Judy McKinley Brewer

Suprachiasmatic Regulation of Circadian Rhythms of Gene Expression in Hamster Peripheral Organs: Effects of Transplanting the Pacemaker

Neurotransplantation of the suprachiasmatic nucleus (SCN) was used to assess communication between the central circadian pacemaker and peripheral oscillators in Syrian hamsters. Free-running rhythms of haPer1, haPer2, and Bmal1 expression were documented in liver, kidney, spleen, heart, skeletal muscle, and adrenal medulla after 3 d or 11 weeks of exposure to constant darkness. Ablation of the SCN of heterozygote tau mutants eliminated not only rhythms of locomotor activity but also rhythmic expression of these genes in all peripheral organs studied. The Per:Bmal ratio suggests that this effect was attributable not to asynchronous rhythmicity between SCN-lesioned individuals but to arrhythmicity within individuals. Grafts of wild-type SCN to heterozygous, SCN-lesioned tau mutant hamsters not only restored locomotor rhythms with the period of the donor but also led to recovery of rhythmic expression of haPer1, haPer2, and haBmal1 in liver and kidney. The phase of these rhythms most closely resembled that of intact wild-type hamsters. Rhythmic gene expression was also restored in skeletal muscle, but the phase was altered. Behaviorally effective SCN transplants failed to reinstate rhythms of clock gene expression in heart, spleen, or adrenal medulla. These findings confirm that peripheral organs differ in their response to SCN-dependent cues. Furthermore, the results indicate that conventional models of internal entrainment may need to be revised to explain control of the periphery by the pacemaker.

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.

Expression of haPer1 and haBmal1 in Syrian Hamsters: Heterogeneity of Transcripts and Oscillations in the Periphery

The molecular biology of circadian rhythms has been extensively studied in mice, and the widespread expression of canonical circadian clock genes in peripheral organs is well established in this species. In contrast, much less information about the peripheral expression of haPer1, haPer2, and haBmal1 is available in Syrian hamsters despite the fact that this species is widely used for studies of circadian organization and photoperiodic responses. Furthermore, examination of oscillating expression of these genes in mouse testis has generated discrepant results, and little is known about gonadal expression of haPer1 and haBmal1 or their environmental control. To address these questions, the authors examined the pattern of haPer1 and haBmal1 in heart, kidney, liver, muscle, spleen, and testis of hamsters exposed to DD. In most organs, Northern blots suggested the existence of single transcripts of each of these messenger RNAs (mRNAs). haPer1 peaked in late subjective day and haBmal1 during the late subjective night. Closer inspection of SCN and muscle haPer1, however, revealed the existence of two major transcripts of similar size, as well as minor transcripts that varied in the 3′-untranslated region. In hamster testis, two haPer1 transcripts were found, both of which are truncated relative to the corresponding mouse transcript and both of which contain a sequence homologous to intron 18 of mPer1. Neither testis transcript contains a nuclear localization signal, and haPer1 transcripts lacked the putative C-terminal CRY1-binding domain. Furthermore, the testis deviated from the general pattern in that haPer1 and haBmal1 both peaked in the subjective night. In situ hybridization revealed that haPer1, but not haBmal1, showed a heterogeneous distribution among seminiferous tubules. Hamster testis also expresses 2 haPer2 transcripts, but no circadian variation is evident. In a second experiment, long-term exposure to DD sufficient to induce gonadal regression was found to eliminate circadian oscillations of both testicular haPer1 transcripts. In contrast, gonadal regression was accompanied by a more robust rhythm of haBmal1.

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 at2h, 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.

Lateralization of the central circadian pacemaker output: a test of neural control of peripheral oscillator phase

