Fertilized eggs of White Leghorn chickens (Gallus gallus domesticus) were obtained from local suppliers (Akebono Farm, Hiroshima, Japan, and Nihon Layer, Gifu, Japan) and incubated at 37.7°C under moderate moisture and quasi-constant darkness. After hatching, the chicks were kept in groups in the same incubator under quasi-constant darkness (Maekawa et al. 2006). This study was carried out in accordance with the Guidelines for the Treatment of Experimental Animals of Tokyo Medical and Dental University and Kitasato University. The experimental protocols described in this article were approved by the Animal Care and Use Committee for Tokyo Medical and Dental University and for Kitasato University.
Chicks were killed with an overdose of anesthetics and were perfused with 4% paraformaldehyde (PFA). The whole brains were post-fixed in the same fixative for 24 h at 4°C, cryoprotected by immersing in 30% sucrose for 48 h, embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan), and frozen in powdered dry ice. Sagittal sections of the left hemisphere, cut at 20- to 40-μm thickness, were made with a cryostat (CM1900; Leica, Nussloch, Germany). We analyzed the left hemisphere exclusively, because it has been suggested that NMDA receptors in the left IMM play an important part in imprinting (McCabe and Horn 1988; McCabe et al. 1992). In addition, we had found that restricted ablation of the left HDCo results in the impairment of imprinting, indicating dominance of the left over the right hemisphere in imprinting (Nakamori et al. 2010).
In situ hybridization was performed as described previously (Maekawa et al. 2007). The chick TrkB gene fragment (GenBank accession no. NM_205231; nt 1780–2870 bp), which contains the intracellular tyrosine kinase domain, was amplified from chick brain cDNAs using the following primers: forward 5′-AGT CCT CTC CAT CAC ATC TC-3′ and reverse 5′-TGG AGT TCA GCG GCA GTT GA-3′. The amplified fragment was cloned into the pBSIISK (−) vector (Stratagene, La Jolla, CA, USA). Similarly, the chick glutamate/aspartate transporter gene fragment (GLAST, GenBank accession no. XM_425011; nt 1047–1752 bp; forward primer, 5′-GCT GTG ATC ATG TGG TAT GCT C-3′ and reverse primer, 5′-CAT TCT CCT CTA TCA CAG AAT TCC C-3′) was prepared and cloned. The chick BDNF fragment (GenBank accession no. NM_001031616; nt 1–741 bp; forward primer, 5′-ATA AAG CTT AGA GTG ATG ACC ATC CTT TTC C-3′ and reverse primer, 5′-TAT TCT AGA CTA TCT TCC CCT TTT AAT GGT T-3′) was prepared and cloned into a pGEM-T-easy vector (Promega, Madison, WI, USA). Plasmids containing vesicular glutamate transporter 2 (VGLUT2) and glutamate decarboxylase 65 (GAD65) cDNA were described previously (Maekawa et al. 2007). Gene-specific sense and antisense digoxigenin- or fluorescein-labeled cRNA probes were generated using a Roche RNA labeling kit (Roche Applied Science, Indianapolis, IN, USA).
For immunohistochemistry, sections were treated as described (Maekawa et al. 2007) and mouse anti-phosphotyrosine antibody (clone 4G10, 1 : 1000; Millipore Corporation, Billerica, MA, USA) was applied to the sections for 12–16 h at 4°C. After washing with phosphate-buffered saline (pH 7.0; PBS) with 0.5% triton X-100, the sections were reacted with a polymer reagent including peroxidase and goat anti-mouse IgG antibody (Dako Envision kit/HRP; DakoCytomation, Glostrup, Denmark) for 1 h at 25°C. Then, they were treated with 0.1% DAB to visualize peroxidase. For double fluorescence immunohistochemistry, mouse anti-phosphotyrosine (1 : 500) and rabbit anti-TrkB (794, sc-12, 1 : 500; Santa Cruz, CA, USA) antibodies, and Alexa 488- and 568-labeled secondary antibodies (A11008 and A11031, respectively, 1 : 1000; Invitrogen, Carlsbad, CA, USA) were used. Images were acquired using a confocal laser microscope (LSM710; Carl Zeiss, Oberkochen, Germany).
