Vincent M. Cassone

Circadian rhythms from multiple oscillators: lessons from diverse organisms

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.

Effects of melatonin on vertebrate circadian systems

In many species of vertebrates the pineal gland and its indoleamine hormone melatonin play central roles in the control of circadian rhythms, whereas in some species, the pineal gland appears to hold little importance. However, recent research indicates that the circadian rhythms of many species of reptiles, birds and mammals, including humans, are synchronized by the administration of exogenous melatonin. These studies have led to questions concerning the role of this hormone in circadian organization in general. Studies of the sites and mechanisms of melatonin action further indicate that melatonin may be an excellent pharmacological tool for research on the cellular mechanisms of circadian clock function and have pointed to the possibility that melatonin or melatonin analogues may be therapeutically useful for the control of circadian clock dysfunctions such as jet lag, shift-work syndrome and sleep disorders.

Melatonin receptors are for the birds: Molecular analysis of two receptor subtypes differentially expressed in chick brain

Two receptors (CKA and CKB) of the G protein-coupled melatonin receptor family were cloned from chick brain. CKA encodes a protein that is 80% identical at the amino acid level to the human Mel1a melatonin receptor and is thus designated the chick Mel1a melatonin receptor. CKB encodes a protein that is 80% identical to the Xenopus melatonin receptor and defines a new receptor subtype, the Mel1c melatonin receptor, which is distinct from the Mel1a and Mel1b melatonin receptor subtypes. A melatonin receptor family consisting of three subtypes is supported by PCR cloning of distinct melatonin receptor fragments from Xenopus and zebrafish. Expression of CKA and CKB results in similar ligand binding and functional characteristics. The widespread distribution of CKA and CKB mRNA in brain provides a molecular substrate for the profound actions of melatonin in birds.

Entrainment of rat circadian rhythms by daily injection of melatonin depends upon the hypothalamic suprachiasmatic nuclei

Although pinealectomy has little effect on the generation of circadian rhythmicity by mammals, daily injections of the pineal hormone melatonin entrain free-running rats [30]. The present study was designed to determine if known components of mammalian circadian organization were necessary for melatonin entrainment. Rats received either lesions to the suprachiasmatic nuclei (SCN), sham-lesions or neurotoxic lesions to brain catecholamines or serotonin. They were then allowed to free-run in constant dim red light (DD) before each received daily injections of either 1 mg/kg melatonin or ethanol:saline vehicle for 90 days. They were allowed to free-run for 30 days afterwards. Rats which received sham-lesions or neurotoxic lesions entrained to melatonin injections but not to vehicle. Rats which received complete SCN lesions were unaffected by melatonin or vehicle. These data suggest that the behavioral effects of melatonin, like those on reproduction in seasonally breeding mammals, depend upon an intact circadian system and the SCN.

Dose-response relationship between light irradiance and the suppression of plasma melatonin in human volunteers

This study tested the capacity of different irradiances of monochromatic light to reduce plasma melatonin in normal humans. Six healthy male volunteers, 24-34 years old, were exposed to 0.01, 0.3, 1.6, 5, or 13 microW/cm2 of 509 nm monochromatic light for 1 h during the night on separate occasions. Light irradiance depressed plasma melatonin in a dose-response pattern. The data indicate that the mean threshold irradiance for suppressing melatonin is between 1.6 and 5 microW/cm2. Individual variations in threshold responses to monochromatic light were observed among the volunteers.

Avian melatonin synthesis: Photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N‐acetyltransferase (arylalkylamine N‐acetyltransferase; AA‐NAT; EC 2.3.1.87). Here we determined that the chicken AA‐NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA‐NAT mRNA was not detected in other tissues. The AA‐NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA‐NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6‐h light pulse late in subjective night in DD. Nocturnal AA‐NAT mRNA levels do not change during a 20‐min exposure to light, whereas this treatment dramatically decreases AA‐NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA‐NAT activity reflect, at least in part, clock‐generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light‐induced decrease in AA‐NAT activity.

