The cytoarchitecture of the prefrontal cortex was studied in ten adult, male mice (strain C57BL/6) of similar weight (approximately 20g). These control mouse brains were kindly donated and immersed fixed by Dr. H. Manji, NIMH, USA. All animal procedures were in a strict accordance with the NIH animal care guidelines. The histological processing of these brains was performed at the laboratory of Dr. Rajkowska. The brains were embedded in 12% celloidin, cut into 40μm serial sections using a sliding microtome and Nissl (1% cresyl violet) stained. Celloidin was chosen as an embedding medium to allow for the preparation of ‘thick’ sections with clear morphology and high contrast of Nissl stained neurons and glial cells. In these immersion-fixed brains, any spots showing pycnotic reaction were not incorporated in this study.
In addition to these ten mice, four adult male mice (C57BL/6 strain) have been stained for dopamine and four adult male mice for acetylcholinesterase (AChE), myelin, and immunohistochemically for SMI, parvalbumin and calbindin: per staining a different set of sections with several consecutive sections stained with Nissl at HBMU's laboratory. The antibodies applied are the dopamine (DA) antibody (Geffard et al., 1984 (link)), SMI-32 antibody (Sternberger Monoclonals Inc., Baltimore, MD, USA: monoclonal antibody to one epitope of non-phosphorylated tau neurofilaments, Lot Number: 11), SMI-311antibody (Pan-Neuronal Neurofilament Marker cocktail of several monoclonal antibodies for several epitopes of non-phosphorylated tau protein, Sternberger Monoclonals Inc., Baltimore, MD, USA: Lot Number 9) [SMI antibodies are presently distributed through Covance Research Products, USA], monoclonal anticalbindin D-28K antibody (Sigma, St. Louis, Mo., USA: Product Number: C-9848, Clone Number: CB-955, Lot Number: 015K4826), and monoclonal anti-parvalbumin antibody (Sigma, St. Louis, Mo., USA: Product Number: P-3171, Clone Number: PA-235, Lot Number: 026H4824). Mice to be stained for DA were intracardially perfused under deep pentobarbital anaesthesia (1 ml/kg body weight, i.p.), with saline followed by fixative. For DA staining the fixative was 5% glutaraldehyde in 0.05 M acetate buffer at pH 4.0. After perfusion the brains were immersed in 0.05 Tris containing 1% Sodium disulfite (Na2S2O5) at pH 7.2 (De Brabander et al. 1992 (link)). Mouse prefrontal cortex was sectioned at 40 μm by a vibratome. These sections were stained overnight in a cold room at 4° C. using the polyclonal primary antibody sensitive for DA raised in the Netherlands Institute for Brain Research (NIBR) (Geffard et al. 1984 (link)) of which the specificity has been demonstrated previously (Kalsbeek et al. 1990 (link)). DA antiserum was diluted 1:2,000 in 0.05 M Tris containing 1% Na2S2O5 and 0.5% Triton X-100, pH 7.2. After overnight incubation the sections were washed three times with Tris buffered saline (TBS) and subsequently incubated in the secondary antibody goat-antirabbit, also raised in NIBR, at 1:100 for 1 h. and, after having been rinsed 3x in TBS, incubated in the tertiary antibody, peroxidase-antiperoxidase, at 1:1,000 for 60 min. Both the secondary and the tertiary antibodies were diluted in TBS with 0.5% gelatine and 0.5% Triton X-100. For visualization the sections were transferred into 0.05% diaminobenzidine 9(DAB; Sigma) with 0.5% nickel ammonium sulphate. The reaction was stopped after a few minutes by transferring the sections to TBS (3x 10 min), then the sections were mounted on slides, air dried, washed, dehydrated and coverslipped. Mice to be stained with antiparvalbumin, anticalbindin and SMI-32 and SMI-311 were fixed with 4% formaldehyde solution in 0.1 M phosphate buffer at pH 7.6. Mouse prefrontal cortex was sectioned at 40 μm by a vibratome. To prevent endogenous peroxidase activity free-floating sections were pretreated for 30 min in a Tris-buffered saline (TBS) solution containing 3% hydrogen peroxide and 0.2% Triton X-100. To prevent non-specific antibody staining, these sections were placed in a milk solution (TBS containing 5% nonfat dry milk and 0.2% Triton X-100) for 1 h. Incubation of the primary antibody, directly after the milk step, was carried out overnight in a cold room at 4%. The primary antibodies were diluted in above-mentioned milk solution: SMI-32 and SMI-311 at 1:1,000, parvalbumin antibody at 1:1,000, and calbindin antibody at 1:250. For the monoclonal SMI-32, SMI-311, parvalbumin and calbindin antibodies, raised in mice, we used peroxidase-conjugated rabbit-antimouse (1:100 in 5% milk solution with 0.2% Triton X-100) as a secondary antibody. Visualization took place in 0.05% diaminobenzidine enhanced with 0.2% nickel ammonium sulphate. The reaction was stopped after a few minutes by transferring these sections to TBS (3x 10 min), after which the sections were rinsed in distilled water, mounted on slides, air dried, washed, dehydrated and coverslipped. Control sections incubated according to the same procedure as described above, but omitting the primary antibody were all negative. All sections have been cut coronally, because the coronal plane offers in general the best view to differentiate between the subareas of the rodent prefrontal cortex (Uylings et al. 2003 (link); Van de Werd and Uylings 2008 (link)).
