nesting task in virgin and pregnant female mice (from Topilko et al. Neuron 2022)

Brain-wide Fos maps identify the peptidergic neurons of the Edinger-Westphal nucleus as selectively more active in pregnant female nest builders (Topilko et al, Neuron 2022)

With this dataset, we searched for the brain activity signature of nesting specific to pregnant females. For this, we performed a screen based on the whole-brain expression of the immediate early gene Fos, using iDISCO+ and ClearMap (Renier et al., 2016). After habituation to handling, half of a cohort was mated. At 14 to 15 days after mating, we further split the groups into two test conditions, in which the mice received either only the control object or fresh nesting material. Mice were perfused 1h after the onset of nesting and processed for 3D Fos immunolabeling and light-sheet imaging. This experiment generated 3 groups (females: virgin, pseudopregnant, and pregnant. males: virgin or mated) with two test conditions each (nestlets or objects). We generated a list of brain regions that were significantly more active in nesting mice over the control condition for each group.

Important information on files:

In this upload, the region-based statistics output from ClearMap are provided as CSV files, and the voxel maps are provided at Tiff files (either self-contained tif, or raw format with mhd headers). Those image stacks are voxelized single cell Fos density data, aligned to the Allen Brain Atlas template (CCFv3). Important: check carefully the orientation and crop of the atlas, using the .raw heatmaps for guidance (files are shown as hemi-brains, right side, ventral side "up" in y axis, midline left on x axis, and rostral side "up" in z axis). Image dimensions should be 279x320x528, a crop from a similar orientation in the CCFv3 25µm reference space.

files with no specific indications are from female mice. Files from male mice have a "males_" prefix.

Methods

Behavior:

At E14-E16 for plugged females, or 1 to 2 weeks following isolation and daily scoring of nests, mice were habituated to a novel home cage suited for video recording for 24h in a video-recording room with separate lighting control. The cages were filled with half of the bedding from the previous home cage. On the dark phase preceding the recording, the nesting material was removed. At the beginning of the following light phase, the live video recording system was turned on and fresh nesting material was added to the cage (~7g = 3 Cotton nestlets (Serlab, France “Nestlets”)). 1 hour following the initiation of nest-building behavior, mice were euthanized with an intra-peritoneal (IP) injection of Pentobarbital 200 mg/kg IP (Euthasol), briefly perfused with PBS (1X) (~10mL) and fixed with intracardiac perfusion of 4% PFA in PBS (1X) (~50mL). The brains were dissected and post-fixed for 2 hours at 4°C in 4% PFA in PBS (1X).

iDISCO+ processing:

Whole brain vasculature staining was performed following the iDISCO+ protocol previously described previously (Renier et al., 2016) with minimal modifications. All the steps of the protocol were done at room temperature with gentle shaking unless otherwise specified. All the buffers were supplemented with 0,01% Sodium Azide (Sigma-Aldrich, Germany) to prevent bacterial and fungal growth.

Perfused brains were dehydrated in an increasing series of methanol (Sigma-Aldrich, France) dilutions in water (washes of 1 hour in methanol 20%, 40%, 60%, 80% and 100%). An additional wash of 2 hours in methanol 100% was done to remove residual water. Once dehydrated, samples were incubated overnight in a solution containing a 66% dichloromethane (Sigma-Aldrich, Germany) in methanol, and then washed twice in methanol 100% (4 hours each wash). Samples were then bleached overnight at 4ºC in methanol containing a 5% of hydrogen peroxide (Sigma-Aldrich). Rehydration was done by incubating the samples in methanol 60%, 40% and 20% (1 hour each wash). After methanol pretreatment, samples were washed in PBS twice 15 minutes and 1 hour in PBS containing a 0,2% of Triton X-100 (Sigma-Aldrich) and further permeabilized by a 24 hours incubation at 37ºC in Permeabilization Solution, composed by 20% dimethyl sulfoxide (Sigma-Aldrich), 2,3% Glycine (Sigma-Aldrich, USA) in PBS-T. In order to start the immunostaining, samples were first blocked with 0,2% gelatin (Sigma-Aldrich) in PBS-T for 24 hours at 37ºC, the same blocking buffer was used to prepare antibody solutions. Primary antibodies (Rabbit-antiFos, Synaptic Systems, 226-003) were incubated for 10 days at 37ºC with gentle shaking, then washed in PBS-T (twice 1 hour and then overnight), and finally newly incubated for 10 days with secondary antibodies. Secondary antibodies raised in donkeys, conjugated to Alexa 647 were used (Life Technologies). After immunostaining, the samples were washed in PBS-T (twice 1 hour and then overnight), dehydrated in a methanol/water increasing concentration series (20%, 40%, 60%, 80%, 100% one hour each and then methanol 100% overnight), followed by a wash in 66% dichloromethane – 33% methanol for 3 hours. Methanol was washed out with two final washes in dichloromethane 100% (15 min each) and finally the samples were cleared and stored in dibenzyl ether (Sigma-Aldrich) until light sheet imaging.

