Hypothalamic circadian organization in birds. I. Anatomy,functional morphology,and terminology of the suprachiasmatic region

In mammals, the “master clock” controlling circadian rhythmicity is located in the hypothalamic suprachiasmatic nuclei (SCN). Until now, no comparable structure has been unambiguously described in the brain of any nonmammalian vertebrate. In birds, early anatomical and lesioning studies described a SCN located in the anterior hypothalamus, but whether birds possess a nucleus equivalent to the mammalian SCN remained controversial. By reviewing the existing literature it became evident that confusion in delineation and nomenclature of hypothalamic cell groups may be one of the major reasons that no coherent picture of the avian hypothalamus exists. In this review, we attempt to clarify certain aspects of the organization of the avian hypothalamus by summarizing anatomical and functional studies and comparing them to immunocytochemical results from our laboratory. There is no single cell group in the avian hypothalamus that combines the morphological and neurochemical features of the mammalian SCN. Instead, certain aspects of anatomy and morphology suggest that at least two anatomically distinct cell groups, the SCN and the lateral hypothalamic nucleus (LHN), bear some of the characteristics of the mammalian SCN.     

Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN.     

Identification of the suprachiasmatic nucleus in birds

Circadian rhythms are generated by an internal biological clock. The suprachiasmatic nucleus (SCN) in the hypothalamus is known to be the dominant biological clock regulating circadian rhythms in mammals. In birds, two nuclei, the so-called medial SCN (mSCN) and the visual SCN (vSCN), have both been proposed to be the avian SCN. However, it remains an unsettled question which nuclei are homologous to the mammalian SCN. We have identified circadian clock genes in Japanese quail and demonstrated that these genes are expressed in known circadian oscillators, the pineal and the retina. Here, we report that these clock genes are expressed in the mSCN but not in the vSCN in Japanese quail, Java sparrow, chicken, and pigeon. In addition, mSCN lesions eliminated or disorganized circadian rhythms of locomotor activity under constant dim light, but did not eliminate entrainment under light-dark (LD) cycles in pigeon. However, the lesioned birds became completely arrhythmic even under LD after the pineal and the eye were removed. These results indicate that the mSCN is a circadian oscillator in birds.     

Physiology of avian circadian pacemakers.

The pineal gland plays a cental role in the circadian organization of birds, although it is clearly only one component in a system with other components that have not yet been positively identified. The relative importance of the pineal and other components may vary from one group of birds to another. In the most thoroughly studied species, the house sparrow, pineal removal abolishes circadian rhythmicity; rhythmicity is restored by transplantation of a donor bird's pineal and the restored rhythm has the phase of the donor. This, and other evidence, argues convincingly that the pineal is a pacemaker in the sparrow circadian system. The pineal of the chicken has circadian rhythms in several biochemical parameters that result in the rhythmic synthesis of melatonin. The activity of one enzyme in this pathway is rhythmic for at least two cycles in organ culture. In view of this result it is interesting that pineal removal does not abolish circadian rhythmicity in chickens. The fact that lesions of the suprachiasmatic nuclei abolish circadian rhythms in sparrows, several mammalian species, and perhaps Japanese quail and reptiles, suggests that vertebrate circadian organization may be based on differentially weighted interactions between the pineal, the suprachiasmatic nuclei, and perhaps other brain regions.     

Paraventricular-subparaventricular hypothalamic lesions selectively affect circadian function