Comparative anatomy of the mammalian hypothalamic suprachiasmatic nucleus.

A detailed analysis of the cytoarchitecture, retinohypothalamic tract (RHT) pro jections, and immunohistochemical localization of major cell and fiber types within the hy pothalamic suprachiasmatic nuclei (SCN) was conducted in five mammalian species: two species of opossum, the domestic cat, the guinea pig, and the house mouse. Cytoarchitectural and immunohistochemical studies were conducted in three additional species of marsupial mammals and in the domestic pig. The SCN in this diverse transect of mammalian taxonomy bear striking similarities. First, the SCN are similar in location, lying close to the third ventricle (3V) dorsal to the optic chiasm (OC), with a cytoarchitecture characterized by small, tightly packed neurons. Second, in all groups studied, the SCN receive bilateral retinal input. Third, the SCN contain immu nohistochemically similar elements. These similarities suggest that the SCN developed char acteristic features early in mammalian phylogeny. Some details of SCN organization vary among the species studied. In marsupials, vaso pressin-like immunoreactive (VP-LI) and vasoactive intestinal polypeptide-like immunoreac tive (VIP-LI) cells codistribute primarily in the dorsomedial aspects of the SCN, while in eutherians, VP-LI and VIP-LI cells are separated into SCN subnuclei. Furthermore, the marsupial RHT projects to the periventricular dorsomedial region, whereas the eutherian RHT projects more ventrally in the SCN into the zone that typically contains VIP-LI perikarya.

Effects of melatonin on neuronal activity in the rat suprachiasmatic nucleus in vitro

The pineal hormone melatonin is believed to act directly on the hypothalamic suprachiasmatic nuclei (SCN), which are principal circadian pacemakers in rodents, although direct demonstration of this is lacking. To determine whether the SCN are sensitive to melatonin electrophysiologically, we investigated the effect of melatonin on rat SCN single-unit activity in an in vitro hypothalamic slice preparation. SCN single-unit activity was inhibited by melatonin superfusion during the late subjective day in a dose-dependent manner but not at other times of day, supporting the view that melatonin acts directly on SCN neurons.

Chickens' Cry2: molecular analysis of an avian cryptochrome in retinal and pineal photoreceptors

We have identified and characterized an ortholog of the putative mammalian clock gene cryptochrome 2 (Cry2) in the chicken, Gallus domesticus. Northern blot analysis of gCry2 mRNA indicates widespread distribution in central nervous and peripheral tissues, with very high expression in pineal and retina. In situ hybridization of chick brain and retina reveals expression in photoreceptors and in visual and circadian system structures. Expression is rhythmic; mRNA levels predominate in late subjective night. The present data suggests that gCry2 is a candidate avian clock gene and/or photopigment and set the stage for functional studies of gCry2.

MELATONIN'S ROLE IN VERTEBRATE CIRCADIAN RHYTHMS

The circadian secretion of melatonin by the pineal gland and retinae is a direct output of circadian oscillators and of the circadian system in many species of vertebrates. This signal affects a broad array of physiological and behavioral processes, making a generalized hypothesis for melatonin function an elusive objective. Still, there are some common features of melatonin function. First, melatonin biosynthesis is always associated with photoreceptors and/or cells that are embryonically derived from photoreceptors. Second, melatonin frequently affects the perception of the photic environment and has as its site of action structures involved in vision. Finally, melatonin affects overt circadian function at least partially via regulation of the hypothalamic suprachiasmatic nucleus (SCN) or its homologues. The mechanisms by which melatonin affects circadian rhythms and other downstream processes are unknown, but they include interaction with a class of membrane-bound receptors that affect intracellular processes through guanosine triphosphate (GTP)-binding protein second messenger systems. Investigation of mechanisms by which melatonin affects its target tissues may unveil basic concepts of neuromodulation, visual system function, and the circadian clock.