c-Fos Expression during Temporal Order Judgment in Mice

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

Specificity of the anti-c-Fos antibodies.

<p>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.</p

Double-staining with c-Fos and other antibodies.

<p>Images were taken from the barrel field of the mice in the test group. (A) c-Fos (green) and GFAP (red). No co-localization was observed. (B) c-Fos (green) and NeuN (red). Most cells were double-stained (yellow). (C) c-Fos (green) and CAMKIIα (red). Some cells were double-stained (arrow heads). (D) c-Fos (red) and GABA (green). Some cells were double-stained (arrows).</p

c-Fos-positive cell density maps (PCDMs) in three task groups.

<p>PCDMs at five different levels (bregma +2, +1, 0, −1, and −2 mm) are shown in rows for each group (columns). In the rightmost column, figures from the brain atlas (Paxinos & Franklin, 2001) are shown for comparison. Abbreviations: M1, primary motor cortex; M2, secondary motor cortex; Pir, piriform cortex; Tu, olfactory tubercle; AOP, anterior olfactory nucleus, posterior; AOM, anterior olfactory nucleus, medial; Cgl, cingulate cortex, area 1; Cg2, cingulate cortex, area 2; S1, primary somatosensory cortex; S1BF, primary somatosensory cortex, barrel field; PV, paraventricular hypothalamic nucleus; RSA, retrosplenial agranular cortex; RSG, retrosplenial granular cortex; MtPA, medial parietal association cortex; LtPA, lateral parietal association cortex; AuD, secondary auditory cortex, dorsal; AuV, secondary auditory cortex, ventral. Scale bar: 3 mm.</p

Laminar analyses in the barrel field of the primary somatosensory cortex.

<p>(A) Representative images of c-Fos immunostaining (c-Fos) and cytochrome oxidase staining (CO) are shown for each of the three groups. (B) c-Fos-positive cell density (ordinate) plotted against layers in the barrel cortex (abscissa). Different symbols show different groups. Error bars show the standard error of the mean. Data were obtained from both hemispheres for each subject. Thus, each symbol represents the mean of ten data points from five subjects of each group. Asterisks show that the mean values were significantly smaller than that of the supragranular c-Fos-positive cell densities of the test group (cross) after a Bonferroni correction for multiple comparisons (α = 0.05).</p