DNA templates of the five NMDAR subunits were provided by Drs. Wada and Jarvis (Wada et al., 2004
). DNA fragments spanning 400–500 bp for NR1, NR2A, NR2B, NR2C, and NR2D were amplified by RT-PCR from zebra finch (Taeniopygia guttata
) brain total RNA. The NR1 probe contained 417 base pairs (bp; GenBank accession No.: AB042756), corresponding to human 705–843 amino acids (aa), and extended from the extracellular loop between putative transmembrane regions III and IV to part of the intracellular C-terminal (Sprengel and Seeburg, 1994
). The chicken NR1 gene has recently been sequenced and shows 85% homology to mammalian NR1 (Zarain-Herzberg et al., 2005
). The NR2A probe (GenBank accession No.: AB042757) contained 521 bp, corresponding to rodent 647–819 aa, and included the extracellular loop between transmembrane regions III and IV (Sprengel and Seeburg, 1994
). The NR2B probe (GenBank accession No.: AB107125) contained 413 bp, corresponding to rodent 801–937 aa, and extended from the extracellular loop between putative transmembrane regions III and IV to the intracellular C-terminal (Sprengel and Seeburg, 1994
). The NR2C probe (GenBank accession No.: AB042758) contained 521 bp, corresponding to rodent 645–817 aa, and comprises whole extracellular loop between transmembrane regions III and IV (Sprengel and Seeburg, 1994
). The NR2D probe (GenBank accession No.: AB042759) contained 521 bps, corresponding to rodent 675–847 aa and including a region similar to that of NR2C (Sprengel and Seeburg, 1994
). The cDNA probes were constructed in pGEM-T Easy Vector (Promega, Madison, WI). The SP6 and T7 promoters in the construct were used to synthesize the digoxigenin- and 35
S-labeled sense and antisense RNA probes, which were extracted with phenol/chloroform (1:1) and then precipitated twice with ethanol to remove the unincorporated nucleotides. The purified cRNA probes were dissolved in DEPC-water and kept at −80°C.
Animals and tissue preparation
Chickens (white leghorn) were purchased from a local breeder (CBT Farms). Chick ages (number of embryos or chickens) were as follows: E7 (10), E10 (16); E12 (12), E14 (12), E18 (8), P0 (12), P16 (4), and adult (older than 4 months; 3). Numbers of animals in which we carried out in situ hybridization studies with 35S-labeled probes and counted silver grains were: E10, 4–6; E12, 4–5; E14, 4–5; E18, 4–5; P0, 4; P16, 3; adult, 3. Numbers varied because the cochlear nucleus was so small that we could not always obtain enough sections of the central regions of NM and NL from one embryo for hybridization with probes against all five subunits.
The time points were selected to correspond to developmental events (see Discussion). Fertilized eggs were incubated in a Marsh automatic incubator (Lyon Electric Company, Chula Vista, CA) at 37°C and 60% humidity. All animal procedures were approved by the University of Maryland Animal Care and Use Committee and followed NIH guidelines.
Embryos up to E18 were anesthetized by cooling and P0 to adult chickens by halothane inhalation, followed by injection of euthasol (Delmarva Laboratories, Inc.). Animals were perfused intracardially with a saline solution (0.9% NaCl), followed by 4% paraformaldehyde in 50 mM phosphate-buffered saline (PBS) for 15 minutes. Dissected brains were postfixed in 4% paraformaldehyde for 8–10 hours. Brains were placed in 30% sucrose solution in 50 mM PBS for 48 hours at 4°C, then embedded in O.C.T. (Sakura Finetek), and stored at −80°C.
In situ hybridization
Twenty-micrometer coronal or oblique (parallel to the frequency axis) sections were cut on a Leica CM 1850 cryostat and mounted on silanated (amine) nuclease-free slides (CEL Associates, Inc.). To make hybridization comparable across all ages, sections from different ages were put on the same slide such that a single slide contained 10 sections with two sections of E10, E12, and E14, and one section of E18, P0, P16, and adult, respectively. All sections were cut and placed on slides, which were stored desiccated at −80°C. We controlled hybridization and autoradiographic conditions to allow accurate comparisons between batches of sections. In all cases, hybridization experiments were carried out with identical probe concentration and strength, solution volumes and hybridization conditions. Similarly, all autoradiography had identical exposure time and development conditions.
