DOI: 10.5061/dryad.fqz612jxv
title: Data from: Evolution of cellular architecture and function of the hippocampus: Insights from the artificial selection experiment

description: Inter-specifically, mammalian species having larger brains built of more
 numerous neurons have higher cognitive abilities (CA), but at the expense
 of higher metabolic costs. It is unclear, however, how this pattern
 emerged, since evolutionary mechanisms act intra-specifically, not
 inter-specifically. Here we tested the existence of the above pattern at
 the species level in the hippocampus- the brain structure underlying CA.
 We used the experimental evolution model system consisting of lines of
 laboratory mice divergently selected for basal metabolic rate (BMR) - a
 trait implicated in the evolution of brain size, its metabolic costs and
 CA. Selection on BMR did not affect hippocampus size. Yet, the high BMR
 mice had superior CA and manifested increased neuronal density, higher
 cytochrome C oxidase density (indexing metabolic costs of neuronal
 activity) and dendritic spine density (indexing connectivity between
 neurons). Thus, our study calls into question generality of patterns of
 the evolution of CA apparent interspecifically. At the species level
 increased CA may arise through re-arrangement of the architecture and
 function of neurons, without a conspicuous increase of their size.
description: <strong>Animals</strong> We used
 3-4-month-old Swiss-Webster female mice from the selection experiment
 carried out at the Faculty of Biology, University of Bialystok. We
 divergently select mice for high/low body mass-corrected Basal Metabolic
 Rate (BMR). In this experiment, we maintain two non-replicated line types:
 the high-BMR (H-BMR) and low-BMR (L-BMR) line type. Individual mice used
 in this study were randomly drawn from a stock of animals not qualified
 for further selection. The divergence achieved in the
 primarily selected trait- BMR is sufficiently large to be confidently
 attributed to the results of the selection, rather than to genetic drift..
 Still, however, a straightforward statistical comparison of both selection
 line types with respect to the traits that are not directly selected, such
 as those analyzed in our study is questionable, because in their case one
 cannot rule out that the differences between the L-BMR and H-BMR mice were
 due to random effects unrelated the applied selection. To remedy this
 problem, we also used female Swiss-Webster mice from four random-bred
 lines maintained concurrently with the selection on BMR. Mice of these
 lines formed the RB line type, serving as a reference to the divergence of
 traits analyzed therein in animals selected for BMR.
 Each mouse used in our study was randomly selected from a
 separate family belonging to a particular line. For information on the
 number of animals used in specific analyzes see electronic Supplementary
 Materials. <strong>Perfusion and tissue
 preparation</strong> Mice were deeply
 anesthetized with sodium pentobarbital (133.3 mg/ml), pentobarbital (26.7
 mg/ml), and morbital, perfused transcardially with PBS (4°C, 50 ml per
 animal) and subsequently with 4% PFA (4°C, 70 ml per animal). Brains were
 dissected, postfixed in 4% PFA (o/n, 4°C), and cryoprotected in 30%
 sucrose solution (4°C) for at least 3 days. Coronal sections containing
 the hippocampus (coordinates following Paxinos and Franklin’s atlas, 2001)
 were cut with the use of a cryostat (-20°C) and kept in PBS (4°C) until
 further handling. <strong>Hippocampus size,
 hippocampal pyramidal area, and neuronal density estimated by Nissl
 staining</strong>
 <strong> </strong>We used 14, 3, and 3 animals from
 the RB, L-BMR, and H-BMR line types, respectively. Out of 14 animals of
 the RB line type, 5, 3, 3, and 3 belonged to the respective replicated
 lines forming this line type. Transverse (coronal) 40
 μm cross-sections of the hippocampus positioned -1.46; -1.82; -2.3 and
 -2.7 mm from bregma (thereafter Position, coordinates according to Paxinos
 and Franklin were mounted on gelatin-coated slides and then dried,
 dehydrated, and stained according to the standard Nissl method: slides
 were washed in 0.1% cresyl violet solution (cresyl violet 0.1g; distilled
 water 50 ml; acetate buffer 0.2 M pH 3.6 50 ml) for 3-10 min and then
 dehydrated through graded alcohols (70, 95, 100, 100%) and xylenes. Next,
 the slides were cleared in xylol and bonded with coverslip by means of
 DePeX (Serva Electrophoresis<sup>TM</sup>).
