Per2 mRNA Levels Research Articles

Tetrodotoxin administration in the suprachiasmatic nucleus prevents NMDA‐induced reductions in pineal melatonin without influencing Per1 and Per2 mRNA levels

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a light-entrainable circadian pacemaker. Neurons in the SCN are part of a circuit that conveys light information from retinal efferents to the pineal gland. Light presented during the night acutely increases mRNA levels of the circadian clock genes Per1 and Per2 in the SCN, and acutely suppresses melatonin levels in the pineal gland. The present study investigated whether the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels requires sodium-dependent action potentials in the SCN. Per1 and Per2 mRNA levels in the SCN and pineal melatonin levels were measured in Syrian hamsters injected with tetrodotoxin (TTX) prior to light exposure or injection of N-methyl-D-aspartate (NMDA). TTX inhibited the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels. TTX did not, however, influence the ability of NMDA to increase Per1 and Per2 mRNA levels, though it did inhibit the ability of NMDA to suppress pineal melatonin levels. These results demonstrate that action potentials in the SCN are not necessary for NMDA receptor activation to increase Per1 and Per2 mRNA levels, but are necessary for NMDA receptor activation to decrease pineal melatonin levels. Taken together, these data support the hypothesis that the mechanism through which light information is conveyed to the pacemaker in the SCN is separate from and independent of the mechanism through which light information is conveyed to the SCN cells whose efferents suppress pineal melatonin levels.

Dark pulse resetting of the suprachiasmatic clock in Syrian hamsters: behavioral phase-shifts and clock gene expression

In mammals, the circadian clock in the suprachiasmatic nuclei (SCN) is mainly synchronized to photic cues provided by the daily light/dark cycle. Phase-shifts produced by light exposure during the night are correlated with rapid induction of two clock genes, Per1 and Per2, in the SCN. Nonphotic stimuli such as behavioral and pharmacological cues, when presented during the subjective day, induce behavioral phase-advances and a down-regulation of Per1 and Per2 expression in the SCN. When applied during the subjective day, dark pulses in continuous light also produce phase-advances. These phase-shifting effects have been interpreted as reflecting either a photic image mirror, nonphotic cues, or a combination of both. Here we evaluated in Syrian hamsters housed in constant light how dark pulses applied in late subjective day affect levels of Per1, Per2 and Cry1 mRNA. Four-hour dark pulses with no access to a wheel produced 1.2±0.4 h phase-advances of locomotor activity rhythm while control manipulation induced non-significant shifts (0.1±0.2 h). Dark pulses transiently down-regulated Per1 and Per2 mRNA levels in the SCN by 40 and 20% respectively, while the levels of Cry1 mRNA remained unaffected. In behaviorally split hamsters in which Per oscillations were asymmetric between the left and right sides of the SCN, dark pulses reduced Per expression in the half-SCN with high Per. This study shows that exposure during the late subjective day to dark pulses independent of wheel-running have nonphotic-like effects on the SCN clock at both behavioral and molecular levels.

Transduction of light in the suprachiasmatic nucleus: evidence for two different neurochemical cascades regulating the levels of Per1 mRNA and pineal melatonin

The suprachiasmatic nucleus (SCN) contains a circadian clock and regulates melatonin synthesis in the pineal gland. Light exposure during the subjective night acutely increases the mRNA levels of the Period (Per)1 gene in the SCN and acutely suppresses melatonin levels in the pineal gland. Activation of N-methyl- d-aspartate (NMDA) receptors in the SCN has been demonstrated to phase-shift the circadian clock in a manner similar to light. We tested the hypothesis that activation of excitatory amino acid (EAA) receptors in the SCN mediates the acute effects of light on Per1 mRNA levels and pineal melatonin. NMDA, injected into the SCN of Syrian hamsters during the night, acutely suppressed melatonin levels in the pineal gland. Both the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) and the α-amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) inhibited the light-induced increase of Per1 mRNA levels in the SCN. In the same animals, however, these antagonists had no effect on the ability of light to suppress pineal melatonin. These results support the hypothesis that EAA receptor activation in the SCN is necessary for the acute effects of light on Per1 mRNA levels. They also indicate that NMDA receptor activation in the SCN is sufficient but may not be necessary for the acute effects of light on pineal melatonin. These data suggest that there may be at least two different neurochemical cascades that transduce the effects of light in the SCN

