Article Figures & Data
Figures
- Figure 1.
There is a circadian rhythm in nuclear CalB content in the SCN. A, Light microscopic (LM; top panels) and electron microscopic (EM; bottom panels) photomicrographs show the changes in localization of CalB-IR as a function of time of day with the nuclei are primarily devoid of CalB during the subjective night (CT22), whereas CalB-IR is seen in both cytoplasm and nucleus during the subjective day (CT6). N, Nucleus; C, cytoplasm. B, Percentage of CalB cells with nuclei devoid of CalB-IR using four different methods. Histograms show the mean ± SEM. C, Histograms show the nuclear versus cytoplasmic ROD of CalB throughout the circadian cycle. Positive values indicate that the staining intensity is greater in the nucleus than in the cytoplasm, and negative values indicate that staining intensity is less in the nucleus than in the cytoplasm. D, Schematic summarizing the temporal relationship between light (600 lux, 30 min)-induced phase shifts of locomotor activity (line graph) and light-induced Per1 mRNA expression in the CalB region (histograms). The phase response curve was determined using a 30 min light pulse (600 lux; 2-5 animals per time point). Histograms depict data as mean ± SEM. *p < 0.05; ***p < 0.001.
- Figure 3.
The role of CalB during the subjective night. A, Schematic depicts the experimental schedule for light pulse administration during subjective night. CT from 0 to 24 hr is indicated as a line above the actogram. Oligodeoxynucleotides and saline were injected into the third ventricle close to the SCN at CT15, and a 30 min light pulse was given at CT19. B-E, Locomotor activity records of individual animals show the effect of CalB-S, saline, VP-AS, and CalB-AS on light-induced phase shifts of locomotor activity rhythm. The phase of the locomotor activity rhythm was assessed visually by drawing a straight line through the onset of activity on successive days before the light pulse and again beginning ∼3 d after the light exposure. The distance between the two lines reveals the magnitude of the phase shift in hours. Scale bars: 3 hr. F, Dose-response of attenuation of photic phase shifting with CalB-AS. G, Quantitative analysis of the phase shift to light after control injections of saline CalB-sense-ODN (CalB-S), CalB-scrambled antisense-ODN (CalB-S-AS), and VP-antisense (VP-AS). Histograms show mean ± SEM. H, Photomicrographs of light-induced expression of Per1 mRNA in the presence of 7.5 nmol of CalB-AS and control injections (saline, CalB-S, 7.5 nmol) during subjective night in representative animals. I, Quantitative analysis of Per1 expression after a subjective night light pulse in each treatment condition. Histograms depict data as mean ± SEM; parentheses give the number of animals per group. **p < 0.01; ***p < 0.001.
- Figure 4.
The role of CalB during the subjective day. A, C, Schematic depicts the experimental schedule for light pulse administration during subjective day. CT from 0 to 24 hr is indicated as a line above the actogram. Oligodeoxynucleotides and saline were injected into the third ventricle close to the SCN at CT2-CT4, and a 30 min light pulse or 8-OH DPAT injection (5 mg/kg) were given at CT6-CT8. B, Sample actograms from animals given CalB-AS pretreatment before a subjective daytime light pulse. Scale bars: 3 hr. D, Representative actogram from an animals that initially received 8-OH-DPAT alone and subsequently received CalB-AS pretreatment, followed by 8-OH-DPAT. E, Effect of CalB-AS on locomotor activity rhythm after light pulse. F, Effect of CalB-AS on locomotor activity rhythm after 8-OH DPAT treatment. G, Photomicrographs of light-induced expression of Per1 mRNA in the presence of 7.5 nmol of CalB-AS and control injections (saline, CalB-S, 7.5 nmol) during subjective day in representative animals. H, Quantitative analysis of Per1 expression after a subjective daytime light pulse in each treatment condition. Histograms depict data as mean ± SEM; parentheses give the number of animals per group.**p < 0.01; ***p < 0.001.
- Figure 2.
CalB-AS treatment decreases CalB protein and mRNA in the SCN. A, Photomicrographs of CalB and VP mRNA in serial sections demonstrating that 7.5 nmol of CalB-AS, but not saline, administered at CT4-CT6 significantly reduced the expression of CalB mRNA sampled 4 hr later but did not affect the expression of VP mRNA. B, Confocal images of CalB-IR demonstrating that 7.5 nmol of CalB-AS, but not saline, administered at CT2-CT4 significantly decreased the OD of Cy2-labeled CalB protein in the nucleus of SCN cells sampled 4 hr later. Histograms depict data as mean ± SEM; parentheses give the number of animals per group. *p < 0.05; **p < 0.01; ***p < 0.001.