The circadian timing system has three principal components: (i) entrainment pathways, (ii) pacemakers, and (iii) efferent pathways from the pacemakers that convey the circadian signal to effector systems. The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal mammalian circadian pacemaker and, although we understand the organization of entrainment pathways to the SCN and the pacemaker itself, we know much less about the functional organization of SCN projections mediating control of effector systems. It is unclear, for example, whether specific subsets of SCN projections control specific effector systems. In this study, we analyzed the effects of lesions ablating the paraventricular hypothalamic nucleus (PVH), with variable extension into the subparaventricular zone (SPVZ) and adjacent structures, on nocturnal pineal melatonin production and rhythms in core body temperature (T_b) and rest-activity (R-A). In accordance with prior work, ablation of the PVH abolishes the nocturnal rise in pineal melatonin. Lesions restricted to the PVH do not affect rhythms in T_b and R-A but lesions extending caudally and ventrally into the SPVZ disrupt the R-A rhythm proportionate to the interruption of caudal SCN projections without affecting the rhythm in T_b. We conclude that pacemaker regulation of the circadian rhythms analyzed in this study is mediated by discrete sets of SCN projections: (i) dorsal projections to the PVH control pineal melatonin production; (ii) rostral projections to the anterior hypothalamic/preoptic areas mediate the T_b rhythm; and (iii) caudal projections to the SPVZ and hypothalamic arousal systems located in the posterior and lateral hypothalamic areas control the rhythm in R-A.     

The luteinizing hormone surge regulates circadian clock gene expression in the chicken ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors' previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary.     

A Coupled Oscillatory Model Mimicking Avian Circadian Regulatory Systems

Much evidence indicates that the pineal gland and thesuprachiasmatic nucleus (SCN) are the primary pacemakers in the housesparrow, Passer domesticus. The interactions between the pineal andSCN predicted by the neuroendocrine loop model indicates that uncouplingwould cause the two oscillators to damp out in constant darkness. Basedupon the original neuroendocrine loop model, a mathematical frameworkof the house sparrow circadian regulatory organization that incorporatesdamping and co-inhibitory coupling has been formulated. The proposedmodel clearly indicates that two coupled oscillators must be 180^°out of the phase for sustaining oscillations. From damping coefficients,which can be determined from experimental data, other parameters suchas external stimuli (interaction coefficient) and characteristicfrequencies can then be computed. Based upon earlier studies and simulations,we conclude that the sparrow pineal gland dampens more rapidly than does theSCN, suggesting that the SCN are probably more important in sparrowsthan previously thought. The model also provides the explanations ofendogenous circadian period (tau) alteration. Finally, we extend this modelto other avian and to mammalian circadian systems. We suggest that avianand mammalian circadian systems may differ in damping coefficients ofpineal glands and the degree of SCN dominance.     

Characterization of melatonin-induced fos-like immunoreactivity in the hypothalamic suprachiasmatic nucleus of the rat.

The hypothalamic suprachiasmatic nucleus (SCN) is primarily responsible for the regulation of circadian rhythmicity. Melatonin, the pineal-derived neurohormone, modulates the rhythmic output of the SCN. Property timed exposure to melatonin is able to induce changes in rhythmic function and thereby entrain circadian rhythms of activity. c-fos is an immediate early gene that is transiently expressed in neurons in response to receptor activation. The ventrolateral portion of the SCN (vSCN) is activated in response to phase-shifting stimuli, an event which is marked by an increase in the expression of c-fos.     

Cellular communication and coupling within the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Importantly, when SCN neurons are removed from the organism and maintained in a brain slice preparation, they continue to generate 24h rhythms in electrical activity, secretion, and gene expression. Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells in the SCN. If we assume that individual cells in the SCN are competent circadian oscillators, it is obviously important to understand how these cells communicate and remain synchronized with each other. Cell-to-cell communication is clearly necessary for conveying inputs to and outputs from the SCN and may be involved in ensuring the high precision of the observed rhythm. In addition, there is a growing body of evidence that a number of systems-level phenomena could be dependent on the cellular communication between circadian pacemaker neurons. It is not yet known how this cellular synchronization occurs, but it is likely that more than one of the already proposed mechanisms is utilized. The purpose of this review is to summarize briefly the possible mechanisms by which the oscillatory cells in the SCN communicate with each other. (Chronobiology International, 18(4)579-600, 2001)     

Expression of clock genes in human peripheral blood mononuclear cells throughout the sleep/wake and circadian cycles

