Role of α2δ3 in Cellular Synchronization of the Suprachiasmatic Nucleus Under Constant Light Conditions.

By the effort to identify candidate signaling molecules important for the formation of robust circadian rhythms in the suprachiasmatic nucleus (SCN), the mammalian circadian center, here we characterize the role of α2δ proteins, synaptic molecules initially identified as an auxiliary subunit of the voltage dependent calcium channel, in circadian rhythm formation. In situ hybridization study demonstrated that type 3 α2δ gene (α2δ3) was strongly expressed in the SCN. Mice without this isoform (Cacna2d3-/-) did not maintain proper circadian locomotor activity rhythms under a constant light (LL) condition, whereas under a constant dark (DD) condition, these mice showed a similar period length and similar light-responsiveness as compared to wild type mice. Reflecting this behavioral phenotype, Cacna2d3-/- mice showed a severely impaired Per1 expression rhythm in the SCN under LL, but not under DD. Cultured SCN slices from Per1-luc transgenic Cacna2d3-/- mice revealed reduced synchrony of Per1-luc gene expression rhythms among SCN neurons. These findings suggest that α2δ3 is essential for synchronized cellular oscillations in the SCN and thereby contributes to enhancing the sustainability of circadian rhythms in behavior.

Involvement of urinary bladder Connexin43 and the circadian clock in coordination of diurnal micturition rhythm.

Nocturnal enuresis in children and nocturia in the elderly are two highly prevalent clinical conditions characterized by a mismatch between urine production rate in the kidneys and storage in the urinary bladder during the sleep phase. Here we demonstrate, using a novel method for automated recording of mouse micturition, that connexin43, a bladder gap junction protein, is a negative regulator of functional bladder capacity. Bladder connexin43 levels and functional capacity show circadian oscillations in wild-type mice, but such rhythms are completely lost in Cry-null mice having a dysfunctional biological clock. Bladder muscle cells have an internal clock, and show oscillations of connexin43 and gap junction function. A clock regulator, Rev-erbα, upregulates connexin43 transcription as a cofactor of Sp1, using Sp1 cis-elements of the promoter. Therefore, circadian oscillation of connexin43 is associated with the biological clock and contributes to diurnal changes in bladder capacity, which avoids disturbance of sleep by micturition.

Accurate determination of S-phase fraction in proliferative cells by dual fluorescence and peroxidase immunohistochemistry with 5-bromo-2'-deoxyuridine (BrdU) and Ki67 antibodies.

To ensure the maintenance of tissues in mammals, cell loss must be balanced with cell production, the proliferative activity being different from tissue to tissue. In this article, the authors propose a new method for the quantification of the proliferative activity, defined as the S-phase fraction of actively cycling cells, by dual labeling with fluorescence and peroxidase immunohistochemistry using BrdU (marker of S-phase) and Ki67 antibodies (marker of G(1)-, S-, G(2)-, and M-phases) after a one-step antigen retrieval. In the generative cell zones of fundic and pyloric glandular stomachs, where the majority of cells were cycling, the authors measured a proliferative activity of 31%. In the epithelium of the forestomach and the skin, where cycling cells are intermingled with G(0) and differentiated cells, proliferative activities were 21% and 13%, respectively. In the adrenal cortex, in which cycling cells were sparsely distributed, the proliferative activity reached 32%. During the regenerative process in the skin after a lesion, the proliferative activity increased in proximity to the wound. The present one-step dual-labeling method has revealed that the proliferative activity is different between tissues and depends on the physiological or pathological state.