Light exposure during daytime modulates expression of Per1 and Per2 clock genes in the suprachiasmatic nuclei of mice.

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master circadian clock in mammals. Nocturnal light pulses that reset the circadian clock also lead to rapid increases in levels of Per1 and Per2 mRNA in the SCN, suggesting that these genes are involved in the synchronization to light. During the day, when light has no phase-shifting effects in nocturnal rodents, the consequences of light exposure for Per expression have been less thoroughly studied. Therefore, the effects of light exposure during the day were assessed on Per1 and Per2 mRNA in the SCN of mice. Expression of Per1 and Per2 was generally increased by 30-min light pulses during the subjective day, with more pronounced effects in the morning. One exception was noted for a transient decrease in Per2 expression after a short light pulse applied at midday. Prolonged light exposure (up to 3 hr) starting at midday markedly increased Per2 expression but not that of Per1. Moreover, the amplitude of the daily variations of both Per and the duration of Per1 peak was increased in mice exposed to a light-dark cycle compared with those transferred to constant darkness. Finally, the amplitude of the daily variations of both Per and the basal level of Per1 were increased in mice under a light-dark cycle compared with animals synchronized to a skeleton photoperiod (i.e., with daily dawn and dusk 1-hr exposures to light). Taken together, the results indicate that prolonged light exposure during daytime positively modulates daily levels of Per1 and Per2 mRNA in the SCN of mice.

Glucose Down-regulates Per1 and Per2mRNA Levels and Induces Circadian Gene Expression in Cultured Rat-1 Fibroblasts

In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified >100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

Is novel wheel inhibition of Per1 and Per2 expression linked to phase shift occurrence?

We studied whether access to a novel running wheel in vivo could reset the suprachiasmatic nuclei (SCN) in vitro. Golden hamsters were transferred to dim red light at Zeitgeber time (ZT) 4, given their first exposure to a running wheel for 3 h, and killed at either ZT7 or ZT9. Using a brain slice preparation, the SCN firing rate rhythm in vitro was advanced relative to controls only in the slices prepared at ZT9 (phase shift: 2.36±0.06 h, n=4) but not ZT7 (−0.26±0.16 h, n=4). Transitions to dim red light or brain slice preparation at ZT7 or ZT9 alone do not shift the rhythm. Hamsters with wheels had significantly lower levels of SCN per1 mRNA compared with controls at ZT7, and lower per2 mRNA when examined at ZT9. We conclude that 3 h of novel wheel access appears to require some extended time in vivo in order for the SCN to be reset, even beyond the time when per1 mRNA levels have been altered.

Neuropeptide Y differentially suppresses per1 and per2 mRNA induced by light in the suprachiasmatic nuclei of the golden hamster.