 The microscope image was captured using a light microscope Leica
 DM1000 LED connected to a Leica ICC50 camera, at 400x and 40x
 magnification for neuron counting and hippocampal measurements,
 respectively. The total cross-sectional surface area
 (µm<sup>2</sup>) of the hippocampus (being a proxy of its
 size), as well as the pyramidal cell layer area
 (µm<sup>2</sup>,<sup>
 </sup><em>sensu</em> ‘superficial’ cell layer) were
 traced manually and then measured at each Position by means of CellSens
 Dimension Desktop (Olympus Corp., Japan). Neuronal
 density was estimated in the pyramidal hippocampal cell layer in CA1, CA2,
 and CA3 regions of the right and left side, in the same cross-sections as
 used for the estimation of the area occupied by the pyramidal cell layer.
 Neurons were counted inside rectangle frames of 3000
 μm<sup>2</sup> (counting frame) localized at random in the
 central part of each of the regions. In sum, four frames (each
 representing one Position) were analyzed for each hippocampal region, then
 measurements were averaged within a given region. To
 obtain an unbiased estimate of the neuron number within the frame we
 followed guidelines formulated by Gundersen (44), that is, only neurons
 crossed by the bottom and right-hand side borders of the frame were taken
 into account. Conversely, all neurons crossed by the left-hand side and
 upper borders of the frame were not accounted for.
 <strong>Neuronal density estimation by DAPI
 staining</strong>
 <strong> </strong>To validate the estimates of
 neuronal density in the pyramidal hippocampal layer obtained by means of
 Nissl staining, we also used DAPI staining. In these analyses, we used a
 separate set of 3, 3, and 4 animals randomly chosen from the RB, H-BMR,
 and L-BMR lines types, respectively. Forty μm coronal
 sections of hippocampus, positioned -2.06 mm from bregma (coordinates
 following Paxinos and Franklin’s atlas, 42), were mounted on
 gelatin-coated slides covered with Vectashield mounting medium containing
 DAPI.  Next, the sections were photographed by means of a Zeiss LSM 780
 confocal microscope, using 63 times magnifying lens for high-resolution
 imaging (Plan Apochromat 63x/1.4 Oil DIC, Zeiss) at identical capturing
 parameters for all images. We assumed that neuronal
 density was equivalent to the density of neuronal nuclei positively
 stained with DAPI. Nuclei were counted by means of ImageJ (NIH) software,
 allowing for the discrimination of neuronal and non-neuronal nuclei by
 their size and shape. The density was estimated in the pyramidal
 hippocampal cell layer, in CA1, and CA3 regions of the right and left side
 of the hippocampus. Due to technical problems with staining, we were
 unable to collect reliable density estimates for the CA2 region. Eight
 non-overlapping frames of 2890 μm<sup>2</sup> (counting
 frames) were picked at random in the central part of each of the regions.
 As in the case of the Nissl staining, we obtained unbiased estimates of
 the neuron following guidelines formulated by Gundersen.
 <strong>Anti-GFAP immunostaining</strong>
 We measured hippocampal astrocyte/neuron ratio (estimated as the
 proportion of the surface area occupied by astrocytes immunostained with
 Anti-GFAP vs. neurons) in a separate set of 18, 3, and 4 animals coming
 from the RB, H-BMR and L-BMR lines types, respectively.