Light sheet microscopy:

The acquisitions were done on a LaVision Ultramicroscope II equipped with infinity-corrected objectives. The microscope was installed on an active vibration filtration device, itself put on a marble compressed-air table. Imaging was done with the following filters: 595/40 for Alexa Fluor-555, and -680/30 for Alexa Fluor-647. The microscope was equipped with the following laser lines: OBIS-561nm 100mW, OBIS-639nm 70mW, and used the 2nd generation LaVision beam combiner. The images were acquired with an Andor CMOS sNEO camera. Main acquisitions were done with the LVMI-Fluor 4X/O.3 WD6 LaVision Biotec objective. The microscope was connected to a computer equipped with SSD drives to speed up the acquisition.

The brain was positioned in sagittal orientation, cortex side facing the light sheet, to maximize image quality and consistency.

A field of view of 1000 x 1300 pixels was cropped at the center of the camera sensor. The light sheet numerical aperture was set to NA-0.03. The 3 light sheets facing the cortex were used, while the other side illumination was deactivated to improve the axial resolution. Beam width was set to the maximum. Laser powers were set to 40-60% (639nm). The center of the light sheet in x was carefully calibrated to the center of the field. z steps were set to 6µm. Tile overlaps were set to 10%. The whole acquisition takes about 1h per hemisphere.

At the end of the acquisition, the objective is changed to a MI PLAN 1.1X/0.1 for the reference scan at 488nm excitation (tissue autofluorescence). The field of view is cropped to the size of the brain, and the z-steps are set to 6µm, and light sheet numerical aperture to 0.03 NA. It is important to crop the field of view to the size of the brain for subsequent alignment steps.

Data analysis:

Tiled acquisitions of Fos-immunolabeled iDISCO+ cleared brains scanned with the light sheet microscope were processed with ClearMap 2 (https://github.com/ChristophKirst/)(Kirst et al., 2020; Renier et al., 2016) to generate both voxel maps of Fos cell densities, as well as region-based statistics of cell counts. The stitching was done with Wobbly Stitcher (Kirst et al., 2020) and the cell detection was run with parameters and filters identical to our previous work (Renier et al., 2016). Stitched images were processed for background removal, on which local maxima were detected to place initial seeds for the cells. A watershed was done on each seed to estimate the volume of the cell, and the cells were filtered according to their volume to exclude smaller artefactual maxima.

The alignment of the brain to the Allen Brain Atlas (March 2017) was based on the acquired autofluorescence image using Elastix (https://elastix.lumc.nl)(Shamonin et al., 2014). Filtered cell’s coordinates were transformed to their reference coordinate in the Allen Brain Atlas common coordinate system (Wang et al., 2020). For voxel maps, spheres of 375µm diameter were drawn on each filtered cell. P-Value maps of significant differences between groups were generated using Mann-Whitney U test (SciPy implementation). Aligned voxelized datasets from each group of animals were manually inspected to identify the regional overlaps of p-value clusters, and volcano-plots of regional counts where generated.

ITK-Snap (Yushkevich et al., 2006) was used to manually annotate the EWcp nucleus, based on a scan of iDISCO+-labeled brain with Ucn1 aligned onto the 25µm template.