The sections were studied at intervals of 80-160 μm, and were examined under the light-microscope at a 63x magnification.
Sections have been processed for acetylcholinesterase staining according to protocol described by Cavada et al. (1995) (link). The sections were incubated overnight in a solution of cupric sulphate and acetate buffer at pH 5 to which acetylthiocholine iodide and ethopropazine were added just before the start of the incubation. After rinsing the sections were developed in a sodium sulphide solution until a light brown color appeared and subsequently intensified in a silver nitrate solution until a dark brown color. Finally, the sections are differentiated after rinsing in a thiosulphate solution, dehydrated and mounted. In all steps the solutions and sections were shaken constantly. The myelin was stained with silver by means of physical development according to Gallyas (1979) (link). The sections were first put into 100% ethanol, and then immersed in a 2:1 solution of pyridine and acetic acid for 30 minutes. After rinsing they were brought into an ammonium-silver-nitrate solution and after rinsing them in 0.5% acetic acid, the sections were immersed in the optimal physical developer solution for room temperature (Gallyas, 1979 (link)) until the sections showed a good stain-intensity, checked under microscope. Then the development of the staining was stopped in 0.5% acetic acid and the sections were dehydrated and mounted with Histomount.
In addition to these ten mice, four adult male mice (C57BL/6 strain) have been stained for dopamine and four adult male mice for acetylcholinesterase (AChE), myelin, and immunohistochemically for SMI, parvalbumin and calbindin: per staining a different set of sections with several consecutive sections stained with Nissl at HBMU's laboratory. The antibodies applied are the dopamine (DA) antibody (Geffard et al., 1984 (link)), SMI-32 antibody (Sternberger Monoclonals Inc., Baltimore, MD, USA: monoclonal antibody to one epitope of non-phosphorylated tau neurofilaments, Lot Number: 11), SMI-311antibody (Pan-Neuronal Neurofilament Marker cocktail of several monoclonal antibodies for several epitopes of non-phosphorylated tau protein, Sternberger Monoclonals Inc., Baltimore, MD, USA: Lot Number 9) [SMI antibodies are presently distributed through Covance Research Products, USA], monoclonal anticalbindin D-28K antibody (Sigma, St. Louis, Mo., USA: Product Number: C-9848, Clone Number: CB-955, Lot Number: 015K4826), and monoclonal anti-parvalbumin antibody (Sigma, St. Louis, Mo., USA: Product Number: P-3171, Clone Number: PA-235, Lot Number: 026H4824). Mice to be stained for DA were intracardially perfused under deep pentobarbital anaesthesia (1 ml/kg body weight, i.p.), with saline followed by fixative. For DA staining the fixative was 5% glutaraldehyde in 0.05 M acetate buffer at pH 4.0. After perfusion the brains were immersed in 0.05 Tris containing 1% Sodium disulfite (Na2S2O5) at pH 7.2 (De Brabander et al. 1992 (link)). Mouse prefrontal cortex was sectioned at 40 μm by a vibratome. These sections were stained overnight in a cold room at 4° C. using the polyclonal primary antibody sensitive for DA raised in the Netherlands Institute for Brain Research (NIBR) (Geffard et al. 1984 (link)) of which the specificity has been demonstrated previously (Kalsbeek et al. 1990 (link)). DA antiserum was diluted 1:2,000 in 0.05 M Tris containing 1% Na2S2O5 and 0.5% Triton X-100, pH 7.2. After overnight incubation the sections were washed three times with Tris buffered saline (TBS) and subsequently incubated in the secondary antibody goat-antirabbit, also raised in NIBR, at 1:100 for 1 h. and, after having been rinsed 3x in