Is the avian circadian system a neuroendocrine loop?

Avian circadian organization is a result of a complex interaction of photoreceptive and oscillatory components. The known components include the pineal gland, the lateral eyes, the suprachiasmatic nuclei (SCN), and extraocular brain photoreceptors. The pathways by which these components integrate circadian rhythmicity suggest a neuroendocrine loop in which the SCN inhibits pineal and ocular oscillators during the course of subjective day via a multisynaptic neuronal pathway which includes the superior cervical ganglia (SCG). During the night, the pineal in turn inhibits SCN activity via its secretion of the hormone melatonin into the blood circulation. This neuroendocrine loop, it is proposed, synchronizes multiple oscillators within each component and maintains the stability and precision of the system.     

The pineal gland influences rat circadian activity rhythms in constant light.

Mammalian circadian organization is believed to derive primarily from circadian oscillators within the hypothalamic suprachiasmatic nuclei (SCN). The SCN drives circadian rhythms of a wide array of functions (e.g., locomotion, body temperature, and several endocrine processes, including the circadian secretion of the pineal hormone melatonin). In contrast to the situation in several species of reptiles and birds, there is an extensive literature reporting little or no effect of pinealectomy on mammalian circadian rhythms. However, recent research has indicated that the SCN and circadian systems of several mammalian species are highly sensitive to exogenous melatonin, raising the possibility that endogenous pineal hormone may provide feedback in the control of overt circadian rhythms. To determine the role of the pineal gland in rat circadian rhythms, the effects of pinealectomy on locomotor rhythms in constant light (LL) and constant darkness (DD) were studied. The results indicated that the circadian rhythms of pinealectomized rats but not sham-operated controls dissociated into multiple ultradian components in LL and recoupled into circadian patterns only after 12-21 days in DD. The data suggest that pineal feedback may modulate sensitivity to light and/or provide coupling among multiple circadian oscillators within 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.     

Pineal regulation of circadian rhythms of 2-deoxy[14C]glucose uptake and 2[125I]iodomelatonin binding in the visual system of the house sparrow,Passer domesticus

The pineal gland and its hormone melatonin are crucial for the generation of circadian rhythms in several species of passerine birds. The sites and mechanisms by which they influence avian behavior are therefore of particular interest. Recent research employing several brain imaging techniques has indicated that the sites of melatonin action within the avian brain are wide-spread within the 4 major visual pathways. In this study, we have investigated whether the avian homologue of the mammalian suprachiasmatic nucleus, the visual suprachiasmatic nucleus (vSCN), and other visually sensitive structures express circadian rhythms of 2-deoxy[¹⁴C]glucose (2DG) uptake and 2[¹²⁵I]iodomelatonin (IMEL) binding in house sparrows,Passer domesticus, under constant environmental conditions in the presence or absence of the pineal gland. The results indicate that 2DG uptake in the vSCN is oscillatory in sham-operated sparrows but damps to arrhythmicity in pinealectomized birds, suggesting this structure contains a damped circadian oscillator independent of pineal input. We have also asked whether IMEL binding is rhythmic under these conditions in the same brains. These results indicate IMEL binding is rhythmic in several structures in the circadian, tectofugal, thalamofugal visual pathways and that pinealectomy increases the level of IMEL binding 2–4 fold suggesting that IMEL binding is down regulated by endogenous melatonin. However, the circadian rhythm of this binding is only gradually abolished, suggesting it too is regulated by a non-pineal circadian clock. These data are discussed in the context of the behavioral neurobiology of avian circadian systems and the neuroendocrine loop model.     

松果体昼夜节律生物钟分子机制的研究进展

在各种非哺乳类脊椎动物中 ,松果体起着中枢昼夜节律振荡器的作用。近来 ,在鸟类松果体中相继发现了几种钟基因 ,如Per、Cry、Clock和Bmal等 ,其表达的时间变化规律与哺乳类视交叉上核 (SCN)的非常相似。钟的振荡由其自身调控反馈环路的转录和翻译组成 ,鸟类松果体和哺乳类SCN似乎具有共同的钟振荡基本分子构架 ;若干钟基因产物作为正向或负向调节子影响钟的振荡 ;昼夜性的控时机制同时也需要翻译后事件的参与。这些过程对钟振荡器的稳定性和 /或钟导引的光输入通路有着重要的调控作用     

Circadian organization is governed by extra-SCN pacemakers

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.     