 Sections from the perfused brain were blocked with NDS (normal
 donkey serum). After the wash, sections were incubated in 0.1% Triton
 X-100 in PBS (PBST) for 15 minutes. Then, after preincubation in
 NDS-buffer (5% NDS in PBST) for 1 hour, the chicken anti-GFAP (Abcam,
 1:800, Cambridge, MA, USA,) antibody (Ab) was added for overnight
 incubation at 4°C. After washing cycles in PBST (3 x 1 min), sections were
 incubated for 2 hours at room temperature with secondary antibody 647
 Alexa Fluor (Jackson ImmunoResearch, West Grove, PA) at 1:400
 concentration to detect GFAP. After the last round of washing with PBST (3
 x 5 min, RT) slices were mounted (Fluoromount-G, Southern Biotech) on
 polysine slides. To control for non-specific binding and imaging artifacts
 ‘no primary antibody’ and ‘no secondary antibody’ immunostainings were
 carried out. Imaging was performed using an Olympus microscope (Olympus
 Corporation, Japan) with RGB Camera Hamamatsu ORCA Flash4.0 V2 and at 20x
 magnification for astrocyte counting. GFAP (astrocyte) immunoreactivity in
 the hippocampus (ROI) was assessed in CA1-CA3 regions using to the ImageJ
 (NIH) macro. <strong>Cytochrome oxidase (CCO)
 activity staining</strong> In this assay, we used
 12, 5, and 5 animals from the RB, H-BM,R, and L-BMR line types,
 respectively. Histochemical reaction for cytochrome oxidase was prepared
 according to the published protocol (45). Forty μm coronal sections of the
 hippocampus (located -2.3 mm from bregma, coordinates according to the
 atlas of Paxinos and Franklin, (42) were incubated ina solution of 0.05 PB
 (phosphate buffer), 1 g sucrose, 50 mg ammonium nickel, 25 mg DAB, 15 mg
 cytochrome C, 10 mg catalase and 250 ul imidazole (amount per 100 ml).
 Slices were incubated for 6 - 8 h in 37°C and then washed (3 x 1 min) in a
 0.05 PB. <strong>Dendritic spine density (DSD)
 analysis</strong> Because of logistic
 limitations, the DSD analysis was restricted to the H-BMR and L-BMR mice
 only (5 animals from each line type). Animals were deeply anesthetized
 with sodium pentobarbital (133.3 mg/ml), pentobarbital (26.7mg/ml), and
 morbital. Their brains were dissected and fixed in 1.5% PFA (1h, RT) and
 moved to ice-cold PBS (10 min, 4°C). Next, they were cut into 140 μm
 sections on a vibratome (Leica VT 1000S, Leica Biosystems Nussloch GmbH,
 Wetzlar, Germany). Slices were processed for Dil staining. Dendritic
 spines were visualized using the lipophilic dye Dil
 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, #D282
 Life Technologies, Warsaw, Poland). Random dendrite labeling was performed
 using 1.6 μm tungsten particles (Bio-Rad, Hercules, CA, USA) coated with
 Dil. Dye was delivered to cells using the Gene Gun (Bio-Rad). After
 staining, slices were fixed with 0.4% paraformaldehyde in
 phosphate-buffered saline (PBS; overnight at 4°C) and placed on
 microscopic slides. Z-stacks of dendrites from the CA1, CA3, and the DG
 regions of the hippocampus were acquired using a Zeiss LSM 880 confocal
 microscope with AiryScan on superresolution mode using 63 times magnifying
 lens for high-resolution imaging (Plan Apochromat 63x/1.4 Oil DIC) (Zeiss,
 Poznań, Poland). Dil emission was excited using a HeNe 594 nm laser. For
 each image, the following parameters were applied: 70 nm pixel size, 300
 nm Z-intervals, averaging 4. Maximum intensity projections of Z-stacks
 covering the dendrite length were analyzed using semiautomatic
 SpineMagick! software. It allowed for marking the dendritic spine head and
 base manually. Next, the software automatically marked spine edges that
 were adjusted manually to fully reflect the spine shape.
 For each animal, 7-10 single dendrites from each hippocampus area
 (one dendrite per neuron per image) were analyzed and dendritic spine
 density was calculated as the number of spines per observation field of
 0.05 μm<sup>2</sup>. There were no stained dendrites in the
 CA2 region, so they were excluded from further analyses.
