Calcium and pituitary adenylate cyclase-activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture

Mammalian circadian clock genes Per1 and Per2 are rhythmically expressed not only in the suprachiasmatic nucleus where the mammalian circadian clock exists, but also in other brain regions and peripheral tissues. The induced circadian oscillation of Per genes after treatment with high concentrations of serum or various drugs in cultured cells suggests the ubiquitous existence of the oscillatory mechanism. These treatments also result in a rapid surge of expression of Per1. It has been shown that multiple signaling pathways are involved in Per1 gene induction in culture cells. We used a dispersed primary cell culture made up of mouse cerebellar granule cells to examine the stimuli inducing the mPer genes and their signaling pathways in neuronal tissues expressing mPer genes. We demonstrated that mPer1, but not mPer2, mRNA expression was dependent on the depolarization state controlled by extracellular KCl concentration in the granule cell culture. Nifedipine treatment reduced mPer1 induction, suggesting that mPer1 mRNA expression depends on intracellular calcium concentration regulated through a voltage-dependent Ca2+ channel. Transient mPer1 mRNA induction was observed after elevating KCl concentration in the medium from 5 mM to 25 mM. This increased expression was suppressed by a calmodulin antagonist, or CaMKII/IV inhibitor, but not by MEK inhibitors. Addition of pituitary adenylate cyclase-activating polypeptide-38 to the medium also induced transient Per1 gene expression. This induction was mimicked by dibutyryl-cAMP and suppressed by a protein kinase A (PKA) inhibitor, but not by MEK inhibitors. These results suggest that Ca2+/calmodulin-dependent protein kinase II/IV- and PKA-dependent pathways are involved in high-KCl and PACAP-induced mPer1 induction, respectively, and neural tissues use multiple signaling pathways for mPer1 induction similar to culture cells.     

Repeated psychosocial stress at night,but not day,affects the central molecular clock

We have recently demonstrated that the outcome of repeated social defeat (SD) on behavior, physiology and immunology is more negative when applied during the dark/active phase as compared with the light/inactive phase of male C57BL/6 mice. Here, we investigated the effects of the same stress paradigm, which combines a psychosocial and novelty stressor, on the circadian clock in transgenic PERIOD2::LUCIFERASE (PER2::LUC) and wildtype (WT) mice by subjecting them to repeated SD, either in the early light phase (social defeat light?=?SDL) or in the early dark phase (social defeat dark?=?SDD) across 19 days. The PER2::LUC rhythms and clock gene mRNA expression were analyzed in the suprachiasmatic nucleus (SCN) and the adrenal gland, and PER2 protein expression in the SCN was assessed. SDD mice showed increased PER2::LUC rhythm amplitude in the SCN, reduced Per2 and Cryptochrome1 mRNA expression in the adrenal gland, and increased PER2 protein expression in the posterior part of the SCN compared with single-housed control (SHC) and SDL mice. In contrast, PER2::LUC rhythms in the SCN of SDL mice were not affected. However, SDL mice exhibited a 2-hour phase advance of the PER2::LUC rhythm in the adrenal gland compared to SHC mice. Furthermore, plasma levels of brain-derived neurotrophic factor (BDNF) and BDNF mRNA in the SCN were elevated in SDL mice. Taken together, these results show that the SCN molecular rhythmicity is affected by repeated SDD, but not SDL, while the adrenal peripheral clock is influenced mainly by SDL. The observed increase in BDNF in the SDL group may act to protect against the negative consequences of repeated psychosocial stress.     

孤儿受体TR3在小鼠睾丸中的定位和表达

本文采用原位杂交和免疫组织化学技术,观察孤儿受体ＴＲ３及其ｍＲＮＡ在小鼠睾丸中的表达及细胞定位。结果表明,在小鼠睾丸中有显著量的孤儿受体ＴＲ３ ｍＲＮＡ和蛋白表达,其表达量在不同曲细精管有明显的差异;孤儿受体ＴＲ３蛋白主要定位于生精细胞,其ｍＲＮＡ在生精细胞特异表达,主要在精原细胞和发育早期的初级精母细胞表达,提示孤儿受体ＴＲ３在小鼠曲细精管精子发生的早期阶段中起着调控作用。     

Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.     

Circadian behaviour in neuroglobin deficient mice

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.     

