Thirty-six common male marmoset monkeys (Callithrix jacchus) were investigated. The animals were obtained from the breeding colony at the German Primate Center (Göttingen, Germany). All investigations and the experiments were performed to the highest ethical standards according to the relevant local, national and international regulations concerning the use of animals. All research projects were carried out only with authorization from the relevant ethics committees. We employed the minimum number of animals required to obtain consistent data and to avoid animal suffering. For the termination of non-human primates, the legal requirements, guidelines and recommendations set by the national authorities were followed strictly. The use of animals including non-human primates in research in Germany is based on the "Tierschutzgesetz" (Animal Protection Law) of the 25th of May, 1998 (BGBl.I Nr.30 v.29.5.1998, S. 1105) and the EU guidelines 86/609/EEG. The proposals for research conducted in this study are approved by the local Government and a specially appointed Ethical Review Committee (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, permit numbers 33.11.425-04-003/08 and 33.11.425-04-026/07).
For expression studies, animals of the following ages were used [25, 26]: postnatal days 1 and 7, and postnatal months 1, 3, 5, 7, 12, and 21 (N = 3 per age). For monocular enucleation (ME), the left eyes of one month-old marmoset monkeys (N = 6; approximate body weight 75 g) were surgically enucleated under general anesthesia. As anesthesia, the animals received 0.1 ml of a premix containing 4 mg/ml alphaxalon and 1.33 mg/ml alphadolon (Saffan®; Schering-Plough Animal Health, Welwyn Garden City, UK), 0.37 mg/ml diazepam, and 0.015 mg/ml glycopyrroniumbromid (Robinul®; Riemser, Germany). Enucleation was carried out as described for pet animals  and the eyehole was filled with Gelastypt® sponge (Sanofi-Aventis, Frankfurt, Germany) as soon as arterial bleeding was no longer visible. The wound was closed with an intracutane suture of vicryl 6-0 (V302H; Ethicon, Norderstedt, Germany). Directly after surgery (day 0) and at days 3 and 5, all animals received an antibiotic (amoxicillin-trihydrate; Duphamox LA®, Fort Dodge, Würselen, Germany). ME animals and six age-matched controls were sacrificed at five months of age .
For in situ hybridization, brains were immediately removed from terminally anesthetized animals [overdose of ketamine (50 mg/ml), xylazine (10 mg/ml), and atropine (0.1 mg/ml)] and the whole visual cortices were quickly dissected (area determined according to [29, 30]), embedded in Tissue Tek (Sakura Finetek, Heppenheim, Germany), flash frozen in liquid nitrogen, and stored at -80°C. Frozen brains were sectioned using a cryostat (Leica CM3050, Bensheim, Germany) and coronal sections (10 μm) of whole visual cortices were thaw-mounted on adhesive silane-coated slides (Histobond, Marienfeld, Laboratory Glassware, Lauda-Königshofen, Germany). For immunohistochemistry, marmosets that were terminally anesthetized (see above), were perfused transcardially with 0.9% saline, followed by 200 ml of fixative containing 4% paraformaldehyde in 0.1 M sodium-phosphate buffer (pH 7.2) for 15 min. The heads were postfixed in the same fixative, and brains were carefully removed from the skulls on the following day. After cryoprotection in 0.1 M phosphate-buffered saline (PBS; 0.1 mM phosphate buffer, 0.9% NaCl, pH 7.2) containing 30% sucrose, serial coronal sections of the entire visual cortices (thickness: 40 μm for expression studies and 60 μm for ME animals and controls) were obtained using a cryostat.
PCR cloning of MHCI transcripts
The isolation of the common marmoset MHC class I heavy chain cDNA sequences was carried out using reverse transcriptase polymerase chain reaction (RT-PCR). One microgram of total brain RNA was reverse transcribed using the oligo (dT) primer and 400 U of reverse transcriptase (Promega, Mannheim, Germany). An aliquot of this reaction was used as a template in a PCR containing primers designed with the Primer3 software . Primers were devised to amplify full-length marmoset MHC class I heavy chain transcripts (accession number: U59637). Primer sequences also included BamHI restriction sites and were as follows: forward 5'-CACGGATCCCACTTTACAAGCCGTGAGAGAC-3', reverse 5'-CACGGATCCCTCCTGTTGCTCTCGGGGGCCTTG-3'. Caja-G*1 (accession number: U59637) was obtained and cloned into the pDrive vector (Qiagen).