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.     

The role of PACAP in the control of circadian expression of clock genes in the chicken pineal gland

Several features of the molecular circadian oscillator of the chicken pineal gland show homology with those in the mammalian SCN. Studies have shown the effects of PACAP on the mammalian SCN, but its effects on the expression of clock genes in the avian pineal gland have not yet been demonstrated. Clock and Cry1 expression was analyzed in pineal glands of chicken embryos after exposure to PACAP-38 in vitro. PACAP reduced expression of both clock genes within 2h. Ten hours after exposure, mRNA contents exceeded that of the controls. Our results support the hypothesis that the molecular clock machinery in the chicken pineal gland is also sensitive to PACAP.     

Multiple redundant circadian oscillators within the isolated avian pineal gland

Summary The avian pineal gland contains a circadian pacemaker that oscillates in vitro. Using a flow-through culture system it is possible to measure melatonin production from very small subsections of an individual gland. We have used this technique to attempt to localize the oscillators in the pineal. Progressive tissue reduction did not affect the rhythmicity of cultured pineals. Multiple pieces (up to eight) from a single pineal all were capable of circadian oscillation — establishing directly that a pineal gland contains at least eight oscillators. All pineal pieces were responsive to light, and single light pulses shifted the phase of the melatonin rhythm. Because pieces equivalent to less than one per cent of the whole gland were rhythmic and because the capacity for oscillation was distributed throughout the gland, an individual pineal appears to be composed of a population of circadian oscillators.     

Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization

The phases of central (SCN) and peripheral circadian oscillators are held in specific relationships under LD cycles but, in the absence of external rhythmic input, may damp or drift out of phase with each other. Rats exposed to prolonged constant light become behaviorally arrhythmic, perhaps as a consequence of dissociation of phases among SCN cells. The authors asked whether individual central and peripheral circadian oscillators were rhythmic in LL-treated arrhythmic rats and, if rhythmic, what were the phase relationships between them. The authors prepared SCN, pineal gland, pituitary, and cornea cultures from transgenic Period1-luciferaserats whose body temperature and locomotor activity were arrhythmic and from several groups of rhythmic rats held in LD, DD, and short-term LL. The authors measured mPer1gene expression by recording light output with sensitive photomultipliers. Most of the cultures from all groups displayed circadian rhythms. This could reflect persistent rhythmicity in vivo prior to culture or, alternatively, rhythmicity that may have been initiated by the culture procedure. To test this, the authors cultured tissues at 2 different times 12 h apart and asked whether phase of the rhythm was related to culture time. The pineal, pituitary, and SCN cultures showed partial or complete dependence of phase on culture time, while peak phases of the cornea cultures were independent of culture time in rhythmic rats and were randomly distributed regardless of culture time in arrhythmic animals. These results suggest that in behaviorally arrhythmic rats, oscillators in the pineal, pituitary, and SCN had been arrhythmic or severely damped in vivo, while the cornea oscillator was free running. The peak phases of the SCN cultures were particularly sensitive to some aspect of the culture procedure since rhythmicity of SCN cultures from robustly rhythmic LD-entrained rats was strongly influenced when the procedure was carried out at any time except the 2nd half of the day.     

Neural mechanisms for entrainment and generation of mammalian circadian rhythms.

The identification of a direct retinohypothalamic tract (RHT) terminating in the supra-chiasmatic nuclei (SCN) has focused attention on the role of these structures in the entrainment and generation of circadian rhythms in mammals. Light effects on circadian rhythms are mediated by both the RHT and portions of the classical visual system. The complex interactions of these systems are reflected both in their direct anatomical connections and in the functional changes in entrainment produced by interruption of either set of projections. Destruction of the RHT/SCN eliminated both normal entrainment and normal free-running circadian rhythms. No circadian rhythms has survived SCN ablation in rodents, but a variety of non-circadian cycles can be generated by lesioned animals. The complex behavioral patterns produced by SCN-lesioned hamsters suggest that circadian oscillators continue to function in these animals, but that their activity is no longer integrated into a single circadian framework. The available evidence indicates that the mammalian pacemaking system comprises a set of independent oscillators normally regulated by the SCN and by light information that is transmitted via several retinofugal pathways.     