Circadian expression of clock genes in human peripheral leukocytes

In mammals, behavioral and physiological processes display 24-h rhythms that are regulated by a circadian system. In the present study, we investigated the possibility that the expression of clock genes in peripheral leukocytes can be used to assess the circadian clock system. We found that Per1 and Per2 exhibit circadian oscillations in mRNA expression in mouse peripheral leukocytes. Furthermore, the rhythms of Per1 and Per2 mRNA expression in peripheral leukocytes are severely blunted in homozygous Cry1/2 double-deficient mice that are known to have an abolished biological clock. We have examined the circadian expression of clock genes in human leukocytes and found that Per1 mRNA exhibits a robust circadian expression while Per2 and Bmal1 mRNA showed weak rhythm. These observations suggest that monitoring Per1 mRNA expression in human leukocytes may be useful for investigating the function of the circadian system in physiological and pathophysiological states.     

Photoperiod Differentially Affects the Expression of Three mPer Genes in the Mouse Heart

To investigate the mechanism that controls circadian rhythms in mammalian peripheral tissues, we housed mice in short days (6 h light: 18 h dark) or long days (18 h light: 6 h dark) and examined the rhythmic expression patterns of the mammalian clock genes mPer1 , mPer2 and mPer3 and a clock-controlled gene Dbp in the mouse heart. Northern blot analyses showed that peak levels of mPer1 mRNA expression in long days were about 50 % higher than those in short days. On the contrary the amplitude of the mPer2 mRNA peak in long days was about 25 % lower than that in short days. We could not find any effect of photoperiod on either the amplitude or waveform of the rhythms of mPer3 and Dbp mRNAs. Photoperiod differentially affected the expression of three mPer genes even in a peripheral tissue of mice.     

MPer1 and mper2 are essential for normal resetting of the circadian clock

Mammalian Per1 and Per2 genes are involved in the mechanism of the circadian clock and are inducible by light. A light pulse can evoke a change in the onset of wheel-running activity in mice by shifting the onset of activity to earlier times (phase advance) or later times (phase delays) thereby advancing or delaying the clock (clock resetting). To assess the role of mouse Per (mPer) genes in circadian clock resetting, mice carrying mutant mPer1 or mPer2 genes were tested for responses to a light pulse at ZT 14 and ZT 22, respectively. The authors found that mPer1 mutants did not advance and mPer2 mutants did not delay the clock. They conclude that the mammalian Per genes are not only light-responsive components of the circadian oscillator but also are involved in resetting of the circadian clock.     

Loss of mPer2 increases plasma insulin levels by enhanced glucose-stimulated insulin secretion and impaired insulin clearance in mice

The existence of peripheral oscillators has been shown, and they are critically important for organizing the metabolism of the whole body. Here we show that mice deficient in mPer2 markedly increase circulatory levels of insulin compared with wild type mice. Insulin secretion was more effectively stimulated by glucose, and alloxan, a glucose analogue, induced more severe hyperglycemia in mPer2-deficient mice. Hepatic insulin degrading enzyme (Ide) displayed an obvious day and night rhythm, which was impaired in mPer2-deficient mice, leading to a decrease in insulin clearance. Deficiency in mPer2 caused increased Clock expression and decreased expression of Mkp1 and Ide1, possibly underlying the observed phenotypes and suggesting that mPer2 plays a role in regulation of circulating insulin levels.     

Neurturin, RET, GFRα-1 and GFRα-2, but not GFRα-3, mRNA are expressed in mice gonads

The gonads are known to produce numerous hormones and also neurotrophins and their receptors. Here we demonstrate expression of glial-cell-line-derived neurotrophic factor (GDNF) family ligands and related receptors in adult mice gonads by in situ hybridization. GDNF mRNA was expressed in the ovary, but was not detectable in testis. Neurturin (NTN), another ligand in this family, gave rise to strong mRNA hybridization signals in a mosaic pattern in the seminiferous tubules of the testis at stages IX-XII and I-II of the cycle. NTN mRNA signals were also found in uterus and the oviduct. In testis, the transducing receptor RET as well as GDNF receptor alpha-1 (GFR)alpha-1 and GFRalpha-2 were distributed in complementary and overlapping patterns, the former at stages XI-XII-I and the latter at stages VII and VIII. GFRalpha-3 could not be detected. Expression of these trophic molecules suggests involvement of GDNF family ligands and related receptor components in reproduction.     

Cloning and characterization of rat casein kinase 1epsilon

Genes differentially expressed in the subjective day and night in the rat suprachiasmatic nucleus (SCN) were surveyed by differential display. A gene homologous to human casein kinase 1epsilon (CK1epsilon) was isolated, which initially appeared to be expressed in the suprachiasmatic nucleus (SCN) in a circadian manner. We here describe the cDNA cloning of the rat CK1epsilon and characterization of the protein products. The rCK1epsilon is predominantly expressed in the brain including the SCN, binds and phosphorylates mPer1, mPer2, and mPer3 in vitro, and translocates mPer1 and mPer3, but not mPer2, to the cell nucleus depending on its kinase activity when coexpressed with these Per proteins in COS-7 cells.