For in situ hybridization, sections were digested with 10 μg/ml proteinase K at 37°C for 10 minutes and fixed in 4% paraformaldehyde for 5 minutes. The sections were then washed three times in PBS, pH 7.5, for 5 minutes each, and acetylated in 0.25% acetic anhydride and 0.1 M triethanolamine in 0.9% NaCl for 10 minutes. After dehydration in a series of graded ethanols, the slides were placed in chloroform for 10 minutes and then in a prehybridization solution containing 50% formamide, 10% dextran sulfate, 10 mM Tris, pH 8.0, 0.3 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1× Denhardt’s solution, and 0.5 mg/ml tRNA at 52°C for 2 hours. Subsequently, the sections were incubated in the hybridization solution containing 0.5 μg digoxigenin-labeled probe/ml or 1.0 × 106
cpm/100 μl for radiolabeled probes at 55°C for 16–18 hours. After briefly rinsing in SSC, the sections were washed twice with SSC for 15 minutes each and incubated in RNase solution with a final concentration of 0.02 mg/ml for 45 minutes. The slides were washed again in 1× SSC and 0.1× SSC for 15 minutes at 42°C. Subsequent procedures for digoxigenin-labeled probes hybridization after SSC washing followed a standard method (Tang et al., 2001
). For 35
S-labeled probe hybridization, the slides were air dried and dipped in NTB2 emulsion diluted 1:1 in water. Sections were exposed at 4°C for 2–3 weeks and then developed with Kodak D-19. Sections were counterstained with 1.0% eosin Y for 15 seconds, followed by 2.0% thionine for 30 minutes. Proteinase K digestion, prehybridization, hybridization, digoxigenin-antibody reactions, and signal development were carried out in a humid environment.
Sections were examined by light microscopy in bright- and darkfield. Digital photomicrographs were captured via Neurolucida system (MicroBrightField, Colchester, VT) and processed in Photoshop CS2 (Adobe Systems) to adjust for brightness and contrast. For each probe, the localization of NMDAR mRNA was verified by comparison of sections hybridized to antisense probe with those hybridized to sense probe in the initial three experiments carried out with new probes. No specific signal was obtained when the sections were treated with sense RNA probes. Silver grains were counted above all well-defined cell bodies, identified by the presence of a clear neuronal profile in both NM and NL. In the NL of E10 and E12 embryos, distinct clusters of silver grains over less well defined cytoplasm in the NL monolayer were also identified as cell bodies, because ribonuclease eliminated most substances stained by thionine and eosin Y in young embryos. For each section, we also counted silver grains in 10 circles just outside each brain section near the cochlear nucleus region. These circles were equal in size to the average NM or NL neuron, and they provided background counts for NM and NL neuronal grain counts. For NM and NL, we counted silver grain density in neurons from chickens killed at seven ages (E10, E12, E14, E18, P0, P16, and adult) for NR1, NR2A, and NR2B and at four ages (E10, E14, P0, and adult) for NR2C and NR2D.
Data were normalized by subtracting background grain counts from neuronal grain counts. We then normalized for increasing cell body size during development by measuring cytoplasm area, obtained from digoxigenin material, and using that area to provide silver grain counts per area of labeled cytoplasm. Because there is tonotopic variation in NMDAR expression during development, all counts were taken from the central region of NM and NL. Counts from the same nucleus were pooled within each animal and presented as means ± SD of the grain density per 100 μm2 of cytoplasm, except for the measures of tonotopic variation within a nucleus (see below). All data from grain counts were statistically analyzed by one-way ANOVA. For post hoc comparisons, Sidak tests were performed (SPSS Inc.). Paired comparisons were made among all age groups. Age pairs that showed a significant difference are presented in the text. All neighboring age groups, such as E10 with E12, were compared. If two successive comparisons showed no significant difference, we compared additional age groups. For example, if there were no significant changes between P0 and P16 and from P16 to adult, we compared the difference between P0 and adult. To show the relative expression levels of NMDAR subunits between NM and NL, grain counts of NM and NL at the same developmental age are presented side by side. Otherwise, data are presented by nucleus and describe NR1, NR2A, NR2B, NR2C, and NR2D expression for each structure.
Tonotopic variation in NR1 hybridization was quantified by measuring silver grain density at E14, because our previous study had shown a tonotopic variation in anti-NR1 expression in NM at this age (Tang and Carr, 2004
). Oblique sections through NM and NL (parallel to the isofrequency axis) were divided into three approximately equal regions, and grain densities in the cells in the rostromedial, central, and caudolateral regions were determined and analyzed as described above.