This work was based on results from 60 barn owls (Tyto alba
) of both sexes with age ranging from embryos to adult. Preliminary data have been presented as abstracts and conference proceedings (Cheng and Carr, 1992
; Carr, 1995
). Due to limited resources, number of owl embryos and owl chicks for each time point was varied and limited (range from 1 to 5; E17 (2), E18 (1), E19 (3), E21 (2), E22 (1), E23 (2), E24 (1), E26 (2), E27 (2), E28 (1), E29 (1), E31 (2), E32/P0 (5), P2 (3), P5 (2), P6 (1), P7 (3), P8 (1), P9 (1), P10 (1), P12 (2), P14 (3), P15 (1), P20 (2), P21 (1), P24 (1), P25 (2), P30 (4), P42 (1), P60 (2), Adult (4)). Owl eggs were incubated in a Lyon Roll-X incubator (Lyon Electric Co., Chula Vista, CA) at 37°C, and owl chicks were hand-raised in the laboratory (Rich and Carr, 1999
). Owl embryos and chicks were staged using the reference data for age determination in Köppl et al. (2005)
. All protocols conformed to NIH and the University of Maryland Animal Care and Use Committee guidelines. All chemicals, except those specified, were from Sigma (St. Louis, MO).
Owls were anesthetized intramuscularly with Ketamine (15 mg/kg), followed by a lethal dose of Pentobarbital (20 mg/kg i.m., Abbott Laboratories, IL). After intracardiac injection of heparin, animals were perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. Brains were then cryoprotected in 30% sucrose and sectioned coronally at 30 μm using a freezing microtome (). Sections were stored in PB for immunohistochemistry. For in situ hybridization, sections were mounted on silane-prep slides. Embryos that were too small for perfusion were anesthetized by cooling, decapitated and fixed by immersion in 4% paraformaldehyde in PB.
Owls (E23, E31, P8, 12, 20, and 30) were anesthetized as above and perfused transcardially with oxygenated avian Tyrodes solution, followed by 1 L of 3% glutaraldehyde, 1% paraformaldehyde in 0.1 M
phosphate buffer at pH 7.4 (Jackson and Parks, 1982
; Carr and Boudreau, 1991
). The brains were postfixed for 8 h, the brainstem was cut in transverse sections on a vibratome, and selected regions were postfixed with 1.0% osmium tetroxide and embedded in Araldite resin. Thin sections were stained with uranyl acetate and triple lead stain. The ultrastructure of NL was studied by first making a camera lucida drawing of an adjacent semithin section. The entire thin section was then viewed on the electron microscope in order to identify major landmarks and any profiles to be examined in serial section.
Owls (n = 2) were injected subcutaneously with 5 mg of 5-bromo-2′-deoxyuridine (BrdU, 5 mg/mL) per 100 g of body weight, and were sacrificed after 8 h as described above. In addition, sections for immunostaining were pretreated with protease (3 μg/mL) and 1 N HCl to remove nuclear histones.
Standard immunohistochemical procedures were followed using the avidin-biotin-peroxidase complex (ABC) method with reagents from Vectastain ABC kits (Vector Labs, Burlingame, CA). Sections were pre-incubated for 1 h in 0.1 M phosphate buffered saline with 4% normal horse serum and 0.4% Triton-X, then incubated for 1–3 days in mouse monoclonal antibodies. Because the availability of owl embryo material was limited, we used antibodies that had been shown to recognize chicken protein. These included antibodies against myelin-associated glycoprotein (α-MAG, 1:200 dilution; No. MAB1567, Chemicon International, Temecula, CA), oligodendrocyte marker (O4, 1:100 dilution; No. MAB345, Chemicon) and proteolipid protein (α-PLP, 1:300 dilution; No. MAB388, Chemicon). The anti-tenascin antibody recognized chicken tenascin (1:300 dilution; M1-B4, Developmental Studies Hybridoma Bank, University of Iowa; Chiquet and Fambrough, 1984). Mouse anti-bromo-2′-deoxyuridine, (α-BrdU, 1:120 dilution; No. B-25315, Sigma) was used to identify BrdU-labeled cells. Sections were incubated for 1 h in biotinylated horse anti-mouse IgG secondary diluted at 1:1500, washed and incubated in ABC for 1 h. Sections were then developed in 0.03% diaminobenzidine tetrahydrachloride and 0.003% hydrogen peroxide in acetate-imidazole buffer with nickel sulfate intensification and then washed. They were mounted onto gelatin-subbed slides, counterstained with Neutral Red, dehydrated, cleared, and coverslipped with Permount.
