Dcx protein-levels in rat brain homogenates and CSF during development.

<p>Olfactory bulb, whole hippocampus, pieces of cerebral cortex and cerebellum, and CSF were analyzed from rats at different developmental stages (postnatal day 5 to 40). A, Dcx-protein levels in rat brain tissue homogenates during postnatal development. B, Dcx-protein levels in CSF during postnatal development (N = 4).</p

Murine Dcx-protein expression using a Dcx-specific immunoassay.

<p>A, Left, Dcx is detected in whole brain homogenates from adult C57BL/6 wt and Dcx-KO mice using a sandwich immunoassay. No signal can be observed in Dcx-KO mice (N = 3). Right, Representative Dcx immunoblot. Dcx can be detected in hippocampal tissue of adult wildtype but not Dcx KO mice. B, expression levels of Dcx-protein in various mouse brain regions (wHp: whole hippocampus, Cx: cortex, Cb: Cerebellum, OB: olfactory bulb, N = 6).</p

Irradiation-induced ablation of neurogenesis.

<p>A, schematic diagram of experimental procedures. Female wistar rats received a high (12 gy) or low (6 gy) irradiation dose or were sham-treated at P10 (N = 10 per group). Mice were sacrificed 7 weeks after treatment. A subset of brains were processed for immunohistochemistry (12 gy: N = 2, 6 gy: N = 3, sham: N = 3). Residual brains were split into hemispheres and dissected for mRNA and protein analysis (N = 6/group). B, Representative images of Dcx-IR in the olfactory bulb of formalin-fixed paraffin embedded (FFPE) sections. Left-to-right: A high density of dendritic Dcx-IR is observed in the olfactory bulb granular layer sham-irradiated animals. Dendritic labeling is reduced with low-irradiation and virtually absent in animals after 12 gy-irradiation. Scale bar: 200 µm. C, bar graphs of Dcx-protein-levels in the olfactory bulb. A dose-dependent decrease in Dcx-protein levels is observed in irradiated animals vs sham-controls. D, bar graphs of Dcx-mRNA-levels in the olfactory bulb. A dose-dependent decrease in Dcx-mRNA levels is observed in irradiated animals vs sham-controls. E, Representative images Dcx-IR in the dentate gyrus of FFPE sections. Upper panel: overview of Dcx-IR in the dentate gyrus. Left-to-right: Dcx-IR is restricted to cells in the dentate gyrus SGZ with dendrites spanning into the granular and molecular layer. Lower panel: higher magnification of the SGZ. F, bar graphs of Dcx-protein-levels in the hippocampus. A slight dose-dependent decrease in DCX-protein levels is observed in irradiated animals. G, bar graphs of Dcx-mRNA-levels in the hippocampus. DCX-mRNA levels do not change significantly between sham and irradiated groups. H, bar graphs of Dcx-protein-levels in the cerebral cortex. I, bar graphs Dcx-mRNA-levels in cerebral cortex. DCX-mRNA levels do not change significantly between sham and irradiated groups. J, bar graphs of Dcx-protein-levels in the cerebellum. K, bar graphs of Dcx-mRNA-levels in cerebellum. Dcx-mRNA levels do not change significantly between sham and irradiated groups. Dunnett’s Multiple Comparisons Test.</p

14-day voluntary running wheel experiment.

<p>In two separate experiments, adult mice with or without access to a running wheel were sacrificed after 2 weeks. The right hemisphere was dissected for either protein or mRNA-analysis while the left hemisphere was used to confirm exercise-induced increase in Dcx-IR via immunohistochemistry. A, Dcx-IR was quantified by calculating the total Dcx-IR area in µm² for four different sections within the dorsal hippocampus (left). Bar graph of hippocampal Dcx-protein-levels in DG and resHp (right). N = 12/group. B, Dcx-IR was quantified by calculating the total Dcx-IR area in µm² for four different sections within the dorsal hippocampus (left). Bar graph of hippocampal Dcx-mRNA-level in DG and resHp (right). N = 15/group. C, Bar graph mRNA-level in DG and resHp for Sox11 and Tbr2/EOMES. N = 15/group. Bonferroni’s Multiple Comparisons Test.</p

Effects of almorexant and SB-334867 on spontaneous locomotor activity of rats during the active phase.

<p>Both almorexant <b>(A)</b> and SB-334867 <b>(B)</b> reduced locomotor activity compared to vehicle (Veh) when administered 3 h after the onset of the dark period. Horizontal locomotor activity was recorded for a period of 30 min. Numbers on the X-axes represent intraperitoneal doses in mg/kg.***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 vs. Veh (one-way ANOVA followed by Dunnett’s analysis). All data are mean±SEM (n = 8 per group).</p

Hourly distribution of W, NR and REM sleep.

