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Mutant neuropeptide S receptor reduces sleep duration with preserved memory consolidation

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Science Translational Medicine  16 Oct 2019:
Vol. 11, Issue 514, eaax2014
DOI: 10.1126/scitranslmed.aax2014
  • Fig. 1 NPSR1-Y206H mutation was identified in a natural short sleep family.

    (A) Pedigree of the family (K50226) carrying the NPSR1 mutation. The self-reported total sleep time per 24-hour day of mutation carriers is indicated. (B) NPSR1-Y206H is localized to the extracellular domain between transmembrane domains 4 and 5 (TM4/5) and is highly conserved among vertebrate NPSR1 orthologs. (C) Schematic of the mutations (Y206H and N107I) in the extracellular loops of NPSR1.

  • Fig. 2 Npsr1-Y206H mice demonstrate reduced sleep time.

    (A) Mouse movement was tracked by ANY-maze under LD 12:12. Total mobile time in 24 hours, light phase, and dark phase were calculated in Npsr1+/+ (n = 19) and Npsr1+/m (n = 15) mice. (B to D) Total sleep (B), NREM sleep (C), and REM sleep (D) time within 24 hours, light phase, and dark phase measured by EEG/EMG were calculated in Npsr1+/+ (n = 14) and Npsr1+/m (n = 12) mice. (E) NREM and REM sleep time were plotted hourly over 24 hours in Npsr1+/+ (n = 14) and Npsr1+/m (n = 12) mice. (F) NREM sleep delta power normalized to the average value during ZT9–12 was plotted hourly in Npsr1+/+ (n = 14) and Npsr1+/m (n = 12) mice over 24 hours. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, two-tailed Student’s t test (A to D); two-way repeated-measures (RM) ANOVA, post hoc Sidak’s multiple comparisons test (E and F). Data are mean ± SEM.

  • Fig. 3 Npsr1-Y206H mice have normal recovery sleep after SD.

    (A) NREM sleep delta power after SD (ZT0–6) normalized to the average NREM delta power during ZT9–12 of the baseline recording was plotted every hour in Npsr1+/+ (n = 14) and Npsr1+/m (n = 12) mice for 18 hours. (B to D) Total (B), NREM (C), and REM (D) sleep were calculated during indicated time periods for Npsr1+/+ (n = 14) and Npsr1+/m (n = 12) mice after 6 hours of SD (ZT0–6). (E) Cumulative NREM and REM sleep loss and gain compared with baseline conditions for the SD experiment in Npsr1+/+ (n = 14) and Npsr1+/m (n = 12) mice. *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed Student’s t test (B to D, left); two-way RM ANOVA, post hoc Sidak’s multiple comparisons test [A, B to D (right), and E]. Data are means ± SEM.

  • Fig. 4 Mutant NPSR1 is more active in vivo.

    (A) p-CREB immunoblots for brain lysates collected at indicated time points from Npsr1+/+ and Npsr1+/m mice. (B) Quantified results of (A) from Npsr1+/+ (ZT2, n = 4; ZT11, n = 5; ZT22, n = 4) and Npsr1+/m mice (ZT2, n = 4; ZT11, n = 5; ZT22, n = 3). (C) Immunoblots for brain lysates collected at ZT11 from Npsr1 +/+ and Npsr1+/m mice after different doses of NPS. (D) Quantified results of (C) from Npsr1+/+ (saline, n = 4; 0.1 nmol NPS, n = 3; 1 nmol NPS, n = 4) and Npsr1+/m mice (saline, n = 4; 0.1 nmol NPS, n = 3; 1 nmol NPS, n = 3). (E) Western blot of p-CREB expression in WT brain lysates collected at ZT11 after SHA 68 (intraperitoneal) and/or NPS (intracerebroventricular) injection. Group sizes: Veh/SHA 68 (50 mg/kg) and saline, n = 3; Veh/SHA 68 (50 mg/kg) and NPS (1 nmol), n = 4. (F) Immunoblots for brain lysates collected at ZT11 from Npsr1−/− mice after saline (n = 4) or 1 nmol NPS injection (n = 4). Quantified results are shown in panels on the right. *P < 0.05, ***P < 0.001, and ****P < 0.0001, ns, not significant. Two-way ANOVA, Sidak’s multiple comparisons test (B and D). One-way ANOVA, Dunnett’s multiple comparisons test (E). Two-tailed Student’s t test (F). Data are means ± SEM.

  • Fig. 5 Calcium imaging of CMT neurons shows increased activity for one subtype in Npsr1-Y206H mice.

    (A) Schematic of calcium imaging setup for recording the activity of CMT neurons in brain slices. (B) Schematic of anatomy for CMT. The injection/recorded area is marked with a pink circle. (C) Representative GCaMP fluorescence traces in different categories of cells that responded differentially to NPS treatment. Group 1, pulse activation; group 2, fast and long-lasting activation; group 3, fast activation and recovery; and group 4, inhibition. (D) Percentage of cells that show different types of response to NPS treatment in Npsr1+/+ (n = 8), Npsr1+/m (n = 7), and Npsr1−/− (n = 3) brain slices. *P < 0.05 and **** P < 0.0001; chi-square test (D). n, number of cells; N, number of animals.

  • Fig. 6 Contextual memory of Npsr1-Y206H mice is resistant to sleep loss.

    (A and B) Mice were trained in contextual fear conditioning at the beginning of the light phase (A) or at the end of the dark phase (B). Freezing response in the trained context was tested 24 (day 1) and 48 (day 2) hours after training. Percentage of time freezing during the 5 min of the test without SD [A, Npsr1+/+ (n = 17) and Npsr1+/m (n = 12); B, Npsr1+/+ (n = 12) and Npsr1+/m (n = 11)] or with SD [A, Npsr1+/+ (n = 10) and Npsr1+/m (n = 10); B, Npsr1+/+ (n = 10) and Npsr+/m (n = 11)] is shown in box and whisker plots. *P < 0.05 and *** P < 0.001. One-way ANOVA multiple comparisons followed by Tukey’s multiple comparisons test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/514/eaax2014/DC1

    Fig. S1. Wake and sleep questionnaires for FNSS.

    Fig. S2. Generation of Npsr1-Y206H mice.

    Fig. S3. Sleep/wake measurements in Npsr1-Y206H and Npsr1 KO mice.

    Fig. S4. EEG data analysis of sleep/wake behavior in Npsr1-Y206H mice.

    Fig. S5. High sleep pressure was observed in Npsr1-Y206H mice.

    Fig. S6. Hyperphosphorylated SNIPPs were observed in Npsr1-Y206H mice after SD.

    Fig. S7. Calcium imaging of LH neurons shows increased activity for one subtype in Npsr1-Y206H mice.

    Data file S1. Raw data.

  • The PDF file includes:

    • Fig. S1. Wake and sleep questionnaires for FNSS.
    • Fig. S2. Generation of Npsr1-Y206H mice.
    • Fig. S3. Sleep/wake measurements in Npsr1-Y206H and Npsr1 KO mice.
    • Fig. S4. EEG data analysis of sleep/wake behavior in Npsr1-Y206H mice.
    • Fig. S5. High sleep pressure was observed in Npsr1-Y206H mice.
    • Fig. S6. Hyperphosphorylated SNIPPs were observed in Npsr1-Y206H mice after SD.
    • Fig. S7. Calcium imaging of LH neurons shows increased activity for one subtype in Npsr1-Y206H mice.

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    Other Supplementary Material for this manuscript includes the following:

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