In situ hybridization
Cryosections (10 μm) of visual cortices were dried at RT for 20 min, fixed in 4% buffered paraformaldehyde (PFA, pH 7.2), rinsed in PBS, acetylated (0.1 M triethanolamine, 0.25% acetic acid anhydride), washed in PBS, and dehydrated through a graded ethanol series. Caja-G plasmid DNA was linearized and riboprobes were synthesized using T7 and SP6 RNA polymerases (Promega, Madison, WI, USA) for the antisense and sense probe, respectively, in the presence of 9.25 MBq of 33P-UTP (Hartmann Analytic GmbH, Braunschweig, Germany; specific activity, 3,000 Ci/mmol) for 1 h at 37°C. Probes were purified using Microspin S-400 HR columns (Amersham Pharmacia, Freiburg, Germany) and hybridization buffer (50% deionized formamide, 10% dextran sulphate, 0.3 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 500 μg/ml tRNA, 0.1 M dithiothreitol, and 1 × Denhardt's solution) was added to yield a final probe activity of 5 × 104 cpm. The hybridization mixture containing the probe was denatured at 70°C for 10 min, cooled to 55°C, and pipetted directly onto sections (80 μl/section). Hybridization was performed for 18 h at 68°C. Sections were subsequently washed in 4 × SSC (0.6 M NaCl, 0.06 M citric acid), 2 × SSC, and 0.5 × SSC for 10 min each at 37°C. After incubation (1 h at 75°C) in 0.2 × SSC, sections were washed twice in 0.1 × SSC, once at 37°C and again at RT, for 10 min each. Finally, sections were dehydrated through graded alcohols, air dried, and exposed to Bio-Max MR film (Amersham Pharmacia) for three days at 4°C. Films were developed and fixed with GBX (Kodak, Rochester, NJ, USA).
Quantitative in situ hybridization
After in situ hybridization, sections were coated with photoemulsion (Kodak NBT2, Rochester, NJ, USA) at 42°C, dried for 90 min at RT, and stored for seven weeks at 4°C in a lightproof container. Exposed slides were developed at 15°C for 5 min (Kodak developer D-19), rinsed twice briefly in H2O, and fixed for 5 min at RT (fixer, Kodak Polymax). Sections were counterstained either with 0.05% toluidine blue in 0.1% disodium tetraborate (Sigma) for expression studies or with methyl green (Sigma) for monocular enucleation studies, cleared in xylol, and coverslipped using mounting medium (Eukitt, Kindler, Freiburg, Germany). Hybridized sections were visualized with a 40× objective (NA = 1.4; Zeiss) under a light microscope (Axioscope, Zeiss) and silver grain quantification was performed on a cell-by-cell basis using ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA). Images were obtained from layer IV neurons and, for each area within the primary visual cortex (demarcated according to [29, 30]) two images were acquired, i.e., one using a green filter to eliminate background staining from methyl green and one using white light to later precisely localize neuronal nuclei. A circular counting mask of 15 μm in diameter was used to delineate the region of interest and was placed over neuronal nuclei during counting. Relative optical density (ROD) threshold intensities were optimized to detect exposed silver grains exclusively. The silver grain density within the region of interest was measured. Grain density was compared between layer IV neurons in primary visual cortices of six animals (three slides per animal, minimum 300 neurons per animal were counted) using Student's t test (GraphPad Prism version 4 for Windows, GraphPad Software, San Diego, CA, USA).
To isolate RNA for RT-PCR, brains were immediately removed from terminally anesthetized animals (see above) and whole occipital lobes containing visual cortices were quickly dissected. Total RNA was isolated from both hemispheres of dissected tissue samples from animals at the age of five months (N = 3 controls and N = 3 unilaterally enucleated animals) using the QIAGEN RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. As the cortices had to be separated during RNA isolation due to their size, both left and right hemispheres of all animals were treated as independent samples. The integrity and quantity of purified RNA was assessed by spectrophotometry. Complementary DNA (cDNA) was synthesized from mRNA transcripts using oligo (dT)12-18 primers and Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany), according to the manufacturer's instructions. The Primer3 software v2.0  was used to design gene-specific primers, with amplicons ranging from 50 to 150 bp in length. The primers used for the detection of MHC class I heavy chain transcripts were: forward 5'-GTGATGTGGAGGAAGAACAGC-3', reverse 5'-CACTTTACAAGCCGTGAGAGA-3' (CajaG*01, accession number U59637). Primers for the detection of GFAP were: forward 5'-AAACGAGTCCCTGGAGAG-3', reverse 5'-TCCTGGTACTCCTGCAAGT-3' (marmoset GFAP, Ensembl transcript number ENSCJAT00000024380). Primers for the detection of c-Fos were: forward 5'-CGAAGGGAAAGGAATAAGAT-3', reverse 5'-GCAGACTTCTCATCTTCCAG-3' (marmoset c-Fos, Ensembl transcript number ENSCJAT00000040535). Primers for the detection of β-actin were: forward 5'-CATCCGCAAAGACCTGTATG-3', reverse 5'-GGAGCAATGACCTTGATCTTC-3' (marmoset ß-actin, accession number DD279463). A quantitative analysis of gene expression was performed using the 7500 Real-time PCR apparatus (Applied Biosystems, Darmstadt, Germany) in combination with Quantitect SYBR green technology (Qiagen). The light cycler was programmed to the following conditions: an initial PCR activation step of 10 min at 95°C, followed by 40 cycling steps (denaturation for 15 s at 95°C, annealing for 30 s at 55°C, and elongation for 60 s at 72°C). Details of the quantitative real-time PCR were described previously . Dissociation curves were generated for all PCR products to confirm that SYBR green emission was detected from a single PCR product . All products were run on 2.5% agarose gels to confirm a single product (Additional file 1). The Caja-G PCR product was additionally sequenced using the BigDye Terminator Sequencing Kit (Applied Biosystems) and forward primer in order to confirm its identity (Additional File 2). The relative abundance of the MHCI-HC, c-Fos and GFAP mRNA transcripts was calculated relative to the mRNA levels of the internal reference gene β-actin and was compared between both left and right cortices of control and enucleated animals using Student's t-test (GraphPad Prism version 4 for Windows).