Hypothalamic regulation of circadian noradrenergic input to the chick pineal gland

Summary While the avian pineal gland contains circadian oscillators and photoreceptors capable of producing circadian rhythms of the hormone melatonin, it is extensively innervated by post-ganglionic fibers of the superior cervical ganglia which release norepinephrine (NE) rhythmically. Norepinephrine turnover is higher during subjective day than during subjective night. In mammals, this rhythmic input, which is higher in subjective night than subjective day, derives from the hypothalamic suprachiasmatic nuclei (SCN) and is essential for rhythmic melatonin production. The present study was designed to determine whether one of two candidates for the avian homologue of the mammalian SCN is necessary for rhythmic NE turnover in the chick pineal gland. Either electrolytic lesions or sham lesions were delivered to the periventricular preoptic nuclei (PPN) or to the visual suprachiasmatic nucleus (vSCN). After recovery, the rates of decline in [NE] were determined following pretreatment with -methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, at mid-subjective day or at mid-subjective night. Birds receiving sham surgeries in either PPN or vSCN and birds receiving lesions of the PPN exhibited rhythmicity in NE turnover. No rhythm of NE turnover could be determined in birds with ablated vSCN.Abbreviations AMPT -methyl-p-tyrosine - DS supraoptic decussation - EBZ ear bar zero (see Methods) - GLv ventral lateral geniculate body - NE norepinephrine - PPN periventricular preoptic nuclei - RH retinohypothalamic projection - SCN suprachiasmatic nuclei - vSCN visual suprachiasmatic nucleus     

Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods

The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. Arylalkylamine N-acetyltransferase (aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague-Dawley rats kept under different light-dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN, the results suggest that the photoperiodic differences accompanying the seasons of the year are imprinted in more than one oscillator and that this may accentuate the important message regarding 'time of year.'     

A Network of (Autonomic) Clock Outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each dependent on the cell‐autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock that produces a coherent output capable of timing all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre‐autonomic and neuro‐endocrine target neurons is controlled by differentially timed waves of vasopressin, GABA, and glutamate release from SCN terminals, among other factors. Together our data indicate that, with regard to the timing of their main release period within the LD cycle, at least four subpopulations of SCN neurons should be discernible. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of four differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure, i.e., the SCN seems to contain neurons that specifically target the liver, pineal gland, and adrenal gland.     

MULTIPLE OSCILLATORS IN THE SUPRACHIASMATIC NUCLEUS

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371–387, 2001)     

Circadian clocks in the mammalian brain

Daily cycles in physiology and behaviour are probably a universal feature of multicellular organisms. These rhythms are predominantly driven by endogenous clocks with a periodicity approximating to one day, i.e. circadian. In mammals, the circadian clock governing activity/ rest, neuroendocrine and autonomic rhythms lies in the hypothalamus, in the suprachiasmatic nuclei (SCN). Intrinsic circadian oscillators are also present in the retina. The SCN "clockwork" is based on a cell autonomous, genetically determined mechanism. Mammalian homologues of a number of Drosophila genes which encode elements of the fly circadian mechanism have recently been identified. In Drosophila, the protein products of these genes interact in a negative feedback loop, establishing a circadian cycle in gene expression. Characterisation of the roles played by putative mammalian clock genes in the SCN, and how the emergent cellular signal imposes order over the entire neuraxis, will provide a fundamental contribution to our understanding of the molecular basis of behaviour. BioEssays 22:23-31, 2000.     

The Avian Pineal Gland

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non‐mammalian species, including birds, the pineal gland contains a self‐sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light‐dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP‐protein kinase‐A system modified the MT release temporarily without phase‐shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.