Immunohistochemical characterization of sleep-active nNOS-ir cells. (A) Triple staining for nNOS (a), NPY (b), and Fos (c) immunoreactivity in mouse cortex during RS. Neurons that express both nNOS and NPY contain Fos (arrows), but neurons that express only NPY do not contain Fos (triangles). (B) Double staining for calretinin (triangles) and Fos (arrow) immunoreactivity in mouse cortex indicating that few calretinin-ir neurons express Fos during RS.

Fos expression in nNOS-ir neurons in mice that were sleep deprived for 6 h or allowed to sleep for 2.5 h after the SD in Experiment 2. (A) In the cerebral cortex, most nNOS neurons did not express Fos during SD (a) but expressed Fos during RS (b). (B) Schematic drawing of Fos/nNOS neuron distribution in representative brain sections at seven levels relative to bregma. Blue circles represent single-stained nNOS neurons and orange squares represent double-stained Fos/nNOS neurons. Few double-stained neurons are found in the cortex after SD (Upper), whereas many double-stained neurons are found in the cortex after RS (Lower). (C) Quantitative analysis demonstrates a significantly greater percentage of Fos-ir cells in all examined regions of the cortex and in the posterior part of the caudate-putamen in mice during RS after SD than during SD itself. *, P < 0.05 compared with corresponding group of sleep-deprived animals. Abbreviations: Cg Cx, cingulate cortex; M Cx, motor cortex; S Cx, somatosensory cortex; I Cx, insular cortex; Pir Cx, piriform cortex; Ent Cx, entorhinal cortex; TeA Cx, TA cortex; V Cx, visual cortex; CPu, a, caudate-putamen, anterior (+1.0 mm from bregma); CPu, p, caudate-putamen, posterior (−1.5 mm from bregma).

Identification of sleep-active neurons in the rat cerebral cortex. (A) During the 2.5-h RS period that followed 6-h SD, wakefulness was significantly reduced (P < 0.05). (B) EEG SWA during NREM sleep was elevated for ≈2 h after the end of SD compared with the baseline level recorded 24 h earlier in the same animals. (C) nNOS-ir neurons (red cytoplasm) in the cerebral cortex did not express Fos (black nuclei) in the SD group (a) but expressed Fos in the RS group (b). (D) Schematic showing Fos⁺/nNOS-ir neuron distribution in two representative brain sections. Blue circles represent single-stained nNOS-ir neurons and orange squares represent double-stained Fos⁺/nNOS-ir neurons. Few double-stained neurons are found in the cortex after SD, whereas many double-stained neurons are found in the cortex after RS. (E) The proportion of Fos⁺/nNOS-ir cells was significantly larger in the RS group than in the SD group in every cortical region and in the caudate-putamen. Data are mean ± SEM. *, P < 0.05 compared with corresponding SD group. Abbreviations: Cg Cx, cingulate cortex; M Cx, motor cortex; S Cx, somatosensory cortex; I Cx, insular cortex; Pir Cx, piriform cortex; CPu, caudate-putamen; PFA, perifornical area.

Sleep parameters and Fos expression in nNOS-ir neurons of mouse and hamster cortex during spontaneous sleep and wakefulness. (A) Mice were killed at four time points across the light/dark cycle; sleep/wake amounts and EEG SWA levels were calculated for the 2.5-h period before they were killed. Statistical comparisons were made among all groups; letters at the top of bars indicate significant differences (P < 0.05) compared with the time bins indicated [from left to right: a–d in mice (A and B) and a–h in hamsters (C)]. Thus, the amount of wakefulness was significantly higher in the dark period (shaded) at ZT14.5 than during the light period (nonshaded) at ZT2.5 or ZT8.5. Conversely, the amounts of NREM and REM sleep were significantly higher during the light (ZT2.5 and ZT8.5) than during the dark at ZT14.5. SWA was significantly lower at ZT8.5 than at ZT2.5, 14.5, or 20.5. (B) Although sleep amounts were similar at ZT2.5 and ZT8.5, the percentage of Fos⁺/nNOS-ir cells was significantly higher at ZT2.5 than at ZT8.5 and thus paralleled the high levels of SWA. Across all groups, the percentage of Fos⁺/nNOS-ir cells in the mouse cortex was highly correlated with NREM delta energy (the relationship is presented here as a semilog plot). (C) In hamsters killed after exposure to constant light for at least 2 weeks, the profile of Fos expression in nNOS-ir cells was similar to that in mice: The highest percentage of Fos⁺/nNOS-ir cells was found at CT3 during the inactive phase when EEG SWA is high and the lowest proportion at CT9–12 when EEG SWA is low. During the active phase, the proportion of Fos⁺/nNOS-ir cells is low at CT15 and increases because sleep debt accumulates late in the active phase (CT21–24). Data are shown as mean ± SEM.