Brains from two sub-adult male giraffes (G. camelopardalis
) (body mass 480 kg and brain mass of 544 g, body mass 450 kg and brain mass of 509 g) and two adult male harbour porpoises (Phocoena phocoena
) (body mass 49 kg and brain mass of 503 g, body mass 55 kg and brain mass of 486 g) were used in the current study. All animals were treated and used according to the guidelines of the University of Witwatersrand Animal Ethics Committee which correspond with those of the NIH for care and use of animals in scientific experimentation. Both giraffes were euthanized with an intravenous overdose of sodium pentobarbital in the late afternoon after being used for unrelated physiological studies. Following euthanasia the heads were removed from the body at the level of the third cervical vertebrae, the common carotid arteries located and a 4 mm inner diameter cannula inserted and secured in place. The heads were then gravity perfused initially with a 10 l rinse of 0.9% saline solution at a temperature of 4 °C followed by 10 l of 4% paraformaldehyde in 0.1 M PB at a temperature of 4 °C for fixation (as per the method described in Manger et al., 2009
). Both harbour porpoises were obtained after being killed according to Greenlandic cultural practices and perfused via
the heart with an initial rinse of 20 l of 0.9% saline solution at a temperature of 4 °C followed by 20 l of 4% paraformaldehyde in 0.1 M PB. The brains were removed from the skull and post-fixed in 4% paraformaldehyde in 0.1 M PB (24 h at 4 °C) and allowed to equilibrate in 30% sucrose in 0.1 M PB. The brains were dissected and the diencephalon frozen in crushed dry ice and coronal sections of 50 μm thickness were made using a sliding microtome.
A one in three series were stained for Nissl, myelin and orexin A. Nissl sections were mounted on 0.5% gelatine coated glass slides and then cleared in a solution of 1:1 chloroform and 100% alcohol overnight, after which the sections were then stained with 1% cresyl violet. The myelin series sections were refrigerated for two weeks in 5% formalin then mounted on 1.5% gelatine coated slides and stained with a modified silver stain (Gallyas, 1979
The sections used for immunohistochemistry were initially treated for 30 min with an endogenous peroxidase inhibitor (49.2% methanol:49.2% 0.1 M PB:1.6% of 30% H2O2), followed by three 10 min rinses in 0.1 M PB. The sections were then preincubated at room temperature for 2 h in a blocking buffer solution containing 3% normal serum (NGS, Chemicon/Millipore), 2% bovine serum albumin (BSA, Sigma) and 0.25% Triton X-100 (Merck) in 0.1 M PB. The sections were then placed in a primary antibody solution (blocking buffer with correctly diluted primary antibody) and incubated at 4 °C for 48 h under gentle shaking. To reveal orexinergic/hypocretinergic neurons, anti-orexin A (AB3704, Chemicon/Millipore, raised in rabbit, against a synthetic peptide corresponding to the C-terminal portion of the bovine Orexin-A peptide) was used at a dilution of 1:2000. This was followed by three 10 min rinses in 0.1 M PB, after which the sections were incubated in a secondary antibody solution for 2 h at room temperature.
The secondary antibody solution contained a 1:1000 dilution of biotinylated anti-rabbit IgG (BA-1000, Vector Labs) in a blocking buffer solution containing 3% NGS and 2% BSA in 0.1 M PB. This was followed by three 10 min rinses in 0.1 M PB after which the sections were incubated in AB solution (Vector Labs) for 1 h. After three further 10 min rinses in 0.1 M PB, the sections were placed in a solution of 0.05% 3,3′-diaminobenzidine (DAB) in 0.1 M PB for 5 min (2 ml/section), followed by the addition of 3 μl of 30% H2O2 to each 1 ml of solution in which each section was immersed. Chromatic precipitation of the sections was monitored visually under a low power stereomicroscope. This process was allowed to continue until the background staining of the sections was appropriate enough to assist with architectonic reconstruction without obscuring any immunopositive neurons. The precipitation process was stopped by immersing the sections in 0.1 M PB and then rinsing them twice more in 0.1 M PB. Omission of the primary or secondary antibody in selected sections was employed as negative controls, for which no staining was evident. In addition to these negative controls that eliminated the possibility of parasitic background staining we ran an additional antibody and peptide inhibition assay, as the novel findings of parvocellular orexinergic neurons in the medial hypothalamus of the giraffe and harbour porpoise needed to be verified. We used an additional orexin-A antibody available from Millipore (AB3098, raised in rabbit, against an 18 amino acid peptide mapping near the amino terminus of mouse Orexin-A) at a dilution of 1:2000 as per the protocol described above. The reason a different antibody was used is due to the fact that no specific inhibition peptide is available for the orexin-A antibody AB3704. The orexin control peptide (AG774, Millipore, specifically for AB3098) was used at a dilution of 1 mg/ ml in the primary antibody solution (see above). This solution was incubated for 3 h at 4 °C prior to being used on the sections. In the case of the AB3098 orexin-A antibody, neurons were observed in the hypothalamus of both the giraffe and harbour porpoise and showed an identical staining pattern to that seen with the AB3704 orexin-A antibody. In the sections where the primary antibody (AB3098) had been inhibited, no staining was evident in either the parvocellular or magnocellular orexinergic regions.
