Figure 1
Locomotor activity and clock period. (A) Representative locomotor activity records of wild-type (wt) (left panel) and synaptophysin knock-out (Syp-/-) male mice (right panel). Black and white bars on top of the plots indicate the 12 h light: 12 h dark cycle (LD). The grey shaded areas filling the panels in the lower part indicate constant darkness (DD). Below the locomotot activity records χ2-periodogram analysis is shown indicating the period length. (B) Period is similar in wild-type (n=17) and Syp-/- male mice (n=18). (C) Total activity is comparable between the two genotypes. (D) Precision of activity onset is comparable between the two genotypes.
Figure 2
Phase shifting response to light pulses and mRNA accumulation in the SCN. (A) Representative actograms of wild-type (wt) and Syp-/- knock-out mice with light pulses at CT10 (top panels), CT14 (middle panels) and CT22 (bottom panels). Blue lines trace activity onset before the light pulse and red lines trace activity onset after the light pulse. (B) Quantification of the phase shifts in Syp-/- knock-out (white bar) and wild-type mice (black bar). Syp-/- knock-out mice show a significantly reduced phase delay in response to light at CT14 (Syp-/- = -34 ± 12 min.; wt = -122 ± 10 min., unpaired t-test p < 0.001 = ***, n = 8 Syp-/- and 12 wt). No difference between the genotypes was observed at CT22 (Syp-/- = 60 ± 12 min.; wt = 45 ± 6 min., n = 7 Syp-/- and 12 wt) and CT10 (Syp-/- = 26 ± 12 min.; wt = 21 ± 8 min., n = 7 Syp-/- and 11 wt). (C) mRNA accumulation in the SCN of Syp-/- knock-out (white bar) and wild-type mice (black bar) before (-) and after (+) a 15 minute light pulse at CT14 or CT22. No differences in expression of cFos, Per1 or Per2 can be observed (n = 3, two-way ANOVA with Bonferroni post-test). Mean values are represented as SEM.
Figure 3
Diurnal changes of the interaction between Synaptophysin (Syp) and Synaptobrevin (Syb). (A) Western blots of proteins from synaptic vesicles prepared from either synaptophysin knock-out animals (Syp-/-), wild-type (wt) littermates or Per2 mutant mice. Antibodies against synaptophysin (α-Syp, left panel) and synaptobrevin (α-Syb, right panel) reveal the presence of synaptophysin (Syp) or synaptbrevin (Syb) with (+) or without (-) chemical cross-linking using disuccimidyl suberate (DSS). Cross-linked samples show in addition to the respective monomers the Syp/Syb complex at 56 kDa and the Syp or Syb dimers. In synaptic vesicles of Syp-/- animals, only Syb and its dimer can be observed. (B) Western blots after cross-linking of proteins show the Syp/Syb interaction over a 12 hour light/12 hour dark cycle. Synaptic vesicles were prepared at the indicated Zeitgeber times (ZT). Antibodies against Syp (left panel) and Syb (right panels) show changes in the Syp/Syb complex over time. SNAP25 served as internal control. (C) Quantification of Syp/Syb complexes using α-Syb antibodies. Data from three independent sets of animals are shown with each sample analyzed at least twice. SNAP25 served as reference for quantification. For each set, values were normalized to the value at ZT0 which was set to 1. While the Syp and Syb monomers and dimers do not change over time, the formation of the Syp/Syb complex appears to be time dependent with maximal values observed during the light period corresponding to the resting phase of mice (ZT6 and ZT12). Values represent the mean of three animals ± S.D. Significance was determined by student’s t-test between ZT6 and ZT18 ( p = 0.06) and ZT12 and ZT18 (p = 0.03).
Figure 4
Transactivation assay of the Syp promoter in NG108-15 neuroblastoma cells. 0.5 μg of a luciferase reporter construct containing a 1.4 kb Syp promoter sequence was co-transfected with 0.2 μg, 0.4 μg, 0.6 μg and 0.8 μg of Bmal1 and Npas2 expression constructs, respectively. As control 0.5 μg the mouse Per1 luciferase reporter construct was used. Duplicates are represented as mean ± SD. One-way ANOVA was performed for statistical analysis. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.