Figure 1.
(A) Strategy for conditional disruption of the murine Prkg1 gene. Depicted are the Prkg1 wild-type locus (+), the loxP-flanked conditional Prkg1 allele (L2), and the excised Prkg1 null allele (L−) obtained after Cre-mediated recombination of the L2 allele. The filled box denotes exon 10 of the murine Prkg1 gene that encodes part of the ATP-binding site. Filled triangles indicate loxP sequences. The open box represents a thymidine kinase (tk)-neomycin resistance (neo) fusion gene. Restriction sites for BamHI, EcoRI, HindIII, NcoI, and NheI are indicated by B, E, H, Nc, and Nh, respectively. (B) X-Gal staining of a brain (sagittal view) of Nes-Cre;R26R reporter mice for Cre activity. (C) Western blot analysis of PRKG1 expression in various brain regions of control and Prkg1BKO mutant mice. The p42/44 mitogen-activated protein kinase (MAPK) was used as loading control. (D) Cre activity (left panels) and PRKG1 expression (middle and right panels) in the dorsomedial hypothalamus (DMH; upper panels) and suprachiasmatic nucleus (SCN, lower panels). Cre activity was monitored by X-Gal staining of cryosections of brains from Nes-Cre;R26R reporter mice. PRKG1 was detected by immunohistochemistry on paraffin sections of brains from control mice (middle panels) and Prkg1BKO mice (right panels).
Figure 2.
Amount and distribution of wakefulness, NREMS, and REMS during baseline and recovery from sleep deprivation.
(A) Time course of mean (±1 SEM) number of minutes spent in each state for consecutive 1 h intervals (Prkg1SMr: filled symbols; control: open symbols) for the 48 h of the experiment. 0–24 h, baseline; 24–30 h, sleep deprivation; 30–48 h, recovery; W, wakefulness; N, NREMS; R, REMS; SD, sleep deprivation. (B) Summary of genotype differences in the relative distribution of the three behavioral states during baseline. Hourly, integrated values are first expressed as % of the total 24 h amount of each state within individuals (see Methods). Mean (±1 SE of the difference) Prkg1SMr – control differences in % accumulated are depicted. Gray lines represent fitted sine functions. Genotype affected the baseline time course of W and N but not of R [2-way ANOVA with factors ‘genotype’ (P = 0.41, 0.89, and 0.010) and ‘time’ (0–24; repeated measures: P<0.0001) and their interaction (P = 0.0012, 0.0007, and 0.064); P-values for W, N, and R, respectively]. (C) Recovery time course of time spent asleep lost during the 6 h SD. Hourly values were first expressed as differences (in min) between corresponding intervals during baseline (30–48 h vs. 6–24 h) within individuals, and then accumulated (see Methods). Values represent mean (±1SEM) accumulation curves for each genotype for N (top) and R (bottom panel). SD affected the amount and time course of time spent asleep [3-way ANOVA with factors ‘genotype’ (P = 0.24 and 0.25), ‘SD’ (recovery vs. baseline; repeated measures; P = 0.0054 and <0.0001) and ‘time’ (6–24 vs. 30–48; repeated measures: P<0.0001); interactions ‘genotype’בSD’ (P = 0.56 and 0.24), ‘genotype’בtime’ (P = 0.0009 and 0.0001), ‘SD’בtime’ (P = 0.042 and <0.0001); P-values for N and R, respectively]. Filled and open bars at the bottom of each panel connect intervals for which accumulated sleep time differed from baseline for Prkg1SMr and control mice, respectively (P<0.05; post-hoc 2-sided paired t-tests). In all panels, gray areas denote the dark periods and gray diamonds indicate 1 h intervals in which values significant differed between genotypes (P<0.05; post-hoc 2-sided t-tests).
Table 1.
Time-spent awake and asleep in baseline.
Figure 3.
Time course of EEG delta power and EEG spectra during NREMS.
(A) Time course of mean (±1SEM) relative levels of EEG delta (1.0–4.0 Hz) power for the 48 h of the experiment (Prkg1SMr: filled symbols; control: open symbols). Values were expressed as % of the lowest levels reached in baseline (8–12 h; see Methods for details). The dynamic range of baseline changes in delta power was greatly reduced in Prkg1SMr mice compared to controls while during recovery from sleep deprivation (SD) this genotype difference disappeared [2-way ANOVA with factors ‘genotype’ (P = 0.0063 and 0.53) and ‘time’ (repeated measures: P<0.0001) and their interaction (P = 0.017 and 0.15); P-values for baseline and recovery, respectively]. Gray areas denote the dark periods. (B) Effect of SD on EEG delta power during the light period. The first 8 values after recovery sleep onset were expressed as % of the first 8 values after sleep (or light) onset in baseline within individual mice. Values represent means±1SEM. (C) Spectral composition of the NREMS EEG during baseline (upper panel; absolute values 0.75–45 Hz at 0.25 Hz resolution, lower panel: % difference between genotypes). EEG power density was markedly lower in Prkg1SMr than control mice in the low delta (0.75–2.25 Hz) frequency range and to smaller extend also in the high delta (3.25–4.75 Hz) and beta (16–21 Hz) ranges. [2-way ANOVA with factors ‘genotype’ (P = 0.0072) and ‘EEG frequency’ (repeated measures, 0.75–45 Hz: P<0.0001) and their interaction (P = 0.0009)]. EEG frequency was plotted on a logarithmic scale to better illustrate differences in the lower frequencies. (D) Upper panel: NREMS EEG spectra during the first interval after sleep onset after SD (solid lines; see A) and the corresponding time-of-day during baseline (dashed lines; see A interval 7). Lower panel: % recovery/baseline change (Black lines: Prkg1SMr, gray lines: control). In all panels, gray diamonds at the bottom indicate times at or frequency bins in which values significant differed between genotypes (P<0.05; post-hoc 2-sided t-tests). In Panels A and D filled and open bars connect intervals for which values differed from baseline for Prkg1SMr and control mice, respectively (P<0.05; post-hoc 2-sided paired t-tests).
