Specimens
We used brains obtained from three cetacean species (harbour porpoise—Phocoena phocoena, minke whale—Balaenoptera acutorostrata, and humpback whale—Megaptera novaeangliae) and 11 artiodactyl species (sand gazelle—Gazella marica, domestic pig—Sus scrofa, Nubian ibex—Capra nubiana, springbok—Antidorcas marsupialis, blesbok—Damaliscus pygargus, greater kudu—Tragelaphus strepsiceros, blue wildebeest—Connochaetes taurinus, dromedary camel—Camelus dromedarius, nyala—Tragelaphus angasii, river hippopotamus—Hippopotamus amphibius, and African buffalo—Syncerus caffer) (Table 1). All artiodactyl brains were perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate buffer through the carotid arteries following euthanasia45. The harbour porpoise specimens were perfusion fixed through the heart following euthanasia, while the minke whale and humpback whale brains were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer. All brains were then stored in an antifreeze solution at – 20 °C until use45. All specimens were taken under appropriate governmental permissions, with ethical clearance provided by the University of the Witwatersrand Animal Ethics Committee (Clearance number 2008/36/1), which uses guidelines similar to those of the National Institutes of Health regarding the use of animals in scientific research and is compliant with ARRIVE guidelines.
Immunohistochemical staining
Blocks of tissue from the anterior cingulate (dorsal to the rostrum of the corpus callosum, in all species apart from the humpback whale where we did not have this tissue block) and occipital cortex (presumably primary visual cortex, from all species) with underlying white matter were taken from each of the specimens. These were placed in a 30% sucrose in 0.1 M phosphate buffer solution at 4 °C until equilibrated. The blocks were frozen in crushed dry ice, mounted on an aluminium stage and sectioned at 50 µm orthogonal to the pial surface. Alternate sections were stained for Nissl (with 1% cresyl violet), UCP1, UCP2, UCP3, UCP4, UCP5, dopamine-ß-hydroxylase (DBH) and tyrosine hydroxylase (TH). To investigate the presence of neural structures immunolocalizing uncoupling proteins, DBH and TH, we used standard immunohistochemical procedures with antibodies directed against UCP1, UCP2, UCP3, UCP4, UCP5, DBH and TH. While immunolocalization for UCP1, UCP4, UCP5, DBH and TH were clear, only occasional cortical neurons were immunopositive for UCP2, and no immunolocalization could be detected for UCP3 in the species studied. It should be noted here that immunostaining for DBH and TH did not work in the humpback whale specimen, perhaps due to the fixation procedure or the conformation of the targeted proteins in this species preventing recognition of the DBH and TH proteins by the antibodies used. Sections used for the Nissl series were mounted on 0.5% gelatine-coated glass slides, cleared in a solution of 1:1 chloroform and absolute alcohol, then stained with 1% cresyl violet to reveal cell bodies. For the immunohistochemical staining, each section was treated with endogenous peroxidase inhibitor (49.2% methanol:49.2% 0.1 M PB:1.6% of 30% H2O2) for 30 min and subsequently subjected to three 10 min 0.1 M PB rinses. Sections were then incubated for 2 h, at room temperature, in blocking buffer (containing 3% normal rabbit serum, NRS, for the UCP1-5 sections/3% normal horse serum, NHS, for the DBH sections/3% normal goat serum, NGS, for the TH sections, plus 2% bovine serum albumin and 0.25% Triton-X in 0.1 M PB). This was followed by three 10 min rinses in 0.1 M PB. The sections were then placed in the primary antibody solution that contained the appropriately diluted primary antibody in blocking buffer for 48 h at 4°C under gentle shacking. The optimal dilutions for the UCP primary antibodies were determined with a series of stains in which the dilution of the primary antibodies ranged from 1:300 through to 1:9600, with any staining in all species being absent at a dilution of 1:4800 irrespective of fixation method. We used antibodies directed against UCP1 (Santa Cruz Biotechnology, C-17, sc-6528, Lot# D0411, goat polyclonal IgG, dilution 1:300, RRID:AB_2304265), UCP2 (Santa Cruz Biotechnology, C-20, sc-6525, Lot# E0211, goat polyclonal IgG, dilution 1:300, RRID:AB_2213585), UCP3 (Santa Cruz Biotechnology, C-20, sc-7756, Lot# A2511, goat polyclonal IgG, dilution 1:300, RRID:AB_2213922), UCP4 (Santa Cruz Biotechnology, N-16, sc-17582, Lot# E2004, goat polyclonal IgG, dilution 1:300, RRID:AB_793648), UCP5 (Santa Cruz Biotechnology, Q-16, sc-50540, Lot# B1207, goat polyclonal IgG, dilution 1:300, RRID:AB_2286101), DBH (Merck-Millipore, MAB308, mouse monoclonal IgG, dilution 1:4000, RRID:AB_2245740) and TH (Merck-Millipore, AB151, rabbit polyclonal IgG, dilution 1:3000, RRID:AB_10000323). This incubation was followed by three 10 min rinses in 0.1 M PB and the sections were then incubated in a secondary antibody solution (1:1000 dilution of biotinylated anti-goat IgG, BA-5000, Vector Labs, for UCP1-5 sections/1:1000 dilution of biotinylated anti-mouse IgG, BA 2001, Vector labs, for DBH sections/1:1000 dilution of biotinylated anti-rabbit IgG, BA-1000, Vector Labs, for TH sections, in a blocking buffer containing 3% NRS/NHS/NGS and 2% BSA in 0.1 M PB) for 2 h at room temperature. This was followed by three 10 min rinses in 0.1 M PB, after which sections were incubated for 1 h in avidin-biotin solution (at a dilution of 1:125, Vector Labs), followed by three 10 min rinses in 0.1 M PB. Sections were then placed in a solution of 0.05% 3,3′-diaminobenzidine (DAB) in 0.1 M PB for 5 min, followed by the addition of 3 ml of 3% hydrogen peroxide to each 1 ml of solution in which each section was immersed. Chromatic precipitation was visually monitored and verified under a low power stereomicroscope. Staining was allowed to continue until such time as the background stain was at a level that would assist architectural reconstruction and matching without obscuring the immunopositive neurons. Development was halted by placing the sections in 0.1 M PB, followed by two more rinses in 0.1M PB. To test for non-specific staining of the immunohistochemical protocol, in selected sections the primary antibody or the secondary antibody were omitted, which resulted in no staining of the tissue. The immunostained sections were then mounted on 0.5% gelatine coated glass slides, dried overnight, dehydrated in a graded series of alcohols, cleared in xylene and coverslipped with Depex. Digital photomicrographs were captured using Zeiss Axioshop and Axiovision software. No pixilation adjustments, or manipulation of the captured images were undertaken, except for the adjustment of contrast, brightness, and levels using Adobe Photoshop 7.
Western immunoblotting
Protein expression for UCP1 and UCP4 was assayed using standard qualitative Western immunoblotting techniques. To verify the specificity of the UCP1 antibody for the UCP1 protein, we tested this antibody with rat brown fat. For the UCP4 antibody protein samples were extracted from the paraformaldehyde fixed tissue using the Qproteome FFPE Tissue Kit (Qiagen, Germany). The tissue blocks analysed here were taken from the anterior cingulate and occipital cortex (as described above) and contained both gray and white matter. 30–40 mg of the sample were incubated in 100 µl of Extraction Buffer EXB Plus (Qiagen, Germany) containing 6% β-mercaptoethanol on ice for 5 min and mixed by vortexing. The samples were boiled for 20 min at 100°C and subsequently incubated at 50°C overnight with agitation at 300 rpm. The samples were then placed on ice for 1 min and centrifuged for 15 min at 14 000g at 4°C. The supernatant was transferred into clean tubes and the protein concentration was determined using the Bradford protein assay kit (Bio-Rad Laboratories, USA). The protein extracts (20 µg) were made soluble in sample buffer comprised of 0.0625 M Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 2.5% β-mercaptoethanol and 0.001% bromophenol blue, boiled at 95°C for 5 min and subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) (Millipore) at 20 V/cm for 1h. Electrophoresis and protein transfer was achieved using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Inc. USA). After the transfer the blots were blocked for 2 h in 1 × Animal-Free Blocker (SP-5030 Vector Labs, USA). The blots were incubated over night at 4°C under gentle agitation in the primary antibody solutions (1:300 goat anti-UCP1, Santa Cruz Biotechnology, sc-6528 or 1:300 goat anti-UCP4, Santa Cruz Biotechnology, sc-17582). The blots were washed for 3 × 10 min in 1 × Animal-Free Blocker and incubated for 1 h at room temperature in HRP-conjugated rabbit anti-goat secondary antibody (1:1000, Dako, USA) for 1 h. This was followed by 3 × 10 min washes with 50 mM Tris buffer, pH 7.2. The protein bands were detected using 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) (Sigma, D5637). The blots were incubated in a solution containing 1mg/ml DAB in 50 mM Tris, pH 7.2 for 5 min at room temperature, followed by the addition of an equal amount of 0.02% hydrogen peroxide solution. Development was arrested by placing the blots in 50 mM Tris (pH 7.2) for 10 min, followed by two more 10 min rinses in distilled water.