Immunocytochemistry for light microscopy
Coronal cryosections (40 μm for expression studies and 60 μm for ME animals) of occipital lobes were collected and washed briefly in PBS before the epitope retrieval step. Epitope retrieval was performed by incubating the sections for 20 min in 10 mM sodium citrate buffer preheated to 80°C. Sections were later brought to RT, washed in PBS, and quenched of endogenous peroxidase activity using 30 min incubation at RT in 0.5% H2O2 in distilled water. Sections were then washed in PBS, blocked for 1 h at RT (3% normal horse serum in PBS), incubated for 16 h at 4°C with mouse monoclonal TP25.99 or Q1/28 IgG [34, 35] kindly provided by S. Ferrone, University of Pittsburgh, USA), 1:300 dilution in 3% normal horse serum in PBS; monoclonal mouse W6/32 , 1:300 dilution in 3% normal horse serum in PBS; monoclonal rabbit anti c-Fos (Cell Signalling Technologies, Beverly, MA, USA); monoclonal anti-GFAP (Sigma) 1:200 dilution in 0.01% Triton-X 100 and 3% normal horse serum in PBS; or control mouse IgG (Sigma), and washed again. For c-Fos, W6/32 and GFAP staining, the epitope retrieval step was not performed. Sections were then incubated with biotinylated horse anti-mouse IgG or donkey anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), 1:200 dilution in 3% normal horse serum in PBS, for 1 h at RT. After washing, sections were incubated with avidin-biotin horseradish peroxidase (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA), 1:100 dilution in 3% normal horse serum in PBS, for 1 h at RT, washed in PBS and then again in 0.05 M Tris/HCl (pH 7.2) prior to DAB detection (DAB detection with or without nickel enhancement was performed according to the manufacturer's instructions; DAB-Kit, Vector Laboratories). Sections were washed in 0.05 M Tris/HCl (pH 7.6) and again in 0.1 M PBS prior to xylol clearance, dehydration, and coverslipping with Eukitt mounting medium (Kindler). For identification of cortical layers, sections adjacent to the ones used for immunocytochemistry were stained with toluidine-blue (Sigma). Digital images of stained sections were acquired using an Axiophot II microscope (Zeiss). Final images were assembled in Corel PhotoPaint X3 and, in case of higher magnifications, were a composition of 4-5 images along the longitudinal axis of the primary visual cortex [29, 30]. Contrast and luminosity were adjusted in Corel PhotoPaint X3 for images obtained from monocularly enucleated animals.
Immunofluorescence and confocal microscopy
Antibodies used in double-labeling experiments were applied sequentially and blocking steps were performed using normal sera of the host species from which the respective secondary antibodies were derived. Cryostat sections (40 μm) of occipital lobes were rinsed in PBS before the epitope retrieval step was performed, as described above. Nonspecific antibody binding sites were blocked with 3% normal serum in PBS for 1 h at RT. Sections were then incubated with mouse monoclonal TP25.99 antibody, 1:300 in 3% normal serum in PBS, for 16 hrs at 4°C, washed, and incubated in secondary antiserum (Alexa 488-coupled goat anti-mouse, Molecular Probes, Invitrogen, Leiden, Netherlands) at a dilution of 1:500 for 4 h in a lightproof container. Sections were then washed and incubated with either rabbit anti-MAP2 antibody (1:200, Synaptic Systems, Göttingen, Germany), rabbit anti-NMDAR1 (1;200, Synaptic Systems), rabbit anti-GFAP (1:500, Synaptic Systems), or rabbit anti-vimentin antibody (1:200, Synaptic Systems) in 3% normal serum in PBS for 16 h at 4°C. For GFAP-vimentin colocalization studies, the epitope retrieval step was not performed and following primary antibodies were used: rabbit anti-vimentin (1:200, Synaptic Systems) and mouse anti-GFAP (1:200, Sigma). Sections were then washed and incubated 4 h at RT in secondary antiserum (Alexa 568-coupled goat anti-rabbit, Molecular Probes, Invitrogen) diluted 1:500 in 3% normal serum in PBS. Thereafter, sections were washed in PBS and floated/mounted on SuperFrost ®Plus slides (Menzel-Gläser GmbH, Braunschweig, Germany) in distilled water, allowed to dry overnight at 4°C, and coverslipped with mounting medium (Aqua-Polymount, Polysciences Inc., Warrington, PA, USA). For control sections, the same procedures were performed omitting the primary antibody.
