Additive effect of mPer1 and mPer2 antisense... : NeuroReport (original) (raw)
INTRODUCTION
The mPer1 gene was identified based upon sequence similarities to the Drosophila period gene (dPer) and shows strong expression within the SCN [1,2]. Following the discovery of the mPer1 gene, two other mouse period genes have been cloned, including _mPer2_[3–5] and _mPer3_[6,7]. The mammalian suprachiasmatic nucleus (SCN) of hypothalamus is the location of a circadian pacemaker that controls various physiological rhythms such as sleep–wakefulness [8,9]. The expression of mRNA of these mPer1, mPer2 and mPer3 in the SCN is in a circadian fashion [2,5–7]. It has been reported that a brief exposure to light at subjective night results in a large and rapid induction of mPer1 expression [10]. We recently demonstrated that light-induced phase delay of locomotor activity at circadian time (CT) 16 was significantly inhibited in mice pretreated with mPer1 antisense ODN 1 h before light exposure [11]. Taken together, it has been suggested that induction of mPer1 gene expression may be one of steps to produce the phase shift. On the other hand, it was recently reported that rapid reduction of mPer1 and mPer2 mRNA in the SCN at CT10 was well associated with non-photic induced phase shifts [12,13].
Light exposure also causes a delayed expression of mPer2 mRNA but not mPer3 mRNA in the SCN [3–5]. Interestingly, recent papers demonstrated a dimerization and nuclear entry of mPer proteins in mammalian cells [14,15]. Recently, it was reported that mPer2 mutant mice showed a low level of mPer2 mRNA in the SCN with a 70–90% reduction of expression of _mPer1_[16]. These reports have suggested the interactions of mPer genes to produce the circadian rhythm and phase shifts. In this study, we asked whether either or both mPer1 and mPer2 genes induction is necessary to produce the phase shift. We examined the effects of mPer1, mPer2 and mPer3 gene ODNs on light-induced phase delays of mouse circadian behavior rhythm. Light-induced phase delays of circadian locomotor rhythm were moderately attenuated by microinjection of mPer1 or mPer2 antisense ODN. Whereas simultaneous injection of mPer1 or mPer2 antisense ODNs prevent the light-induced phase delays.
MATERIALS AND METHODS
Male BALB/c mice (5 weeks old) purchased from Charles River Japan were housed for 2–3 weeks under a 12:12 h light:dark cycle. Under anesthesia with hydrazine (10 mg/kg, i.p.) and ketamine (5 mg/kg, i.p.), guide cannulas (stainless steel, o.d. 0.7 mm) were chronically implanted into the brain regions 1 mm above the lateral ventricle (posterior 0.5 mm and 1.0 mm lateral from bregma and 2.1 mm below of the surface of the skull) of both sides according to the mouse brain atlas [17] and used for microinjection of ODNs into the lateral ventricle. For assessment of locomotor activity rhythm, implanted mice (9–10 weeks of age) were housed individually, and their locomotor activity rhythms were recorded on personal computers. Locomotor activities were measured by area sensors (FA-05 F5B Omron, Japan) with a thermal radiation detector system [10]. Animals were allowed to entrain before being released into constant darkness (DD). After free-running for 10–20 days in DD, implanted mice were randomly assigned ODNs and 60 min after ODN injection, each animal was exposed to a light pulse (20 lux) for 15 min at CT16 (CT12 = onset time of locomotor activity). After treatment, animals were returned to constant darkness. Each group received repeated intracerebroventricular (i.c.v.) injections (3–4 times for each animal) after at least 14 days intervals. Injections were performed randomly into the right or left ventricle. A 5 μl aliquot of each substance (1–8 nmol) was unilaterally injected into the lateral ventricle via the injection cannulae (o.d. 0.35 mm) extending 1.0 mm below the tip of the guide cannulae. To ascertain that ODNs were administered exactly into the cerebral ventricle, mice were injected with 5 μl of saturated fast green, and brain sections were examined macroscopically after experiments. The phase of the rhythms was assessed visually by applying a straight edge to the onset of activity on successive days before the light pulse and again 7 days after light exposure [10].