TBS, incubated in the tertiary antibody, peroxidase-antiperoxidase, at 1:1,000 for 60 min. Both the secondary and the tertiary antibodies were diluted in TBS with 0.5% gelatine and 0.5% Triton X-100. For visualization the sections were transferred into 0.05% diaminobenzidine 9(DAB; Sigma) with 0.5% nickel ammonium sulphate. The reaction was stopped after a few minutes by transferring the sections to TBS (3x 10 min), then the sections were mounted on slides, air dried, washed, dehydrated and coverslipped. Mice to be stained with antiparvalbumin, anticalbindin and SMI-32 and SMI-311 were fixed with 4% formaldehyde solution in 0.1 M phosphate buffer at pH 7.6. Mouse prefrontal cortex was sectioned at 40 μm by a vibratome. To prevent endogenous peroxidase activity free-floating sections were pretreated for 30 min in a Tris-buffered saline (TBS) solution containing 3% hydrogen peroxide and 0.2% Triton X-100. To prevent non-specific antibody staining, these sections were placed in a milk solution (TBS containing 5% nonfat dry milk and 0.2% Triton X-100) for 1 h. Incubation of the primary antibody, directly after the milk step, was carried out overnight in a cold room at 4%. The primary antibodies were diluted in above-mentioned milk solution: SMI-32 and SMI-311 at 1:1,000, parvalbumin antibody at 1:1,000, and calbindin antibody at 1:250. For the monoclonal SMI-32, SMI-311, parvalbumin and calbindin antibodies, raised in mice, we used peroxidase-conjugated rabbit-antimouse (1:100 in 5% milk solution with 0.2% Triton X-100) as a secondary antibody. Visualization took place in 0.05% diaminobenzidine enhanced with 0.2% nickel ammonium sulphate. The reaction was stopped after a few minutes by transferring these sections to TBS (3x 10 min), after which the sections were rinsed in distilled water, mounted on slides, air dried, washed, dehydrated and coverslipped. Control sections incubated according to the same procedure as described above, but omitting the primary antibody were all negative. All sections have been cut coronally, because the coronal plane offers in general the best view to differentiate between the subareas of the rodent prefrontal cortex (Uylings et al. 2003 (link); Van de Werd and Uylings 2008 (link)).
The sections were studied at intervals of 80-160 μm, and were examined under the light-microscope at a 63x magnification.
Sections have been processed for acetylcholinesterase staining according to protocol described by Cavada et al. (1995) (link). The sections were incubated overnight in a solution of cupric sulphate and acetate buffer at pH 5 to which acetylthiocholine iodide and ethopropazine were added just before the start of the incubation. After rinsing the sections were developed in a sodium sulphide solution until a light brown color appeared and subsequently intensified in a silver nitrate solution until a dark brown color. Finally, the sections are differentiated after rinsing in a thiosulphate solution, dehydrated and mounted. In all steps the solutions and sections were shaken constantly. The myelin was stained with silver by means of physical development according to Gallyas (1979) (link). The sections were first put into 100% ethanol, and then immersed in a 2:1 solution of pyridine and acetic acid for 30 minutes. After rinsing they were brought into an ammonium-silver-nitrate solution and after rinsing them in 0.5% acetic acid, the sections were immersed in the optimal physical developer solution for room temperature (Gallyas, 1979 (link)) until the sections showed a good stain-intensity, checked under microscope. Then the development of the staining was stopped in 0.5% acetic acid and the sections were dehydrated and mounted with Histomount.