For cell counting, three or more chicks in each condition were used, and three sections that included the VW, HDPe, IMM, hyperpallium apicale, and nidopallium were selected per brain. The HDCo is located about 2.5–3.5 mm caudally from the rostral surface and 2.0–3.0 mm from the dorsal surface. The HDPe is located about 0–1.0 mm rostrally from the lateral ventricle and 2.0–3.0 mm from the dorsal surface. Therefore, with reference to the chick brain atlas (Puelles et al. 2007), the sections corresponding to L1.68–1.92 were selected for analysis. Images were acquired using a Zeiss microscope (Axio Imager A1; Carl Zeiss) equipped with Olympus DP25 digital camera and DP2-BSW software (Olympus Corporation, Tokyo, Japan). The light microscopy images were transferred to a graphics program (Adobe Photoshop CS4; Adobe Systems Incorporated, San Jose, CA, USA) in which the brightness and contrast were adjusted. To compare the number of positive cells between conditions, we first determined the threshold level in 256-shade grayscale, which adequately reflected the positive cells. This threshold level was kept constant for all samples designed to be compared. We manually counted the number of assemblies of black pixels in a 225 × 300-μm square (1040 × 1392 pixels) of the HDCo and a 178 × 534-μm square (680 × 2040 pixels) of the HDPe. Experimenters were not aware of the experimental condition of the chick from which the sections were derived. Considering the size of the cells, assemblies that contained fewer than 300 pixels were not counted. The number of cells involved in each assembly was confirmed and determined by the experimenter with reference to the original photograph. We compared the number of positive cells counted by the experimenter with the number counted as described above, examined the correlation, and confirmed insignificant differences between these two values.
Cell culture and transfection
HEK293T cells were grown in Dulbecco's modified Eagle medium (Sigma-Aldrich, MO, USA) supplemented with 10% fetal calf serum, 2% chick serum, 100 U/mL penicillin and streptomycin (Sigma-Aldrich), and 2 mM l-glutamine (Sigma-Aldrich). An IRES2–EGFP cassette was excised from the pIRES2–EGFP plasmid (PT3267-5; Clontech, CA, USA) with SmaI and NotI, and inserted into the EcoRV/NotI site of pCAGGS (Niwa et al. 1991), yielding pCX-IE. The chick BDNF fragment cloned into the pGEM-T-easy vector (see Histology section above) was excised with EcoRI, blunted, and inserted into the XhoI-cut and blunted pCX-IE, yielding pCX-BDNF-IE. HEK293T cells were then transfected with pCX-BDNF-IE or pCX-IE using FuGene6 (Roche). After 48-h culture, the cells were lysed in the extraction buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 100 mM NaF, 100 μM phenylarsine oxide, 10 mM Na4P2O4, 1% protease inhibitor cocktail (Sigma-Aldrich), and 1% phosphatase inhibitor cocktail (Sigma-Aldrich)] and stored at −80 °C until use.
Chick brains were sagittally sliced at 1-mm thickness using a brain slicer (Muromachi Kikai, Tokyo, Japan) and the left HDCo was dissected from the slice, weighed, and then stored at −80°C until use. Frozen tissue was then homogenized in the extraction buffer, sonicated, centrifuged at 16 000 g for 5 min at 4°C, and the supernatant was collected. Protein content was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) with bovine serum albumin as a standard. The protein (5 μg) was separated on 15% sodium dodecyl sulfate –polyacrylamide gel by electrophoresis, and blotted onto Immobilon-P membrane (Millipore). Then the membrane was soaked in 0.2% tween 20/tris-buffered saline containing 5% skim milk (Difco, Becton-Dickinson, Franklin Lakes, NJ, USA), and incubated with polyclonal anti-BDNF (N-20, 1 : 500; Santa Cruz, CA, USA) or anti-β actin (sc-47778, 1 : 1000; Santa Cruz) antibody at 4°C overnight. After three washes, the blot was reacted with a secondary antibody conjugated with horseradish peroxidase (1 : 1000). Signals were detected using SuperSignal West Femto (Thermo Fisher Scientific). Images were acquired using a detector (Image Quant™ LAS 4000 mini; GE Healthcare, Little Chalfont, UK) with Image Quant LAS 4000 Control Software.
Analysis of imprinting behavior
Behavioral imprinting experiments were done as previously described (Maekawa et al. 2006). Briefly, a chick was placed in a running wheel and an imprinting stimulus was presented to the chick by one of the two liquid crystal monitors (15-inch Flex Scan L367, EIZO; Nanao) on either side of the wheel, perpendicular to its axis. The images were generated using a visual stimulus generator system (VSG; Cambridge Research Systems) and each image bounced left and right horizontally on the screen. The chick's movements toward or away from the image presented on the display were recorded (Muromachi Kikai, Tokyo, Japan).
During the training, a chick was placed in the running wheel and exposed to a training image (a red circle, unless otherwise indicated) presented on the monitor. Depending on the experiment, duration of training was selected: 1 h (30 min presentation on one monitor followed by 30 min on the other monitor), 20 min, or 15 min (presentation only on one monitor). Training that lasts 1 h can induce imprinting behavior (Nakamori et al. 2010). For the control training, chicks were put into the apparatus for the same duration as the training group without any image on the black screen. We trained chicks once on P1 (24–48 h after hatching), unless otherwise specified. After the training, they were returned to the same incubator as before the training.