Stereological analysis
Using a design-based stereological approach we analysed immunohistochemically stained sections in the grey matter of the anterior cingulate and occipital cortex, as well as the underlying white matter from these regions of 14 cetartiodactyl species. Regions of interest (ROI) were drawn from similar locations across species as supported by published anatomical descriptions of the cetacean and artiodactyl brain. Using a light microscope equipped with a motorized stage, digital camera, MicroBrightfield system (MBF Bioscience, USA) system and StereoInvestigator software (MBF Bioscience, version 2018.1.1; 64-bit), we quantified UCP1-immunoreactive neuron densities in the grey matter, UCP4-immunoreactive glia densities in the grey and white matter, and DBH- and TH-immunoreactive bouton densities in the grey and white matter of these cortical regions. Separate pilot studies for each immunohistochemical stain was conducted to optimise sampling parameters, such as the counting frame and sampling grid sizes, and achieve a coefficient of error (CE) below 0.127,46,47,48,49. In addition, we measured the tissue section thickness at every sampling site, and the vertical guard zone was determined according to tissue thickness to avoid errors/biases due to sectioning artefacts27,46,47,48,49. Supplementary Tables S1–S4 provide details of the parameters used for each neuroanatomical region and stain and between the species in the current study. To estimate the ROI total number, we used the ‘Optical Fractionator’ probe.
UCP1- and UCP4-immunoreactive neuron and glia densities were obtained by sampling the cortical areas of interest and subjacent white matter with the aid of an optical disector. The cortex and white matter were outlined separately at low magnification (2X), and the optical disector was performed at 40X. UCP-immunoreactive neuron and glia density was calculated as the total number of UCP-immunoreactive neurons and glia divided by the product of surface area (x, y), the tissue sampling fraction, and the sectioned thickness (50 µm). The tissue sampling fraction was calculated as the ratio of the optical disector height to mean measured section thickness. Given that overall cell density per unit volume is known to vary with differences in brain size, we calculated the percentage of UCP-immunoreactive neurons or glia, expressed as the ratio of UCP-immunoreactive neurons or glia to total neuronal or glial density for each region of interest, to standardize the data for cross species comparison. Using Nissl-stained sections we obtained estimates of neuronal and glial densities within the cortex and glial density within the white matter using optical disector probes combined with a fractionator sampling scheme46. A pilot study determined the optimal sampling parameters and grid dimensions to place disector frames in a systematic-random manner. For DBH and TH bouton densities, ‘spot’ densities were calculated by multiplying the ROI area by the cut section thickness, and then using the generated volume as the denominator to the ROI estimated number. For all tissue sampled the optical fractionator was used while maintaining strict criteria, e.g. only complete boutons were counted, 63 X oil immersion, and obeying all commonly known stereological rules. The stereologic analyses presented here resulted in sampling an average of 118 counting frames per region of interest with a total of 13,053 counting frames investigated.
Statistical analyses
We hypothesized that the percentage of cortical neurons immunoreactive to UCP1 were significantly different between artiodactyls and cetaceans. To test this hypothesis, we compared the proportion of UCP1 expression in the anterior cingulate and occipital cortex of 16 cetartiodactyls. For the anterior cingulate cortex, we sampled a total of 1109 sampling sites (~ 100 sites per species) within the artiodactyl group and found that 36.83% of sampled cortical neurons were immunoreactive to UCP1. In comparison our cetacean sample consisted of 723 sampling sites (~ 145 sites per species), with 87.28% of the sampled cortical neurons immunoreactive to UCP1. For the occipital cortex, we sampled a total of 1 038 sites (~ 94 sites per species) within the artiodactyl group and found that 34% of sampled cortical neurons within the occipital cortex were immunoreactive to UCP1. The cetacean sample consisted of 723 sampling sites (~ 145 sites per species), and we found that 92.36% of the sampled cortical neurons were immunoreactive to UCP1.