To evaluate the contribution of neural pathways to the determination of the circadian oscillator phase in peripheral organs, we assessed lateralization of clock gene expression in Syrian hamsters induced to split rhythms of locomotor activity by exposure to constant light. We measured the ratio of haPer1, haPer2, and haBmal1 mRNA on the high vs. low (H/L) side at 3-h intervals prior to the predicted activity onset (pAO). We also calculated expression on the sides ipsilateral vs. contralateral (I/C) to the side of the suprachiasmatic nucleus (SCN) expressing higher haPer1. The extent of asymmetry in split hamsters varied between specific genes, phases, and organs. Although the magnitude of asymmetry in peripheral organs was never as great as that in the SCN, we observed significantly greater lateralization of clock gene expression in the adrenal medulla and cortex, lung, and skeletal muscle, but not in liver or kidney, of split hamsters than of unsplit controls. We observed fivefold lateralization of expression of the clock-controlled gene, albumin site D-element binding protein (Dbp), in skeletal muscle (H/L: 10.7 +/- 3.7 at 3 h vs. 2.2 +/- 0.3 at 0 h pAO; P = 0.03). Furthermore, tyrosine hydroxylase expression was asymmetrical in the adrenal medulla of split (H/L: 1.9 +/- 0.5 at 0 h) vs. unsplit hamsters (1.2 +/- 0.04; P < 0.05). Consistent with a model of neurally controlled gene expression, we found significant correlations between the phase angle between morning and evening components (psi(me)) and the level of asymmetry (H/L or I/C). Our results indicate that neural pathways contribute to, but cannot completely account for, SCN regulation of the phase of peripheral oscillators.

Endocrine disruption in human placenta: expression of the dioxin-inducible enzyme, CYP1A1, is correlated with that of thyroid hormone-regulated genes.

CONTEXT Thyroid hormone (TH) is essential for normal development; therefore, disruption of TH action by a number of industrial chemicals is critical to identify. Several chemicals including polychlorinated biphenyls are metabolized by the dioxin-inducible enzyme CYP1A1; some of their metabolites can interact with the TH receptor. In animals, this mechanism is reflected by a strong correlation between the expression of CYP1A1 mRNA and TH-regulated mRNAs. If this mechanism occurs in humans, we expect that CYP1A1 expression will be positively correlated with the expression of genes regulated by TH. OBJECTIVE The objective of the study was to test the hypothesis that CYP1A1 mRNA expression is correlated with TH-regulated mRNAs in human placenta. METHODS One hundred sixty-four placental samples from pregnancies with no thyroid disease were obtained from the GESTE study (Sherbrooke, Québec, Canada). Maternal and cord blood TH levels were measured at birth. The mRNA levels of CYP1A1 and placental TH receptor targets [placental lactogen (PL) and GH-V] were quantitated by quantitative PCR. RESULTS CYP1A1 mRNA abundance varied 5-fold across 132 placental samples that had detectable CYP1A1 mRNA. CYP1A1 mRNA was positively correlated with PL (r = 0.64; P < .0001) and GH-V (P < .0001, r = 0.62) mRNA. PL and GH-V mRNA were correlated with each other (r = 0.95; P < .0001), suggesting a common activator. The mRNAs not regulated by TH were not correlated with CYP1A1 expression. CONCLUSIONS CYP1A1 mRNA expression is strongly associated with the expression of TH-regulated target gene mRNAs in human placenta, consistent with the endocrine-disrupting action of metabolites produced by CYP1A1.

Central Control of Circadian Phase in Arousal-Promoting Neurons

Cells of the dorsomedial/lateral hypothalamus (DMH/LH) that produce hypocretin (HCRT) promote arousal in part by activation of cells of the locus coeruleus (LC) which express tyrosine hydroxylase (TH). The suprachiasmatic nucleus (SCN) drives endogenous daily rhythms, including those of sleep and wakefulness. These circadian oscillations are generated by a transcriptional-translational feedback loop in which the Period (Per) genes constitute critical components. This cell-autonomous molecular clock operates not only within the SCN but also in neurons of other brain regions. However, the phenotype of such neurons and the nature of the phase controlling signal from the pacemaker are largely unknown. We used dual fluorescent in situ hybridization to assess clock function in vasopressin, HCRT and TH cells of the SCN, DMH/LH and LC, respectively, of male Syrian hamsters. In the first experiment, we found that Per1 expression in HCRT and TH oscillated in animals held in constant darkness with a peak phase that lagged that in AVP cells of the SCN by several hours. In the second experiment, hamsters induced to split their locomotor rhythms by exposure to constant light had asymmetric Per1 expression within cells of the middle SCN at 6 h before activity onset (AO) and in HCRT cells 9 h before and at AO. We did not observe evidence of lateralization of Per1 expression in the LC. We conclude that the SCN communicates circadian phase to HCRT cells via lateralized neural projections, and suggests that Per1 expression in the LC may be regulated by signals of a global or bilateral nature.

Effects of the duper mutation on circadian responses to light.

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τDD) by 2 hours when expressed on a tau mutant (τss) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τss background) showed weak entrainment, while τss animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τss hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.