Imprinting performance was evaluated 2 h or 48 h later. The chicks were put into the running wheel again. After a 5-min adaptation period (black monitor, no image presentation), a red circle was presented for 5 min, followed by a blue square for 5 min on the same monitor. The direction and number of wheel revolutions were recorded. We calculated the preference score (PS) as described previously (Maekawa et al. 2006) using the following formula as an index of success of visual imprinting: PS = The number of wheel revolutions toward the display during 5 min/The total number of wheel revolutions during 5 min. Inactive chicks that rotated the wheel fewer than 22.5 revolutions during the 15-min evaluation period were excluded from the analysis (Maekawa et al. 2007). When the PS value for a red circle was significantly larger than chance (0.5) and the difference between the PS for the red circle and the blue square was regarded as statistically significant (p < 0.05), we judged the chick to be imprinted. These criteria are based on the fact that imprinting behavior is a following response specific for the imprinting stimulus.
In this study, the order of image presentation at evaluation was constant. This is based on our observation that showed this order did not affect imprinting performance. When the novel stimulus was presented before the imprinting stimulus, the PS values were 0.40 ± 0.29 and 0.73 ± 0.26, respectively (p < 0.01).
For the analysis of phosphotyrosine and TrkB expression, chicks were trained with a red square for 1 h. Evaluation was performed 1 h later with a red and blue square sequentially, and killed immediately.
Human recombinant BDNF (rBDNF; mature BDNF containing 119 amino acids) was a generous gift from Dainippon Sumitomo Pharma Co. Ltd. (Osaka, Japan). PBS or rBDNF (125 ng/μL in PBS), both containing 0.01% Evans blue dye and 1% bovine serum albumin (Sigma-Aldrich), was injected with a syringe (Hamilton Company, Reno, NV, USA; 2 μL/chick) using the methods described previously (Nakamori et al. 2010). Chick was anesthetized by brief exposure to diethyl ether and the head was held in a horizontal position and a free-hand injection was performed at the left HDCo or HDPe. Left HDCo was positioned 7 or 8 mm rostrally from bregma for P1 and P7 chicks, respectively, 2 mm to the left of midline of the skull, and at a 2-mm depth from the skull surface. The left HDPe was positioned 2.5 mm rostrally from bregma, 2 mm to the left of the midline, and at a 2-mm depth from the skull surface for P1 chicks. These positions were determined by geographic features on the skull surface seen through the skin. The depth of the injection was governed by a plastic sleeve on the 26s gauge needle (Hamilton). Twenty minutes after the injection, the imprinting training for 15 min (weak training) or 1 h was performed for P1 and P7 chicks, respectively, and imprinting behavior was evaluated 2 h after the end of the training.
A Trk receptor family antagonist, K252a (ENZO Life Science, Farmingdale, NY, USA; 50 μM, 50% dimethyl sulfoxide in PBS, 47 ng/chick; Rattiner et al. 2004; Niculescu et al. 2008), or a control solvent, both containing 0.01% Evans blue dye was injected by the same method into the HDCo (2 μl/chick). Five minutes after the injection, imprinting training was performed for 20 min, and 48 h later, the imprinting performance was evaluated. It was shown previously that K252a injected in the brain is no longer effective after 48 h (Rattiner et al. 2004).
At the end of the experiment, the chicks were deeply anesthetized, the brain was dissected, and the blue-stained area in the brain was inspected in each slice. We excluded all data obtained from chicks that lacked staining with the blue dye in the target area, or that showed staining that extended to areas other than the target area.
All data in this article are expressed as mean ± SEM. The number of animals used is indicated in each figure or the legends. Normal distribution of the data was checked by chi-square test and confirmed unless otherwise specified below. After confirming equality of variances by the Bartlett test, we used one-way anova, followed by Scheffé's F test to compare the values between conditions (Fig. 2e and g). For Fig. 2d, as data were not normally distributed, Kruskal–Wallis and Bonferroni tests were used. After confirming equality of variances by F-test, a Student's t-test was used to compare the number of positive cells between the control and training groups (Fig. 3d–f). A one-sample t test was used to examine whether or not the PS value was significantly larger or smaller than chance (0.5; indicated by #; Figs 4c, d and 5b). To compare the PS between the red circle and blue square, a paired t-test was used except for one case in which data were not normally distributed and a Mann–Whitney U-test was used (Fig. 4d control). Two-way repeated measure anova was also used to analyze the effects of variables (Fig. 4c, d and 5b). Differences were regarded as statistically significant at p < 0.05 (*, #) and p < 0.01 (**, ##).