<p>W, NR and REM sleep for 6 h prior to and 18 h after administration of SB-334867 <b>(A)</b>, EMPA <b>(B)</b>, and almorexant <b>(C)</b> as compared to zolpidem (ZOL) and vehicle. Shaded area represents the dark phase; vertical dotted line in each panel indicates the time of injection. <b>(A)</b> Hourly amounts of wakefulness following SB 334867. <b>(A’)</b> Hourly amounts of NR sleep following SB 334867. <b>(A’’)</b> Hourly amounts of REM sleep following SB 334867. <b>(B)</b> Hourly amounts of wakefulness following EMPA. <b>(B’)</b> Hourly amounts of NR sleep following EMPA. <b>(B’’)</b> Hourly amounts of REM sleep following EMPA. <b>(C)</b> Hourly amounts of wakefulness following almorexant. <b>(C’)</b> Hourly amounts of NR sleep following almorexant. <b>(C’’)</b> Hourly amounts of REM sleep following almorexant. Data represent the mean±SEM (n = 8 rats per group). *, <i>p</i><0.05. For detailed statistical results, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039131#pone.0039131.s009" target="_blank">Text S1</a>.</p

Average hourly LMA and relative T_core. LMA and relative T_core for 6 h prior to and 18 h after administration of SB-334867 (A), EMPA (B), and almorexant (C) as compared to zolpidem (ZOL) and vehicle.

<p>Shaded area represents the dark phase; vertical dotted line in each panel indicates the time of injection. (A) Average hourly LMA following SB-334867. (A’) The average hourly T_core following SB-334867. (B) The average hourly LMA following EMPA. (B’) The average hourly T_core following EMPA. (C) The average hourly LMA following almorexant. (C’) The average hourly T_core following almorexant. Data represent the mean±SEM (n = 8 rats per group). *, <i>p</i><0.05. For detailed statistical results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039131#pone.0039131.s009" target="_blank">Text S1</a>.</p

Latency to the onset of NR and REM sleep following administration of SB-334867. (A)

<p>, EMPA <b>(B)</b>, and almorexant <b>(C)</b> as compared to zolpidem (ZOL). * = significantly different from vehicle (p<0.05); <b>+</b> = significantly different from ZOL (p<0.05) (One-way repeated measures ANOVA followed by paired two-tail <i>t</i> tests; n = 8 per group). Data represent the mean±SEM.</p

Cumulative time in NR and REM sleep over the first 2, 4 and 6 h following drug administration.

<p><b>(A–C)</b> Cumulative time spent in NR sleep following SB-334867 (<b>A</b>), EMPA (<b>B</b>) and almorexant <b>(C)</b> compared to zolpidem (ZOL). <b>(A’–C’)</b> Cumulative time spent in REM sleep for the same drug treatments. (One-way repeated measures ANOVA followed by paired two-tail <i>t</i> tests; n = 8 per group). Data represent the mean±SEM. *, significantly different from vehicle; <b>+</b>, significantly different from ZOL.</p

Time-course of HCRT1R and HCRT2R occupancies by almorexant.

<p><b>(A,B)</b> Representative autoradiograms showing [3H]SB-674042 (5 nM) binding to HCRTR1 <b>(A)</b> and [3H]EMPA (1 nM) binding to HCRTR2 <b>(B)</b> in rat coronal brain sections. For both receptors, total binding (<i>TB</i>) was maximal in control animals (not injected) sampled at time 0 (<i>t0</i>). For HCRTR1 <b>(A)</b>, a clear signal was evident in the locus coeruleus (<i>LC</i>), which could be displaced by co-incubation with an excess of cold SB-674042 (10 µM) (non-specific binding, <i>NSB</i>). In contrast to vehicle administration (<i>Veh, 2 h</i>), almorexant (30 mg/kg injected intraperitoneally at ZT18) attenuated such specific signal after 2 h (<i>Almo, 2 h</i>), but not after 12 h (<i>Almo, 12 h</i>). For HCRTR2 <b>(B)</b>, signal was observed in various brain regions, including the tuberomammillary nuclei (<i>TMN</i>), cerebral cortex (<i>CC</i>), field CA3 of the hippocampus (<i>CA3</i>), retrosplenial cortex (<i>RSC</i>), dorsal raphe nuclei (<i>DRN</i>), pontine nuclei (<i>Pn</i>) and parabigeminal nuclei (<i>PBG</i>). [3H]EMPA could be displaced by co-incubation with an excess of Cp5 (10 µM) (<i>NSB</i>). HCRTR2 binding became minimal 2 h after almorexant (<i>Almo, 2 h</i>), but not after Vehicle (<i>Veh+2 h</i>), administration. After 12 h (<i>Almo, 12 h</i>), HCRTR2 binding was intermediate. Scale bars, 2 mm. <b>(C)</b> Time course of HCRTR1 and HCRTR2 occupancies by almorexant. Receptor occupancy was calculated by measuring the specific binding at various time points in the <i>LC</i> for HCRTR1, and in the <i>TMN</i> and <i>DRN</i> for HCRTR2. *, <i>p</i><0.001 versus time 0; (#), <i>p</i><0.05 (TMN only), #, <i>p</i><0.05 (TMN) or <i>p</i><0.01 (DRN), vs. time 30 min (one-way ANOVA followed by Dunnett’s analysis). <b>(D)</b> Almorexant plasma concentrations. Data represent the mean±SEM (n = 5 rats per group).</p