Processing of brain tissue and calculation of brain region
volumes
We quantified the volumes of brain regions based on 3D reconstructions
of serial-sectioned tissue as published in Lázaro et al. (2018b).
Briefly, brains were cut sagittally in half and the two hemispheres were
weighed to the nearest 0.001 g. Then we fixated them for two weeks in
PBS/4% paraformaldehyde and then transferred them to PBS/0.1% sodium
azide at 4 °C for long- term storage. We used the left hemispheres for
all volume reconstructions. Before sectioning, we immersed the
hemispheres in a series of PBS/10, 20 and 30% sucrose for
cryoprotection. We cut the tissue in the coronal plane on a freezing
sliding microtone (Reichert- Jung Hn-40) to obtain series of 30 µm-thick
sections, of which we mounted every fifth section on microscope slides
and stained them with 0.5% cresyl violet. We measured the following
brain regions: olfactory bulb, neocortex, rhinal and piriform cortices,
caudoputamen, amygdala, nucleus accumbens, thalamus, hypothalamus,
hippocampus, dentate gyrus, CA1, CA2, CA3, subiculum and cerebellum and
the total hemi-sphere (see Lázaro et al. (2018b) for details). To
outline each region on the sections we use the software Neurolucida (MBF
Bioscience, Williston, VT, USA) and we applied the Cavalieri principle
to calculate the volume of each region based on the sum of the outlined
areas multiplied by the section thickness and inter-section distance.
This calculation was made automatically in Neurolucida Explorer. All
data from Radolfzell were previously published in Lázaro et al. (2018b).
We accounted for shrinkage of tissue during the histological process
with a correcting factor. This correcting factor was calculated for each
brain as the quotient between the original hemisphere volume –
determined by dividing the fresh hemisphere mass by the specific gravity
of brain tissue (Stephan, 1960) – and the final volume of the
hemisphere as determined by our reconstruction. The correction factor
for each brain was then applied to the brain regions of that specimen.
We also size corrected brain region volumes by the upper tooth row
(which does not change in length over the year) obtained from X-ray
images (Lázaro et al. , 2017).