Article Figures & Data
Figures
- Fig. 1.
CREB phosphorylation in the cerebral cortex after sleep and waking. A, The experimental conditions chosen to distinguish changes associated with sleep (S), waking (W), circadian factors, and the sleep-deprivation procedure (SD). A 12 hr light/dark cycle is indicated by the horizontal bar. Three hours (3h) and 8 hr (8h) of S,SD, and W are shown.B, Anti-P-CREB staining in coronal sections of parietal cortex (layers II–VI) from a rat that slept for 3 hr (S) and a rat that was sleep-deprived for 3 hr (SD). Scale bar, 100 μm. C, Mean number (±SEM) of P-CREB-immunoreactive neurons in entorhinal (ent), parietal (par), temporal (te), and occipital (occ) cortex after 3 or 8 hr of S (n = 4/group), 3 or 8 hr of SD (n = 4/group), and 3 or 8 hr of spontaneous W (n = 3 after 3 hr; n = 2 after 8 hr). The sampled area was 500 μm wide across all cortical layers. The number of P-CREB-immunoreactive neurons was significantly higher inSD and W with respect to S(Mann–Whitney U test, *p < 0.01). No differences were found between SD andW. For each condition, there were no differences between data from the 3 and 8 hr groups, which were therefore pooled.
- Fig. 2.
Arc expression in the cerebral cortex after sleep and waking. A, cDNA microarrays showing cortical Arc mRNA levels (arrowheads) after 3 hr (3h) or 8 hr (8h) of sleep (S; n = 7), sleep deprivation (SD; n = 7), and waking (W; n = 6).B, Densitometric analysis performed by scanning the microarrays with a PhosphorImager. The y-axis values refer to signal intensity (arbitrary units). C,In situ hybridization for Arc mRNA in brain sections of a representative rat killed after 8 hr ofS and of a rat killed at the same circadian time after 8 hr of SD. Scale bar, 1.5 mm. D, Arc levels measured with immunocytochemistry in the parietal cortex of rats after 3 hr of S and 3 hr of W. Scale bar, 100 μm. E, Mean number (±SEM) of Arc-immunoreactive neurons in parietal cortex after 3 hr ofS (n = 8), SD(n = 8), and W(n = 5). The sampled area was a 500-μm-wide cortical column spanning all layers (Mann–Whitney Utest, *p < 0.01). F, Double labeling in the parietal cortex of a rat that was sleep deprived for 3 hr. Arc immunoreactivity (black cells;left) does not colocalize with parvalbumin immunoreactivity (white cells; right). Scale bar, 50 μm.
- Fig. 3.
Arc expression in the parietal cortex as a function of the duration of waking. A, B, Arc levels measured with immunocytochemistry in cortical layers V (A) and VI (B) after 1 hr (1h), 3 hr (3h), and 9 hr (9h) of sleep deprivation (SD). Scale bar, 200 μm.
- Fig. 4.
BDNF expression in the cerebral cortex after sleep and waking. A, RPA showing corticalBDNF mRNA levels after 8 hr of sleep (S;n = 7), sleep deprivation (SD;n = 7), and waking (W;n = 6). A β-actinantisense riboprobe was used to normalize the amount of sample RNA.Lane 1 (from left), Molecular weight markers. Lane 2, BDNF andβ-actin riboprobes hybridized with 10 μg of yeast RNA, incubated without RNase mixture. Most of the signal is the full-length BDNF (arrow) andβ-actin (arrowhead) probes. Lanes 3–8, BDNF andβ-actin probes hybridized under conditions of excess probe with 2 μg of pooled RNA. S,lanes 3 and 4; SD, lanes 5and 6; W, lanes 7 and 8. The protected fragments are 407 bp for BDNF and 250 bp for β-actin. B, Densitometric analysis performed by scanning the RPA gel with a PhosphorImager. The y-axis values refer to signal intensity (arbitrary units). Relative to S,BDNF mRNA levels were higher in both SDand W rats (t test, p= 0.021 and 0.028, respectively). C, In situ hybridization for BDNF mRNA in brain sections of representative rats killed after 8 hr of Sand 8 hr of SD. Scale bar, 1.5 mm. D, BDNF levels (picograms per milligram of protein) measured with ELISA in the cerebral cortex of rats after 8 hr of S,SD, and W. Each columnrepresents an individual rat (mean of 3 measurements ± SEM). Animals were divided into two groups on the basis of their age: 8–9 weeks (left) and 15–16 weeks (right). BDNF levels for all rats were measured simultaneously on the same assay. BDNF levels were higher inSD and W rats than in Srats (F = 21.11; p < 0.01).E, Densitometric analysis showing changes inTrkB mRNA levels, as measured by RPA, after 8 hr ofS (n = 7), SD(n = 7), and W(n = 6). The y-axis values refer to signal intensity (arbitrary units). Relative to S, cortical TrkB mRNA levels were significantly higher after 8 hr of SD (t test,p = 0.032) but not after 8 hr of W(t test, p = 0.058).
- Fig. 5.
BDNF and Arcexpression in the cerebral cortex after lesion of the noradrenergic system of the locus coeruleus. A, RPA showing corticalBDNF and Arc mRNA levels after 8 hr of sleep deprivation in control rats who received a saline injection (C; n = 4; lanes 1–3from left) and in rats treated with DSP-4 to destroy the noradrenergic innervation of the cerebral cortex (n= 4; lanes 4–6). Aβ-actin antisense riboprobe was used to normalize the amount of sample RNA. B, Densitometric analysis performed by scanning the RPA gel with a PhosphorImager. They-axis values refer to signal intensity (arbitrary units). BDNF and Arc mRNA levels were higher in C than in DSP-4 rats (t test,p = 0.030 and 0.00, respectively). C, Left, Coronal brainstem section of a representative rat in which the left locus coeruleus was destroyed by a local injection of 6-OHDA (the arrow indicates the right locus coeruleus). Tyrosine hydroxylase immunostaining, used to identify noradrenergic neurons and fibers in the locus coeruleus, is abolished on the side of the lesion, whereas it is intact on the other side. Scale bar, 1.2 mm.Right, A rostral coronal brain section immunostained against tyrosine hydroxylase from the same animal showing the almost complete depletion of noradrenergic fibers in the left cerebral cortex and hippocampus 2 weeks after the lesion. The noradrenergic innervation on the right side of the brain is intact. Scale bar, 2 mm. D, Arc immunostaining in a coronal section of parietal cortex adjacent to that shown in C. Arc expression is high on the intact side (right) after 3 hr of sleep deprivation, but it is as low as in sleep on the side where the noradrenergic innervation had been destroyed (left). Scale bar, 100 μm.
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