To test if the respective underlying proportions were different between the sample groups, we conducted statistical hypothesis testing using the Two-Proportions Z-test as implemented in the R Programming language. Our Null hypothesis (Ho) stated that there is no significant difference between the proportions of artiodactyl immunoreactive UCP1 sampled cortical neurons (π1) and the proportions of cetacean UCP1 sampled cortical neurons (π2)—that is, π1 − π2 = 0. The alternate hypothesis (H1) stated that there is a significant difference in these proportions such that π1 − π2 ≠ 0, with one of the proportions being either less than or greater than the other. We thus conducted a two-sided hypothesis test, with the significance level (α) set at 0.05 (i.e., P-values less than, or equal to, α, would reject the null hypothesis in favour of the alternate hypothesis). Based on these analyses the proportion of immunolabelled UCP1 cortical neurons were found to be significantly different between the groups, with cetaceans having a significantly higher proportion of UCP1-immunoreactive neurons in the anterior cingulate cortex (χ2 = 51.69; df =1, P = 6.49 × 10−13, 95% confidence interval = − 0.122; − 0.067) and occipital cortex (χ2 = 56.30; P = 6.21 × 10−14, 95% confidence interval = − 0.114; − 0.060).
We used a two sample T-test (as implemented in R) to test for significant differences in noradrenergic bouton density between cetaceans and artiodactyls. Cetaceans were found to have significantly higher mean DBH-immunoreactive bouton densities in the anterior cingulate cortex as compared to artiodactyls (t = − 3.595; df =15, P = 0.011). Cetaceans were also found to have significantly higher mean DBH-immunoreactive bouton densities in the occipital cortex as compared to artiodactyls (t = − 4.546; df =15, P = 0.002). Similarly, we tested for significant differences in mean DBH bouton density in the underlying cortical white matter of cetaceans and artiodactyls. We did not find any significant differences in DBH-immunoreactive bouton density for the anterior cingulate (t =− 0.597; df =15, P = 0.585) or occipital cortex (t = − 0.08; df =15, P = 0.941).
To test for the effect of confounding variables on the significant differences observed in DBH bouton density in the cortex, we used an analysis of covariance controlling sequentially for the effect of cortical neuron density, cortical glia density and brain mass. Our analyses revealed that after adjusting for the density of cortical neurons cetaceans still had significantly higher DBH-immunoreactive bouton density in the anterior cingulate cortex (adjusted mean = 10.176) in comparison to artiodactyls (adjusted mean = 8.176) (F = 5.222; df =13, P = 0.041). Adjusting for the covariate cortical neuron density, resulted in a similar result for the occipital cortex (adjusted mean = 14.678) in comparison to artiodactyls (adjusted mean = 10.395) (F = 14.05; df =13, P = 0.00278). When controlling for the density of cortical glia, cetaceans also had significantly higher DBH-immunoreactive bouton densities in the anterior cingulate cortex (adjusted mean = 10.62) in comparison to artiodactyls (adjusted mean = 8.01) (F = 9.72; df =13, P = 0.00889). Similar results were found for the occipital cortex, with cetaceans having significantly higher DBH-immunoreactive bouton density (adjusted mean = 14.471) compared to artiodactyls (adjusted mean = 10.395) (F = 11.2; df =13, P = 0.00581). When controlling for brain mass, cetaceans were also found to have a significantly higher DBH-immunoreactive bouton densities in the anterior cingulate (adjusted mean = 11.36) in comparison to artiodactyls (adjusted mean = 7.75) (F = 11.06; df = 13, P = 0.00604) as well as in the occipital cortex (cetacean adjusted mean = 15.406, artiodactyls adjusted mean = 10.055) (F = 11.85; df = 13, P = 0.00488).
Source: Ecology - nature.com
