1 software package (Noldus Software, Wageningen, The Netherlands). The distance moved in a cage was calculated in 30-min time bins. Locomotor activity, the sleep–wake cycle and histamine release time series were
initially examined for the presence of statistically significant periods with lengths from 3 to 30 h by use of the Lomb–Scargle VE-822 manufacturer method (Ruf, 1999) implemented in lsp software (Refinetti et al., 2007). Identified periods were subjected to a multiple cosinor analysis (Bingham et al., 1982; Libre Office Calc, The Document Foundation) to obtain their mesor, orthophase and amplitude values. To verify the applicability of cosinor analysis, all of the time series were tested for zero amplitude and sinusoidality (whenever applicable). The parameters of periodicity in the population rhythm were separately estimated and tested for significance with the cosinor procedure. Cross-correlation Bafetinib analysis was performed with spss 15.0 (SPSS, Armonk, NY, USA). The correlation between histamine release and power spectrum frequencies was computed for individual mice with Spearman correlation coefficients. To obtain average correlation coefficients, the values were subjected to Fisher Z-transformation.
They were then averaged and reverse transformed. If no periodicity was detected, the data sets were compared by the use of two-way anova with time and strain as factor variables, and P ≤ 0.05 was considered to be significant. For the measurement of histamine and 1-methylhistamine concentrations and HDC and HNMT activities, samples were collected every 4 h for two consecutive days (as described above), and then approximated by use of a multiple cosinor procedure with a major period set to 24 h and a first harmonic of 12 h. When a period was considered to be non-significant, it was removed from the model, and the time series was further examined by use of a single cosinor model.
The significance levels SPTLC1 were set to P ≤ 0.05 in all experiments, unless otherwise stated. The temporal pattern of hdc transcript expression in C57BL/6J mice was assessed with quantitative radioactive in situ hybridization. It was measured in E2/E3 and E4/E5 subpopulations of histaminergic neurons in the TMN region of the hypothalamus at 4-h intervals over a period of 24 h. No significant periodicity in mRNA expression was found in either group [E2/E3, F2,27 = 2.15 (P = 0.137); E4/E5, F2,27 = 0.96 (P = 0.38); Fig. 1]. The average expression levels [mean ± standard deviation (SD)] were 0.168 ± 0.028 μCi/g/pixel for the E2/E3 group, and 0.117 ± 0.017 μCi/g/pixel for the E4/E5 group. The activity of both enzymes was measured in hypothalamic, striatal and cortical samples of C57BL/6J mice. The enzymatic activity of HDC showed no 24-h periodicity in any structures analysed (Table 1), as estimated by multiple cosinor analysis. It was approximately three-fold higher in hypothalamic samples than in the other regions.