Figure 4.
Circadian phenotype of Prkg1BKO and Prkg1SMr mutants.
(A) and (C) Representative activity recordings (actograms) of Prkg1BKO (A) and Prkg1SMr (C) mutant mice and their respective controls whose wheel-running activity is plotted as vertical bars in a double-plot format. Each horizontal line represents two 24 h periods; the second (right) half of each line is repeated on the first (left) half of the following line. Mice were entrained to LD 12∶12 and subsequently released into DD (A, upper panels, and C) or LL (A, lower panels). Black and white bars on top depict the distribution of dark and light periods during initial LD, shaded areas in the actograms indicate actual dark periods during the experiment. LD, light-dark cycle; DD, constant darkness; LL, constant light. (B) and (D) Theoretical probability distributions for period lengths between 10 and 35 hours (chi square periodograms) of the activity data obtained under DD (B, upper panels, and D) or LL (B, lower panels) for the same animals whose actograms are shown in (A) and (C). The X coordinate of the peak indicates the internal period length of the free-running rhythm, the Y coordinate is a measure of the robustness of the rhythm. Note that Y axis scales are the same for adjoining periodograms to make differences more readily visible. (E)–(G) Total activity values (E), internal period length tau (F) and amplitude of chi square periodograms (G) for Prkg1BKO and Prkg1SMr mutants and their respective controls in LD (only for activity), DD and LL (only for Prkg1BKO mutants and controls). n = 18 for Prkg1BKO mutants, n = 20 for the respective controls for LD and DD; n = 9 for Prkg1BKO mutants, n = 11 for controls for LL; n = 6 for Prkg1SMr mutants and the respective controls. ** p<0.01, * p<0.05 as determined by unpaired t-test. LD, light-dark cycle; DD, constant darkness; LL, constant light.
Figure 5.
Prkg1BKO have a weakened circadian oscillator.
(A) Onset errors of control and Prkg1BKO mutant mice kept in LD 12∶12 of the indicated intensities, DD and LL were determined from the actograms. They are expressed as the average deviation of the actual onset from lights off for LD and from a regression line drawn through all onsets for DD and LL. n = 5 for both genotypes for LD 50 and 10 lx, n = 11 for controls, n = 9 for Prkg1BKO mutants for all other conditions. ** p<0.01, * p<0.05 as determined by unpaired t-test. LD, light-dark cycle; DD, constant darkness; LL, constant light. (B) Control and Prkg1BKO mutant mice were subjected to a lighting schedule mimicking chronic jet lag for 18 days (4 h forward phase shift every two days, see diagram on top: black and white bars indicate distribution of dark and light phases). Total wheel-running activity was quantified before the jet lag, directly after and two months after the treatment. n = 11 for controls, n = 9 for Prkg1BKO mutants. * p<0.05 as determined by unpaired t-test. (C) Daytime activity was determined for control and Prkg1BKO mutant mice kept in LD 12∶12 of the indicated intensities. Activity recorded during the light phase is plotted as percentage of total activity. n = 5 for both genotypes for 50 and 10 lx, n = 20 for controls and n = 18 for Prkg1BKO mutants for 400 lx. *** p<0.001, * p<0.05 as determined by unpaired t-test. (D) Control and Prkg1BKO mutant mice were kept in LD 12∶12. Bouts of inactivity during the activity phase, e.g. the period of more or less continuous activity, were quantified from the actograms. n = 19 for controls, n = 18 for Prkg1BKO mutants. ** p<0.01, * p<0.05 as determined by unpaired t-test. (E) The average distribution of activity over one day was analyzed for control and Prkg1BKO mutant mice kept in LD 12∶12. Black and white bars on top depict the distribution of dark and light periods during one day. Data is expressed as average counts/minute over a ten-minute interval. The inset graph shows ZT 0.33–11.33 on a magnified scale. Gray diamonds indicate statistically significant differences between genotypes (p<0.05 as determined by unpaired t-test). n = 19 for controls, n = 18 for Prkg1BKO mutants; ZT, Zeitgebertime. (F) Relative drinking activity during the light phase of Prkg1BKO and Prkg1SMr mice as compared to their respective litter-matched controls. Values are expressed as % of total drinking volume consumed during the light phase. *** p<0.001, ** p<0.01 as determined by unpaired t-test. n = 8 for both Prkg1BKO mutants and the respective controls, n = 10 for Prkg1SMr mutants, n = 21 for the respective controls. Note that the absolute drinking volume during 24 hours was not different between genotypes. (G) Representative recordings of drinking activity over six days for all four genotypes. Black and white bars on top depict the distribution of dark and light periods during the recording. For better orientation, those parts of the diagrams recorded during darkness are additionally shaded.