Shin-Ichi T. Inouye

Additive effect of mPer1 and mPer2 antisense oligonucleotides on light-induced phase shift

It is well known that light induces both mPer1 and mPer2 mRNA in the suprachiasmatic nucleus. We have reported that mPer1 antisense oligonucleotides (ODNs) inhibited the light-induced phase delays of mouse locomotor rhythm. In this study, we asked whether both or either mPer1 or mPer2 expression is necessary to induce the phase shift. We examined the effects of inhibition of mRNA expression on light-induced phase delays of mouse circadian behavior rhythm. Light-induced phase delays were moderately attenuated by microinjection of mPer1 or mPer2 antisense ODN, but not by mPer3 antisense or mPer1, mPer2 scrambled ODNs, whereas following simultaneous injection of both mPer1 and mPer2 antisense ODNs they disappeared. The present results suggest that acute induction of mPer1 and mPer2 gene play an additive effect on photic entrainment.

Mouse dexamethasone-induced RAS protein 1 gene is expressed in a circadian rhythmic manner in the suprachiasmatic nucleus

We identified the Dexamethasone-induced RAS protein 1 (Dexras1) gene as a cycling gene in the suprachiasmatic nucleus (SCN). Investigation of the whole brain using in situ hybridization demonstrated the localization of the expression of the gene in the SCN, thalamus, piriform cortex and hippocampus. However, rhythmic expression of the gene was observed only in the SCN. The rhythmic change in gene expression during 1 day was approximately five-fold, and the maximum expression was observed during subjective night. Real-time PCR using the SCN, paraventricular nucleus and cortex confirmed these results. Next, we analyzed the expression of the Dexras1 gene in the SCN of cryptochrome (Cry) 1 and 2 double knockout mice. We found that the rhythmic expression disappeared. The results indicate that Dexras1 rhythmicity and levels are dependent upon CRYs. This is the first time that the G protein, which may be involved in the input pathway, has been isolated as a cycling gene in the SCN.

A NEW REGULATORY INTERACTION SUGGESTED BY SIMULATIONS FOR CIRCADIAN GENETIC CONTROL MECHANISM IN MAMMALS

Knowledge of molecular biological systems is increasing at an amazing pace. It is becoming harder to intuitively evaluate the significance of each interaction between the molecules of the complex biological systems. Hence, we need to develop an efficient computational method to explore the biological mechanisms. In this study, we employed a hybrid functional Petri net in order to analyze mammalian circadian genetic control mechanisms, which consists of feedback loops of clock genes and generates endogenous near 24 h rhythms in mammals. We constructed a computational model based on the available biological data, and by using Genomic Object Net, we performed computer simulations of the time courses of clock gene transcription and translation. Although the original model successfully reproduced most of the circadian genetic control mechanisms, two discrepancies remained despite a wide selection of the parameters. We found that addition of a hypothetical path into the original model result in successful simulation of time courses and phase relationships among clock genes. This also demonstrates the usefulness of the hybrid functional Petri net approach to biological systems.

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA‐positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double‐labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA‐containing neurons co‐expressed either vasoactive intestinal peptide (VIP), gastrin‐releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA‐containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2‐expressing neurons in the SCN.

Day-night variation of pituitary adenylate cyclase-activating polypeptide (PACAP) level in the rat suprachiasmatic nucleus

Adenylate cyclase-activating polypeptide (PACAP) is synthesized in the retinal ganglion cells which terminate on vasoactive intestinal polypeptide neurons in the suprachiasmatic nucleus (SCN), the location of circadian clock. To examine whether PACAP exhibits daily variations in the rat SCN, we measured endogenous PACAP contents throughout the day under 12:12 h light-dark or constant dark conditions. PACAP level was low during the light periods, high during the dark periods, and was stable under constant dark conditions. In the periventricular nucleus of the hypothalamus and cerebral cortex, PACAP content did not show any significant variation throughout the day. Our findings suggest that PACAP content in the SCN may be changed by lighting conditions. Thus, PACAP-containing neurons may play certain roles in the entrainment of circadian rhythms.

Pituitary adenylate cyclase-activating polypeptide rhythm in the rat pineal gland

Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated to stimulate melatonin synthesis in the rat pineal gland. Circadian rhythms of melatonin concentration are well known. However, it has not been clarified whether PACAP contents in the pineal gland show circadian rhythm. In this study, we measured PACAP contents in the rat pineal gland throughout the day under 12:12 h light-dark cycle or constant dark conditions. A significant fluctuation was observed in the PACAP content under light-dark conditions but not under constant darkness. On the other hand, the pituitary gland showed no significant variation throughout the day under either conditions. These observations suggest that PACAP may participate in the modulation of melatonin synthesis depending on light conditions in the pineal gland.