Sample preparation:
Mice were transferred to constant darkness for one extra daily cycle, and at ZT15, were given saline, both mPer1 and mPer2 antisense ODNs (4 nmol for each) or their scrambled ODNs (4 nmol for each). Mice then received light treatment (20 lux, 15 min) at ZT16. At ZT17.5, mice were deeply anesthetized with ether and intracardially perfused with 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde (PFA). Brains were removed, post-fixed in 0.1 M PB containing 4% PFA for 24 h at 4°C and transferred into 20% sucrose in PB for 72 h at 4°C. Brain slices (40 μm) including the SCN were cut using a cryostat (Microm, HM505E, Germany) and placed in 2× SSC until processing for hybridization.
In situ hybridization:
The effect of mPer1 and mPer2 antisense ODNs on mPer1 and mPer2 expression in the SCN was examined by in situ hybridization. Slices were treated with 1 μg/ml proteinase K in 10 mM Tris–HCl buffer (pH 7.5) containing 10 mM EDTA for 10 min at 37°C followed by 0.25% acetic anhydride in 0.1 M triethanolamine and 0.9% NaCl for 10 min. The slices were then incubated in the hybridization buffer (60% formamide, 10% dextran sulfate, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.6 M NaCl, 1× Denhardt's solution (0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin), 0.2 mg/ml tRNA, 0.25% sodium dodecyl sulphate) containing 33P-labeled cRNA probes for 16 h at 60°C. Radioisotope (RI: α[33P]UTP; New England Nuclear, USA) -labeled antisense cRNA probes were made from linearized cDNA templates kindly provided by Dr Okamura (Kobe University). After two washes in 2× SSC/50% formamide, slices were treated with RNaseA (10 μg/ml) for 30 min at 37°C and washed twice in 2× SSC/50% formamide. The radioactivity of each slice visualized on BioMax MR film (Kodak) was analyzed using a microcomputer interface to an image analysis system (MCID, Imaging Research Inc., Canada) after conversion into optical density by 14C-autoradiographic microscales (Amersham, Buckinghamshire, UK). For data analysis, we subtracted the intensities of the optical density in the corpus callosum from those in the SCN, and regarded this value as the net intensity for SCN area. The intensity values of sections from the rostral to the caudal part of the SCN (4–5 sections per mouse brain) were then summed; the sum was considered to be a measure of the amount of mPer1 or mPer2 mRNA in this region.
Phosphotioate ODNs:
Published sequences of mPer1 and of mPer2 were used to design antisense ODNs targeted to the region of the mRNA containing the initiation ATG. Briefly, the sequences of the antisense ODN of mPer1, mPer2 and mPer3 are 5′-TAGGGGACCACTCATGTCT-3′, 5′-CACGTATCCATTCATGTCG-3′ and 5′-TCCACAGG GATCCATCCCG-3′, respectively. The sequences of the scrambled ODN of mPer1 and mPer2 are 5′-CCGTT AGTACTGAGCTGAC-3′ and 5′-ACCATGTTACCTGAC CTGT-3′, respectively. The scrambled ODNs contain an equal GC content to the antisense ODNs of mPer1 and mPer2. All ODNs were phosphotioated and purified by HPLC to reduce the possible toxicity.
Statistics:
Results are expressed as the mean ± s.e.m. The significance of differences between groups was determined by one-way analysis of variance followed by Fisher's protected least significant difference (PLSD) test or Student's _t_-test.
RESULTS
Previously, we obtained the BALB/c mouse phase– response curve to light, which is the response of the locomotor activity circadian rhythm to brief standard light exposure as a function of the phase in the circadian cycle [10]. Light exposure at CT16 caused a large phase delay (2 h delay) and exposure at CT0 caused a small phase advance (0.5 h advance). Phase delays induced by light exposure at CT16 were dose-dependently attenuated by injections of mPer1 (ANOVA, F(3,24) = 3.92, p < 0.05) or _mPer2_ (ANOVA, F(3,26) = 3.26, _p_ < 0.05) antisense ODNs 60 min before light exposure, but not by _mPer3_ antisense ODN (F(1,10) = 0.11, _p_ > 0.05;Fig. 1, Fig. 2a). Each injection of 4 nmol mPer1 or mPer2 antisense ODN reduced the phase delays to the control level (saline-injected non-light group, p > 0.05, Student's _t_-test).