description: # **Evolution of cellular architecture and function of the hippocampus:
 Insights from the artificial selection experiment**
 [https://doi.org/10.5061/dryad.fqz612jxv](https://doi.org/10.5061/dryad.fqz612jxv) **Description of the data and file structure** The Excel file ‘RSBL_Data_File’ contains all raw data used in the analyses in the manuscript accepted in Biology Letters. Subsequent folders contain the following data sets: * **Hippocampal area**: total cross-sectional surface area (µm2) of the hippocampus- the brain structure underlying cognitive abilities. Hippocampal area is used as a proxy of its size. **Missing data denoted as '.'** **Line type**: code of the line type **Subline:** code of the replicated subline within line type **ID** code of an animal **Side** code of the brain side (Left, Right) **P146, P182, P230, P270** cross-sectional surface area (µm2) of the hippocampus area positioned at  -1.46, -1.82, -2.30, -2.70 mm respectively from bregma * **Pyramidal Cell Area**: pyramidal cell layer area (µm2, *sensu* ‘superficial’ cell layer- Slomianka, L, Amrein, I, Knuesel, I, Sørensen, JCh, Wolfer, DP. 2011 Hippocampal pyramidal cells: the reemergence of cortical lamination Brain Struct Funct 216, 301–317.) Pyramidal cells are the primary excitation units of the mammalian hippocampus. **Missing data denoted as '.'** **Line type**: code of the line type **Subline:** code of the replicated subline within line type **ID** code of an animal **Side** code of the brain side (Left, Right) **P146, P182, P230, P270** cross-sectional surface area (µm2) of the hippocampus pyramidal cell layer area positioned at  -1.46, -1.82, -2.30, -2.70 mm respectively from bregma * **Neuronal Density Nissl**: Neuronal density in the pyramidal hippocampal cell layer, estimated by the Nissl staining. Neuronal density is the proxy of neuronal size. **Missing data denoted as '.'** **Line type**: code of the line type **Subline:** code of the replicated subline within line type **ID** code of an animal **Region** hippocampal region (CA1-CA3) **Side** code of the brain side (Left, Right) **P146, P182, P230, P270** neuronal density of the cross positioned at   -1.46, -1.82, -2.30, -2.70 mm respectively from bregma * **Neuronal Density DAPI**: Neuronal density in the pyramidal hippocampal cell layer, estimated by the DAPI staining. **Missing data denoted as '.'** **Line type**: code of the line type **ID** code of an animal **Region** hippocampal region (CA1-CA3) **Side** code of the brain side (Left, Right) **Neurons** number of neurons * **Astrocyte to Neuron**: Astrocyte/Neuron ratio, expressed as the percent of the GFAP signal positive cross- sections of hippocampus. Astrocyte/Neuron ratio informs about the capacity of nourishment of neurons and formation of new connections between them. **Missing data denoted as '.'** **Line type**: code of the line type **Subline:** code of the replicated subline within line type **ID** code of an animal **Side** code of the brain side (Left, Right) **CA1** astrocyte/neuron ration at hippocampal region CA1 **CA2** astrocyte/neuron ration at hippocampal region CA2 **CA3** astrocyte/neuron ration at hippocampal region CA3 * **Spine Density**: Dendritic spine density in hippocampus. The number of dendritic spines in key areas of the hippocampus is a proxy of neuronal connectivity.  **Line type**: code of the line type **ID** code of an animal **Region:** hippocampal region (CA1-CA3) **Spine_Dens**  the number of spines per observation field of 0.05 μm2 * **CCO**: Cytochrome C oxidase density (expressed as the intensity of color in 8- bit scale of gray) of hippocampus—a proxy of metabolic costs of neuronal activity. **Missing data denoted as '.'** **Line type**: code of the line type **Subline:** code of the replicated subline within line type **ID:** code of an animal **CA1_R** Cytochrome C oxidase density in the right side of hippocampal region CA1 **CA2_R**  Cytochrome C oxidase density in the right side of hippocampal region CA2 **CA3_R** Cytochrome C oxidase density in the right side of hippocampal region CA3 **DG_R**  Cytochrome C oxidase density in the left side of hippocampal region DG **CA1_L** Cytochrome C oxidase density in the left side of hippocampal region CA1 **CA2_L** Cytochrome C oxidase density in the left side of hippocampal region CA2 **CA3_L** Cytochrome C oxidase density in the left side of hippocampal region CA3 **DG_L** Cytochrome C oxidase density in the left side of hippocampal region DG **Sharing/Access information** N/A. All data and software codes used in the analyses are deposited therein.