The rhythmic expression of circadian clock genes in the neurons of the suprachiasmatic nucleus (SCN) underlies the manifestation of endogenous circadian rhythmicity in behavior and physiology. Recent evidence demonstrating rhythmic clock gene expression in non-SCN tissues suggests that functional clocks exist outside the central circadian pacemaker of the brain. In this investigation, the nature of an oscillator in peripheral blood mononuclear cells (PBMCs) is evaluated by assessing clock gene expression throughout both a typical sleep/wake cycle (LD) and during a constant routine (CR). Six healthy men and women aged (mean±SEM) 23.7±1.6 yrs participated in this five-day investigation in temporal isolation. Core body temperature and plasma melatonin concentration were measured as markers of the central circadian pacemaker. The expression of HPER1, HPER2, and HBMAL1 was quantified in PBMCs sampled throughout an uninterrupted 72 h period. The core body temperature minimum and the midpoint of melatonin concentration measured during the CR occurred 2:17±0:20 and 3:24 ±0:09 h before habitual awakening, respectively, and were well aligned to the sleep/wake cycle. HPER1 and HPER2 expression in PBMCs demonstrated significant circadian rhythmicity that peaked early after wake-time and was comparable under LD and CR conditions. HBMAL1 expression was more variable, and peaked in the middle of the wake period under LD conditions and during the habitual sleep period under CR conditions. For the first time, bi-hourly sampling over three consecutive days is used to compare clock gene expression in a human peripheral oscillator under different sleep/wake conditions.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)     

Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators.

The circadian timing system in mammals is composed of a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and slave clocks in most peripheral cell types. The phase of peripheral clocks can be completely uncoupled from the SCN pacemaker by restricted feeding. Thus, feeding time, while not affecting the phase of the SCN pacemaker, is a dominant Zeitgeber for peripheral circadian oscillators. Here we show that the phase resetting in peripheral clocks of nocturnal mice is slow when feeding time is changed from night to day and rapid when switched back from day to night. Unexpectedly, the inertia in daytime feeding-induced phase resetting of circadian gene expression in liver and kidney is not an intrinsic property of peripheral oscillators, but is caused by glucocorticoid signaling. Thus, glucocorticoid hormones inhibit the uncoupling of peripheral and central circadian oscillators by altered feeding time.     

哺乳动物昼夜节律组构中的下丘脑视交叉上核和松果腺

哺乳动物下丘脑视交叉上核（SCN）是昼夜节律最主要的起搏器,控制着机体的生理和行为的节律。它具有自身内在的节律性,同时也受光照周期信号和一些内源性化学物质的调节。检查腺分泌裉黑素（MEL）受SCN的调控,MEL通过作用于SCN上高亲和性MEL受体,启动第二、第三信使系统,调整SCN的昼夜节律活动。这种调整具有时间敏感性。     

Daily rhythms of Fos expression in hypothalamic targets of the suprachiasmatic nucleus in diurnal and nocturnal rodents.

Little is known about the differences in the neural substrates of circadian rhythms that are responsible for the maintenance of differences between diurnal and nocturnal patterns of activity in mammals. In both groups of animals, the suprachiasmatic nucleus (SCN) functions as the principal circadian pacemaker, and surprisingly, several correlates of neuronal activity in the SCN show similar daily patterns in diurnal and nocturnal species. In this study, immunocytochemistry was used to monitor daily fluctuations in the expression of the nuclear phosphoprotein Fos in the SCN and in hypothalamic targets of the SCN axonal outputs in the nocturnal laboratory rat and in the diurnal murid rodent, Arvicanthis niloticus. The daily patterns of Fos expression in the SCN were very similar across the two species. However, clear species differences were seen in regions of the hypothalamus that receive inputs from the SCN including the subparaventricular zone. These results indicate that differences in the circadian system found downstream from the SCN contribute to the emergence of a diurnal or nocturnal profile in mammals.