Double-plotted representative examples of locomotor activity records of mice with injections of mPer1 or mPer2 or both antisense ODN before light exposure. (a) Mice were injected with saline at CT15, and then light was delivered at CT16. (b, c) Mice were injected with mPer1 ODN (4 nmol) or mPer2 ODN (4 nmol) at CT15, respectively then light was delivered at CT16. (d) Mice were simultaneously injected with mPer1 (4 nmol) and mPer2 (4 nmol) ODN at CT15, and then light was applied at CT16. Time of day is indicated horizontally and consecutive days are arranged vertically in the conventional double plot format. Arrowheads indicate the day of injection. Change in time of activity onset, indicated by transverse lines added to the figure, was used to assess effects of each injection on phase.
Effect of mPer antisense ODNs on light-induced phase delays of locomotor activity. (a) Mice were injected with vehicle (saline), mPer1, mPer2, mPer3 or both mPer1 and mPer2 antisense ODNs 60 min before light exposure at CT16 (20 lux, 15 min). (b) Mice were injected with the scrambled ODNs of mPer1 or mPer2 60 min before light exposure at CT16 (20 lux, 15 min). (c) Mice were injected with saline, mPer1 or mPer2 antisense ODNs at CT15 without light exposure. Numbers in columns indicate the number of animals. Data represent mean ± s.e.m. *p < 0.05, **p < 0.01 vs light only group (Fisher's PLSD test).
In the next experiment, we injected both mPer1 and mPer2 ODNs simultaneously. Co-injections of mPer1 and mPer2 antisense ODNs strongly and dose-dependently inhibited the light-induced phase delay (ANOVA, F(3,24) = 8.34, p < 0.01;Fig. 1d;Fig. 2a). There were no significant differences between simultaneous injection of 2 nmol (_p_ > 0.05) or 4 nmol (p > 0.05) of mPer1 and mPer2 antisense ODNs and saline group in phase delay (Student's _t_-test vs saline-injected non-light group). In this experiment, control studies are needed to rule out non-specific effects on light-induced phase delay. Injection of saline, mPer1 and mPer2 scrambled ODNs did not attenuate the light-induced phase delay (Fig. 2b). In addition, injection of saline, mPer1, mPer2 or mPer3 antisense ODNs at CT15 without light exposure did not cause any phase shifts (Fig. 2c).
In our previous study we demonstrated that administration of 6 nmol mPer1 antisense ODN significantly reduced the expression of mPer1 mRNA (68% of random ODN treatment) using RT-PCR [11]. In the present experiment, we examined the inhibitory effect of simultaneous administration of mPer1 and mPer2 antisense ODNs on light-induced expression of mPer1 and mPer2 mRNA in the SCN using in situ hybridization method. Light exposure at CT16 significantly increased the mPer1 (350% of non-light group) expression and mPer2 (250% of non-light group) expression 90 min after light exposure (Fig. 3a,b). The light-induced expression of mPer1 and mPer2 (p < 0.05 vs scrambled, respectively) in the SCN was significantly reduced by co-administrations of mPer1 antisense and mPer2 antisense ODNs (4 nmol for each mPer1 and mPer2) 60 min before light exposure.
Effect of simultaneous injection of mPer1 and mPer2 antisense or scrambled ODNs on light-induced mPer1 and mPer2 mRNA expression in the suprachiasmatic nucleus. (a) Representative example of in situ hybridization of mPer1 (left) and mPer2 (right) in the SCN. Mice were injected with saline, mPer1 and mPer2 antisense or scrambled ODNs 60 min before light exposure at CT16 (20 lux, 15 min), and some mice were injected with saline at CT15 without light exposure. (a) Saline without light; (b) Saline with light; (c) mPer1 and mPer2 scrambled ODNs (4 nmol each) with light; (d) mPer1 and mPer2 antisense ODNs (4 nmol each) with light. Bar = 0.7 mm/. (b) Quantitative analysis of effect of antisense or scrambled ODNs on hybridization signals of mPer1 (left) and mPer2 (right) in the SCN. The columns indicate averaged values of gene expression. Numbers in columns indicate the number of animals. *p < 0.05, **p < 0.01 vs. non-light saline group (Fisher's PLSD test). #p < 0.05, ##p < 0.01 vs. light/saline or scrambled/light ODNs group (Fisher's PLSD test).