Identification of genes that express in response to light exposure and express rhythmically in a circadian manner in the mouse suprachiasmatic nucleus

Most biological phenomena, including behavior and metabolic pathways, are governed by an internal clock system that is circadian (i.e., with a period of approximately 24 h) and is reset by light exposure from outside. In order to understand the molecular basis of the resetting mechanism of the clock, we attempted to isolate light-inducible transcripts in the suprachiasmatic nucleus, where the master clock resides, using a new gene expression profiling procedure. We identified 87 such transcripts, successfully cloned 60 of them and confirmed their light inducibility. Six of the 60 were already known to be light inducible and 17 are protein-coding transcripts registered in the public database that were not known to be light inducible. Induction is subjective night specific in most of the transcripts. Interestingly, 6 of the transcripts exhibit rhythmic expression in a circadian manner in the suprachiasmatic nucleus.

Phase dependent response of vasoactive intestinal polypeptide to light and darkness in the suprachiasmatic nucleus.

Responsiveness of the vasoactive intestinal polypeptide (VIP) content to light and darkness in the rat suprachiasmatic nucleus (SCN) was examined by enzyme immunoassay of micropunched tissues. VIP content in the SCN has been shown to decrease monotonically in animals maintained in illumination. Decreases in VIP content in the SCN in response to both 6-h light and dark pulses depended on the phase of the circadian cycle when the pulses were applied. Light imposed at circadian time (CT) 18 or CT 22 was more effective in suppressing VIP levels than light exposure of the same intensity imposed at CT 0 or CT 6. Darkness interrupting continuous light was more effective at around CT 0 and less effective at around CT 12. These results suggest that VIP responsiveness to light and darkness in the SCN is regulated by the circadian clock in different ways and are correlated with phase-dependent phase shifts in the activity rhythm after light and dark pulses.

Luminance-dependent decrease in vasoactive intestinal polypeptide in the rat suprachiasmatic nucleus

Light responsiveness of the vasoactive intestinal polypeptide (VIP) content in the suprachiasmatic nucleus (SCN) of the rat with pupils dilated by atropine was examined by enzyme immunoassay. After exposure to 6 h light at 3-1000 lux VIP levels in the SCN decreased as a monotonic function with a working range from 3 to 300 lux. At 12 h, 30 lux light decreased the VIP content to the minimum level that was attained by 300 lux light exposure in 6 h, suggesting that brighter illumination decreases VIP levels more rapidly, but light at a luminance of 0.05 lux for 3 days did not suppress VIP levels. These results suggest that VIP in the SCN codes visual information on luminance with a small working range and a relative high threshold.

Temporal profile of circadian clock gene expression in a transplanted suprachiasmatic nucleus and peripheral tissues

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is the master oscillator that regulates the circadian rhythms of the peripheral oscillators. Previous studies have demonstrated that the transplantation of embryonic SCN tissues into SCN‐lesioned arrhythmic mice restores the behavioral circadian rhythms of these animals. In our present study, we examined the clock gene expression profiles in a transplanted SCN and peripheral tissues, and also analysed the circadian rhythm of the locomotor activity in SCN‐grafted mice. These experiments were undertaken to elucidate whether the transplanted SCN generates a dynamic circadian oscillation and maintains the phase relationships that can be detected in intact mice. The grafted SCN indeed showed dynamic circadian expression rhythms of clock genes such as mPeriod1 (mPer1) and mPeriod2 (mPer2). Furthermore, the phase differences between the expression rhythms of these genes in the grafted SCN and the locomotor activity rhythms of the transplanted animals were found to be very similar to those in intact animals. Moreover, in the liver, kidney and skeletal muscles of the transplanted animals, the phase angles between the circadian rhythm of the grafted SCN and that of the peripheral tissues were maintained as in intact animals. However, in the SCN‐grafted animals, the amplitudes of the mPer1 and mPer2 rhythms were attenuated in the peripheral tissues. Our current findings therefore indicate that a transplanted SCN has the capacity to generate a dynamic intrinsic circadian oscillation, and can also lock the normal phase angles among the SCN, locomotor activity and peripheral oscillators in a similar manner as in intact control animals.