In Situ Hybridization and Autoradiography
The chicken PLP cDNA clone used in this study was provided by Dr. Klaus Nave. In this clone the chicken cDNA fragments corresponding to the PLP coding region (822 bp) were subcloned into the pBluescript KS+ phagemid vector (Nave et al., 1987
). The cDNA was linearized using HindIII and 35
S-labeled antisense RNA probes were generated by in vitro
transcription using T7 polymerase. The antisense riboprobe contain exon 3B of PLP and is specific for PLP-mRNA positive oligodendrocytes. Nonspecific sense riboprobes were also generated for control experiments. For in situ
hybridization, sections were mounted on RNAase-free silane-prep slides. After pretreatment with proteinase K (50 μ
g/mL), and acetic anhydride (10 μ
g/mL), sections were hybridized with riboprobes in hybridization buffer (Life Technologies, Rockville, MD) at 65°C for overnight. Sections were subsequently washed with SSC buffer (1 mM
NaCl, 1% SDS, 10 mM
Na Citrate) at 42°C. For emulsion autoradiography, slides were dipped in photographic emulsion (NTB2, Kodak, Rochester, NY) and exposed before being developed and counterstained. The sense control was run in parallel with antisense experiments, and was used to monitor the level of background noise during autoradiography. None of the control experiments showed any labeling in the tissue sections.
To quantify the progress of myelination, the numbers and hybridization intensity of PLP-mRNA positive oligodendrocytes were measured from both rostro-medial (high frequency coding) NL and caudo-lateral (low frequency coding) NL during postnatal development. The number of oligodendrocytes, represented by the silver grain clusters under darkfield illumination, were counted as follows: An eyepiece grid (10 × 10), which covers an area of 187.5 μm × 187.5 μm under the 40× objective, was placed over NL. For each section, number of oligodendrocytes within NL were counted under both brightfield and darkfield microscopy sequentially, and position of oligodendrocytes related to the grid area are marked and compared. Only those that were identified under both conditions were recorded as positive. Measurements were obtained from each postnatal week up to one month posthatch. Since the dimension of NL also expands during the first month posthatch, we expressed changes in terms of oligodendrocyte cell density, which is determined by the number of oligodendrocytes per grid area, over time.
The hybridization intensity of PLP mRNA was represented by the optical density of silver grain clusters measured with NIH Image (version 1.61, NIH, Bethesda, MD) and a Scion LG-3 frame grabber (Scion Corp., Frederick, MD) connected to an Olympus OLY-750 camera and BX60 microscope (Olympus America, Melville, NY). Images of silver grain clusters were captured under 40× darkfield microscopy and inverted to brightfield images for uncalibrated optical density measurements. All images were taken under same darkfield illumination, with the camera gain control set at 18 dB. Under NIH Image, the boundary of individual silver grain cluster, which corresponds to an oligodendrocyte, was drawn using the freehand selection tool, and optical density of the average gray value (mean) in the selection was analyzed. Optical densities of average background value were sampled across various positions and were consistent along the rostro-caudal NL and across the NL border. The intensity level of each oligodendrocyte was calculated by first subtracting the background from the mean optical density in the selection (background-corrected mean), and then normalized to the cell area.