Confocal microscopy was performed using a laser-scanning microscope (LSM 5 Pascal, Zeiss) with an argon 488 nm laser and a helium/neon 543 nm laser (in multiple-tracking mode). High magnification, single optical plane images of layer IV neurons of the primary visual cortex [29, 30] or of the subcortical white matter in the occipital lobes were obtained at a resolution of 1,024 × 1,024 with an Apochromat 63× oil objective (NA = 1.4) and immersion oil (Immersol, Zeiss; refractive index = 1.518). The control sections (no primary antibodies) displayed only background fluorescence (data not shown).
Brains were immediately removed from terminally anesthetized animals (see above), and the whole visual cortices were quickly dissected. Whole visual cortex samples were homogenized with a Dounce homogenizer (tight pestle) in ice-cold homogenization buffer consisting of 50 mM Tris/HCl pH 7.4, 7.5% glycerol, 150 mM NaCl, 1 mM EDTA, 1% Triton-X 100, and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). After homogenization, the samples were centrifuged at 4,000× g for 20 min at 4°C. The resulting supernatant was centrifuged again until it was clear. Protein concentration was measured using the Bio-Rad DC Protein assay (Bio-Rad Laboratories, Hercules, CA, USA).
Protein preparations were electrophoresed in 12.5% SDS gels under reducing conditions. Proteins were subsequently transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) via semidry electroblotting for 2 h at 1 mA/cm2 in transfer buffer containing 25 mM Tris-base, 150 mM glycine, and 10% (v/v) methanol. After the transfer, the blotted membranes were blocked with 5% (w/v) milk powder and 0.1% Tween-20 in PBS for 1 h at RT and were then incubated with either monoclonal TP25.99 (1:1,000) or monoclonal anti-SNAP-25 (1:1,000, Synaptic Systems) antibodies overnight at 4°C. After washing three times for 5 min in PBS/0.1% Tween-20, blots were incubated for 1 h at RT with horseradish peroxidase-coupled goat anti-mouse IgG (1:4,000, Santa Cruz Biotechnology, Santa Cruz, USA). Prior to visualization, blots were washed in PBS/0.1% Tween-20 (3 × 5 min) and once more in PBS. Signals were visualized using SuperSignal West Dura enhanced luminescence substrate (Pierce Biotechnology, Rockford, IL, USA). Membranes were subsequently stripped in a mixture of β-mercaptoethanol and SDS in PBS and incubated with monoclonal anti-β-actin antibody (1:4000, Sigma). For controls, the same procedures were performed using the mouse IgG (Sigma) instead of a primary antibody. Control IgG yielded no signals in the Western blot (data not shown). For quantification, blots were visualized with MCID Basic software (Imaging Research Inc., St. Catherines, Ontario, Canada) so that nonsaturating bands were obtained. Quantification was performed using the gel analysis plug-in of ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA). Values obtained for MHCI and SNAP-25 were normalized to ß-actin values.
Image analysis of sections from monocularly enucleated animals
Digital images of immunostained tissue sections were acquired using an Axiophot II microscope (Zeiss). Images were taken from the V1 areas of right visual cortices. Variations in MHCI immunoreactivity through layer IV were measured as described previously . Briefly, images were normalized and black-white inverted using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA) and regions within layer IV that encompassed the thickness of the entire layer IV (2 mm in length and approximately 1 mm in thickness) were defined as the regions of interest. For identification of cortical layers, sections adjacent to the ones used for immunocytochemistry were stained with toluidine-blue (Sigma). The density profile of layer IV in the primary visual cortex (region demarcated according to [29, 30]) was obtained using ImageJ. Values were averaged (using the four closest neighbors) and plotted as a function of the distance along layer IV parallel to the pia mater. After delineating the borders of immunoreactive patches, their width was measured using ImageJ and compared using Student's t-test (GraphPad Prism version 4 for Windows).