Neuropeptide Y (NPY), present in an input pathway to the suprachiasmatic nuclei (SCN), can block the effects of light on circadian rhythms. The authors have studied this interaction using an in vitro brain slice technique. Effects of NPY on light-induced period1 and period2 mRNA in the SCN were examined in vitro following a light pulse during early subjective night. Golden hamsters (n = 91) were housed under a 14:10 LD cycle and then moved to constant dim red light for 3 days. Hamsters were exposed to a 5-min light pulse previously shown to induce phase shifts and prepared for in vitro application of NPY. Hypothalamic slices containing the SCN were maintained in vitro for 40 min to 4 h after the light pulse, then quick-frozen. Sections were evaluated by in situ hybridization with [35S]-labeled cRNA probes for per mRNA. Rapid light induction of both per1 and per2 by 40 min and 1 h after the light pulse, respectively, was apparent, with NPY inhibition of this response significant by at least these same time points. However, although striking suppression of per2 mRNA by the NPY continued through the peak for per2 at 2 h, per1 mRNA levels rebounded quickly to equal the per1 induction peak at 1 h and mirrored the control light induction pattern for per1 thereafter. Delaying NPY to 30 min after slice preparation demonstrated that NPY is capable of suppressing peak per1 levels. These results confirm the feasibility of measuring light-induced gene expression in the SCN in vitro. A differential regulation of per1 and per2 transcription might be of critical importance for the modulation of circadian responses to light.

THREE DAYS OF NOVEL WHEEL ACCESS DIMINISHES LIGHT-INDUCED PHASE DELAYS IN VIVO WITH NO EFFECT ON PER1 INDUCTION BY LIGHT

The mammalian circadian clock, located in the hypothalamic suprachiasmatic nuclei, synchronizes endogenous behavioral and physiological rhythms to a 24h period through responses to two types of stimuli: photic (light) and nonphotic (behaviorally induced arousal and/or increases in activity). Photic stimuli can block nonphotic effects and vice versa, although the mechanisms and levels of interactions between these two stimuli types are unknown. Here, we investigated whether 3 d of access to a novel running wheel alters the phase shift to light in vivo, and whether this effect could be seen on induction by light of the circadian gene per1. Through measurement of running wheel activity of golden hamsters, access to a new wheel for 3 d was shown to diminish photic phase delays with no effect on phase advances. As seen using in situ hybridization, however, there was no effect on levels of light-induced per1 mRNA. This study indicates a possible role for this paradigm as a model of interactions between photic and nonphotic stimuli.

Neuropeptide Y rapidly reduces Period 1 and Period 2 mRNA levels in the hamster suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN) contains the main circadian clock. Neuropeptide Y (NPY) that is released from the intergeniculate leaflet of the lateral geniculate body to the SCN, acts in the SCN to advance circadian phase in the subjective day via the NPY Y2 receptor. We used semi-quantitative in situ hybridization to determine the effect of NPY on circadian clock genes, Period 1 ( Per1) and Period 2 ( Per2), expression in SCN slices. Addition of NPY to the brain slices in the subjective day resulted in reduction of Per1 and Per2 mRNA levels 0.5 and 2 h after treatment. NPY Y1/Y5 and Y2 agonists decreased Per1 within 0.5 h. These results suggest that NPY may induce phase shifts by mechanisms involving or resulting in reduction of Per1 and Per2 mRNA levels.

Circadian expression of Period 1, Period 2, and Arylalkylamine N-acetyltransferase mRNA in the rat pineal gland under different light conditions

Period 1 (Per1), 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) mRNA levels were determined by semi-quantitative in situ hybridization in rat pineal glands. In agreement with previous reports, AA-NAT mRNA levels were rhythmic in light:dark (LD) cycles and the rhythm persisted in constant dim light (DLL). Per1 and Per2 mRNA also showed significant variations both in LD and DLL. AA-NAT and Per1 mRNA levels showed very similar patterns of variations in LD and DLL to one another, whereas Per2 showed a different pattern of expression from AA-NAT and Per1. Exposure to 30 min of light did not affect the expression of the three genes, while exposure to a longer light pulse (1 or 2 h) decreased AA-NAT and Per1 mRNA levels; Per2 mRNA levels were also decreased but only temporarily. Our results demonstrate that Per1 and Per2 expression in the rat pineal is under circadian control, and suggest Per1 may be regulated by the same mechanism which controls the expression of AA-NAT gene. Per2 seem to be controlled by a different mechanism.

Neural regulation of circadian rhythms.

Neural regulation of circadian rhythms.