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c-Fos expression during temporal order judgment in mice.

Wada M, Higo N, Moizumi S, Kitazawa S - PLoS ONE (2010)

Bottom Line: The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex.Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project.The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Juntendo University School of Medicine, Tokyo, Japan. m-wada@juntendo.ac.jp

ABSTRACT
The neuronal mechanisms for ordering sensory signals in time still need to be clarified despite a long history of research. To address this issue, we recently developed a behavioral task of temporal order judgment in mice. In the present study, we examined the expression of c-Fos, a marker of neural activation, in mice just after they carried out the temporal order judgment task. The expression of c-Fos was examined in C57BL/6N mice (male, n = 5) that were trained to judge the order of two air-puff stimuli delivered bilaterally to the right and left whiskers with stimulation intervals of 50-750 ms. The mice were rewarded with a food pellet when they responded by orienting their head toward the first stimulus (n = 2) or toward the second stimulus (n = 3) after a visual "go" signal. c-Fos-stained cell densities of these mice (test group) were compared with those of two control groups in coronal brain sections prepared at bregma -2, -1, 0, +1, and +2 mm by applying statistical parametric mapping to the c-Fos immuno-stained sections. The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex. Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project. The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.

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Specificity of the anti-c-Fos antibodies.Immunostaining of the primary somatosensory cortex with an N-terminus antibody, SC-52 (A), and an internal epitope antibody, SC-253 (B), is shown with a control of staining without primary antibody (C). The samples were prepared from neighboring sections in one subject.
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pone-0010483-g002: Specificity of the anti-c-Fos antibodies.Immunostaining of the primary somatosensory cortex with an N-terminus antibody, SC-52 (A), and an internal epitope antibody, SC-253 (B), is shown with a control of staining without primary antibody (C). The samples were prepared from neighboring sections in one subject.

Mentions: For single-labeling of cells that expressed c-Fos protein, the avidin-biotin peroxidase method was used with a polyclonal rabbit antibody specific for c-Fos (SC-52; Santa Cruz Biotechnology, Santa Cruz, CA). The sections were first treated with 0.01% H2O2 in methanol for 20 min to destroy endogenous peroxidases and then incubated for 1 h in 0.1% Triton-X 100 and 1.5% normal goat serum to minimize nonspecific labeling. Next, the tissue sections were incubated for 72 h at 4°C in a 1∶4000 dilution of anti-c-Fos antibody (SC-52 in 0.01M PBS with 0.5% bovine serum albumin). The sections were washed, placed for 1.5 h in a 1∶200 dilution of biotinylated goat anti-rabbit antibody (Vectastain, ABC Elite kit, Vector Laboratories, Burlingame, CA), washed again, and then placed for 1 h in a 1∶200 dilution of avidin–biotin complex (Vectastain, ABC Elite kit). The peroxidase activity was visualized with a nickel-enhanced coloring solution (0.2 mg/ml diaminobenzidine: DAB, 0.02% H2O2, 0.03% nickel ammonium in Tris-buffered saline). After washing with distilled water, the sections were dehydrated through a series of 70, 90, 99, and 100% ethanol (1 min at each concentration), transferred to xylene for three washes of 1 min each, and then covered with coverslips and mounting medium (MP-500, Matsunami Glass Ind., Ltd., Osaka, Japan). Figure 2A shows cell nuclei stained with the c-Fos antibody (SC-52, N-terminus epitope). The specificity of the antibody (SC-52) had been previously confirmed by Western blot analysis (data sheet from Santa Cruz Biotechnology). We additionally confirmed in neighboring sections that another anti-c-Fos antibody that combines with an internal epitope (SC-253; 1∶4000, Santa Cruz Biotechnology) yielded similar results (Fig. 2B). We further confirmed that there was no nonspecific labeling in the absence of the primary c-Fos antibody (Fig. 2C).


c-Fos expression during temporal order judgment in mice.

Wada M, Higo N, Moizumi S, Kitazawa S - PLoS ONE (2010)

Specificity of the anti-c-Fos antibodies.Immunostaining of the primary somatosensory cortex with an N-terminus antibody, SC-52 (A), and an internal epitope antibody, SC-253 (B), is shown with a control of staining without primary antibody (C). The samples were prepared from neighboring sections in one subject.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864740&req=5

pone-0010483-g002: Specificity of the anti-c-Fos antibodies.Immunostaining of the primary somatosensory cortex with an N-terminus antibody, SC-52 (A), and an internal epitope antibody, SC-253 (B), is shown with a control of staining without primary antibody (C). The samples were prepared from neighboring sections in one subject.
Mentions: For single-labeling of cells that expressed c-Fos protein, the avidin-biotin peroxidase method was used with a polyclonal rabbit antibody specific for c-Fos (SC-52; Santa Cruz Biotechnology, Santa Cruz, CA). The sections were first treated with 0.01% H2O2 in methanol for 20 min to destroy endogenous peroxidases and then incubated for 1 h in 0.1% Triton-X 100 and 1.5% normal goat serum to minimize nonspecific labeling. Next, the tissue sections were incubated for 72 h at 4°C in a 1∶4000 dilution of anti-c-Fos antibody (SC-52 in 0.01M PBS with 0.5% bovine serum albumin). The sections were washed, placed for 1.5 h in a 1∶200 dilution of biotinylated goat anti-rabbit antibody (Vectastain, ABC Elite kit, Vector Laboratories, Burlingame, CA), washed again, and then placed for 1 h in a 1∶200 dilution of avidin–biotin complex (Vectastain, ABC Elite kit). The peroxidase activity was visualized with a nickel-enhanced coloring solution (0.2 mg/ml diaminobenzidine: DAB, 0.02% H2O2, 0.03% nickel ammonium in Tris-buffered saline). After washing with distilled water, the sections were dehydrated through a series of 70, 90, 99, and 100% ethanol (1 min at each concentration), transferred to xylene for three washes of 1 min each, and then covered with coverslips and mounting medium (MP-500, Matsunami Glass Ind., Ltd., Osaka, Japan). Figure 2A shows cell nuclei stained with the c-Fos antibody (SC-52, N-terminus epitope). The specificity of the antibody (SC-52) had been previously confirmed by Western blot analysis (data sheet from Santa Cruz Biotechnology). We additionally confirmed in neighboring sections that another anti-c-Fos antibody that combines with an internal epitope (SC-253; 1∶4000, Santa Cruz Biotechnology) yielded similar results (Fig. 2B). We further confirmed that there was no nonspecific labeling in the absence of the primary c-Fos antibody (Fig. 2C).

Bottom Line: The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex.Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project.The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Juntendo University School of Medicine, Tokyo, Japan. m-wada@juntendo.ac.jp

ABSTRACT
The neuronal mechanisms for ordering sensory signals in time still need to be clarified despite a long history of research. To address this issue, we recently developed a behavioral task of temporal order judgment in mice. In the present study, we examined the expression of c-Fos, a marker of neural activation, in mice just after they carried out the temporal order judgment task. The expression of c-Fos was examined in C57BL/6N mice (male, n = 5) that were trained to judge the order of two air-puff stimuli delivered bilaterally to the right and left whiskers with stimulation intervals of 50-750 ms. The mice were rewarded with a food pellet when they responded by orienting their head toward the first stimulus (n = 2) or toward the second stimulus (n = 3) after a visual "go" signal. c-Fos-stained cell densities of these mice (test group) were compared with those of two control groups in coronal brain sections prepared at bregma -2, -1, 0, +1, and +2 mm by applying statistical parametric mapping to the c-Fos immuno-stained sections. The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex. Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project. The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.

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