DISCUSSION
We previously reported that exposing BALB/c mice to light at CT16 produced large phase delays in circadian locomotor rhythm [10,11]. Therefore, in this experiment, we examined the effect of mPer antisense ODNs on the phase delays induced by light pulses at CT16. Light-induced phase delays of circadian activity rhythms were dose-dependently attenuated by treatment with mPer1 antisense ODN as well as mPer2 antisense ODN. mPer2 induction was observed in the SCN 90 and 120 min after brief light exposure at CT16 and CT14, respectively [5,7]. Although a significant reduction of light-induced phase delays was observed by a treatment with each ODN, a maximal reduction was almost 40–50% even after high dose of each ODN. The present result indicates that induction of either mPer1 or mPer2 is insufficient to produce the full size of phase delays. Therefore, we injected both ODNs simultaneously to see the additive role of mPer1 and mPer2 expression. As shown in results, co-administration of mPer1 and mPer2 ODNs strongly and completely attenuated the light-induced phase delays. Our recent paper demonstrated that the extent of clock resetting and the magnitude of mPer1 induction are positively correlated [10]. Thus, the present results disagree with our previous paper. Unfortunately in a previous paper, we did not examine whether the degree of mPer2 induction correlates with the relative amount of mouse phase delays. Therefore, it is important to examine the total amount of mPer1 or mPer2
In the present experiment, simultaneous injection of 4 nmol mPer1 and mPer2 antisense ODN reduced to 75% of mPer1 and mPer2 mRNA expression induced by light exposure. Although we did not detect the protein production of mPer1 or mPer2 after light exposure, we can estimate that a 25% reduction of mPer1 and mPer2 mRNA may be enough reduction to prevent the light (20 lux, 15 min)-induced phase delays in mouse behavior. Although the reduction of light-induced phase delay by antisense ODN in vivo may be a result of the inhibition of light-induced acute induction of mPer1 and mPer2 but not mPer3 gene in the SCN, the present results indicate that the change mRNA level are not as robust as one might expect on the basis of the behavioral effect of ODNs. Thus, the present results are different from our previous data [10]. The result of specific hybridization of the antisense ODN to its complementary mRNA causes disrupting of the translation of mRNA into protein [18]. Therefore, it is important to examine the correlation between the amount of phase shifts and total amount of per1 and per2 after injection of ODNs, because both light-induced and circadian expressions of per1 are observed in the SCN [19,20]. We hope that mPer1 mutant mice are produced and characterized, and combinations of the mPer1 mutant with other _mPer2_[16] and _mPer3_[21] mutants should be studied.
In contrast to mPer1 and mPer2 antisense ODNs, mPer3 antisense ODN did not block light-induced phase delays of mouse circadian rhythms. Recent papers have demonstrated that induction of mPer3 mRNA is never observed in the SCN after light exposure at any circadian time, even at high intensity of light [6,7]. The present results combined with the above papers suggest that phase shifts of overt rhythm is independent of the expression of mPer3 mRNA. The lack of effects of mPer antisense ODN treatment on locomotor activity implies a lack of damage or toxicity associated with ODNs. In addition, simultaneous injections both ODNs did not affect the locomotor activity itself. We demonstrated that mPer1 sense and scrambled ODNs did not produce any inhibition of light-induced phase delays [11], ruling out the possibility that effects exerted by mPer1 and mPer2 antisense ODNs are caused by sequence-independent effects on circadian clock centers.
CONCLUSION
Light exposure has been reported to produce mPer1 and mPer2 mRNA expression in the SCN. However, we do not know whether both or each induction of mPer1 and mPer2 mRNA is necessary to cause the light-induced phase shift of behavioral rhythm. Therefore, we injected either ODN or both ODNs of mPer1 and mPer2 genes to examine the role of mPer1 and mPer2 gene expression in light-induced phase delays. Co-injections of both ODNs strongly blocked light-induced phase delays of mouse locomotor rhythms. In summary, the present results suggest that induction of mPer1 and mPer2 genes plays an additive role in light-induced phase shifts of mouse circadian locomotor rhythm.
Acknowledgements:
This study was partially supported by grants to S. S. from Japanese Ministry of Education, Science, Sports and Culture (11170248,11145240) and The special Coordination Funds of the Japanese Science and Technology Agency.
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Keywords:
Antisense oligonucleotides; Circadian clock; mPer; Suprachiasmatic nucleus
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