The immunohistochemically stained sections were mounted on 0.5% gelatine coated slides and left to dry overnight. The sections were then dehydrated in graded series of alcohols, cleared in xylene and cover slipped with Depex. All sections were examined under low power using a stereomicroscope and the architectonic borders of the sections were traced according to the Nissl and myelin stained sections using a camera lucida. The immunostained sections were then matched to the traced drawings, adjusted slightly for any differential shrinking of the stained sections and immunopositive neurons were marked. The drawings were then scanned and redrawn using the Canvas 8 (Deneba) drawing program.
The number of orexinergic (OxA+) positive cells was determined with stereological techniques through the complete hypothalamus in all 4 brains. A Nikon E600 microscope with three axis motorized stage, video camera, Neurolucida interface and Stereo-Investigator software (MicroBrightfield Corp.) was used for the stereological counts. In an attempt to achieve the most accurate estimation of OxA+ neurons, a pilot study was first conducted in an individual of each species. The pilot study determined the best counting frame size and grid size and these parameters were then used for all individuals of each species investigated. A 200 μm × 186 μm counting frame and a 1259 μm × 1220 μm sampling grid were employed in each individual (). Only orexinergic neurons with clearly visible nuclei were marked in the sampling grids. For the calculation of total neuron numbers, we measured section thickness in a random sample of 20 sections from each individual in the regions where orexinergic neurons were present and used these measurements to calculate the species average mounted thickness. The ‘optical fractionator probe’ function of the software computationally determined the number of parvocellular OxA+ cells, the number of magnocellular OxA+ cells and the total number of OxA+ in the hypothalamus of each individual using the following formula:
– was the total estimated neuronal number, Q
– was the number of neurons counted, SSF – was the section sampling fraction (in the current study this was 0.5), ASF – is the area sub fraction (this was the ratio of the size of the counting frame to the size of the sampling grid), and TSF – was the thickness sub fraction (this was the ratio of the dissector height relative to cut section thickness). In order to determine TSF we used the average mounted section thickness calculated for each individual (), subtracted the total vertical guard zones (10 μm) to give dissector height and used the ratio of dissector height to cut section thickness (50 μm) to provide TSF for each individual. A function in the stereology programme called the “nucleator probe” facilitated the estimation of the mean cross-sectional area, volume and length of the orexinergic positive cells. Only neurons with a distinct nucleus were chosen for analysis. The “nucleator probe” was employed in conjunction with the optical fractionator and stereology procedures for systematic random sampling to identify cells (Gundersen, 1988
). In total, seven probes were used in the current study namely: the optical fractionators, optical fractionator using number weighted section thickness, physical dissector, physical fractionator, Schmitz nearest neighbour, Cavalieri estimator for area and volume, and combination of planes with the optical fractionator for absolute length. As a measure of variability, the standard deviations/error (SD/SE) together with the mean values were given. In the present study, a permutation t
-test (10 000 permutations) was used for all reasonable paired comparisons of volume, area and length; corrected for multiple tests using the sequential Bonferroni method (Holm, 1979
). Additionally, counts were tested using the following equation:
against the t
-distribution, where Y1
is the first count and Y2
is the second count (Sokal and Rohlf, 1998
). Signficance values were adjusted again using the sequential Bonferroni method. Statistically significant differences were considered at α
Stereological parameters used for Giraffa camelopardalis and Phocoena phocoena.