Ethics statement
The study was conducted according to the guidelines of the ARRIVE. All experiments were performed according to German guidelines on animal welfare under the supervision of the local ethics committees and were approved by the Landesamt fürGesundheit und Soziales, Berlin; Permit numbers: G0170/15, T0160/14 andT0078/16.
Animals and tissue
Etruscan shrews used in this study were housed in terraria containing a layer of dry soil, moss, and broken flowerpots. Crickets were provided as a food source, with water ad libitum. The detailed housing conditions were as previously described 10. The adult rat (6 months) photographed next to the stuffed shrew was used in another experimental study and was obtained from Janvier Laboratories (Le Genest-Saint-Isle, France).
For coronal brain slices, animals were euthanized by isoflurane and then perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). After postfixation in 0.1 M PB, the brains were immersed in 30% sucrose solution for cryoprotection until they sank to the bottom of the vial. All brains were embedded in a mixture of egg yolk, and 30% sucrose supplemented by 0.75 mL glutaraldehyde and mounted on a cryostat (Lecia 2035 Biocut) to obtain 30- or 40-µm-thick coronal sections. Brains were then mounted on glass slides for Nissl staining 11.
Torpor behavior
Etruscan shrews were housed in a reverse 12 hr light/dark cycle for convenience. Torpor occurred preferentially at long intervals after the last feeding at the beginning of the dark cycle. After ~8 hours fasting, Etruscan shrews start to enter torpor with a significant decrease in body temperature and activity. The surface body temperature of the Etruscan shrews was measured manually, taking thermal images using FLIR C5 thermal camera.
Stereology
Coronal brain slices from Etruscan shrews and rats were examined with StereoInvestigator software (MBF Bioscience, Wilistron, VT), employing an Olympus (Tokyo, Japan) BX5 1 microscope with an MBFCX9000 camera (MBF Bioscience) mounted on the microscope. The microscope was equipped with a motorized stage (LUDL Electronics, Hawthorne, CA) and a z-encoder (Heidenhain, Schaumburg, IL).
For estimating the hemispheric volume, we used Stereoinvestigator/Neurolucida software to draw the contour of the pia in every fourth slice of the Etruscan shrew and every twelfth slice of the rat. Contours of the MPA were drawn in each relevant slice for both Etruscan shrews and rats. We used Neurolucida/neuroexplorer software to calculate the cumulative surface area of all contours, and volumes were calculated by multiplying by the corresponding thickness of the slides.
For cell counting in the MPA, we employed a standard stereological sampling scheme called the optical fractionator method (MBL Stereoinvestigator). Our region of interest (MPA) was identified and outlined at low magnification, and neurons were identified by their shape and staining intensity at high magnification. For counting, we evaluated the contours of the MPA in each slice containing the MPA in Etruscan shrews and for every second slice in rats. Optical dissectors were randomly placed on a series of sections, and we manually labelled the number of nucleoli that came into focus and lay within the defined lines of the dissector. This method provided an unbiased estimation because the number of neurons is estimated directly, without referring to neuron densities. We specifically used 15 x 15 µm counting frames and counted on average 0-4 neurons per frame.
Statistics
Statistics were performed in excel and Matlab. Bar graphs were generated in Matlab.