The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehydebased photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

Daily rhythms in many behavioral, physiological, and biochemical functions are generated by endogenous oscillators that function as internal 24-hour clocks. Under natural conditions, these oscillators are synchronized to the daily environmental cycle of light and darkness. Recent advances in locating circadian pacemakers in the brain and in establishing model systems promise to shed light on the cellular and biochemical mechanisms involved in the generation and regulation of circadian rhythms.

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The purification and chemical properties of thymosin, obtained from bovine thymus tissue, are described. The biological activity of the thymic hormone has been assessed by a newly developed rosette assay, which permits measurement of thymus-dependent lymphoid cells. Thymosin activity is associated with a physicochemically homogeneous protein of molecular weight 12,600. The hormonal activity is evident in in vitro incubation assay, after injection into adult thymectomized mice, and in prolonging survival of neonatally thymectomized mice and the reconstitution of their response to a skin allograft.The extraction, partial purification, and assay of a thymic factor, which has been termed thymosin, from calf thymic tissue has been described by this laboratory (1). Thymosin-containing fractions lowered the incidence, in neonatally thymectomized mice, of a wasting disease characterized by an atrophy of lymphoid tissue and high mortality (2) and a failure of development of cell-mediated immune responses, including the capacity of host cells to elicit a normal graftversus-host response (3) and to reject histoincompatible skin grafts (4). In addition, thymosin administration to newborn normal mice accelerated the ontogenesis of cell-mediated immunity, as measured by the capacity of spleen cells from the treated animals to elicit a normal graft-versus-host response (5) and by the development of resistance to progressive tumor growth after inoculation with Moloney sarcoma virus (6). The addition of thymosin preparations in vitro to discrete populations of lymphoid stem cells from bone marrow rapidly converted such cells into immunologically competent cells, as measured by a graft-versus-host assay (5) and by the appearance of cells with characteristics of T-cells (7) in the rosette bioassay.In this preliminary communication, we describe the further purification and characterization of thymosin. We have isolated from thymus glands of calves a carbohydrate-and lipid-free homogeneous protein that, in preliminary assays, has several of the biological activities that we have reported for crude fractions (8,9). The purification and activity are based upon a modification of a new rosette-forming cell assay described recently (7). A complete paper on the chemistry and additional properties of thymosin will be reported in a separate communication (Guha, A. et MATERIALS AND METHODSAnimals. CBA/J and A/J male mice, 60 days old, were purchased from Jackson Laboratories, Bar Harbor, Me. CBA/ Wh mice of the same age were raised in our own colony. All animals received food and water ad libitum until killed.Chemicals and Reagents. Azathioprine was generously provided by Burroughs-Wellcome as its sodium salt. Sephadex G-150 (40-120 ,um) was purchased from Pharmacia Chemicals, Inc.; Cellex E (Ecteola; 0.43 meq/g exchange capacity) and Biogel HTP were obtained from Bio-Rad Laboratories. All other chemicals were of analytical or reagent grade and were used without further purification.Rosette Assays. Two types of rosette assays for ...

The avian pineal gland, like that of mammals, displays a striking circadian rhythm in the synthesis and release of the hormone melatonin. However, the pineal gland plays a more prominent role in avian circadian organization and differs from that in mammals in several ways. One important difference is that the pineal gland in birds is relatively autonomous. In addition to making melatonin, the avian pineal contains photoreceptors and a circadian clock (thus, an entire circadian system) within itself. Furthermore, avian pineals retain their circadian properties in organ or dispersed cell culture, making biochemical components of regulatory pathways accessible. Avian pinealocytes are directly photosensitive, and novel candidates for the unidentified photopigments involved in the regulation of clock function and melatonin production, including melanopsin, pinopsin, iodopsin, and the cryptochromes, are being evaluated. Transduction pathways and second messengers that may be involved in acute and entraining effects, including cyclic nucleotides, calcium fluxes, and protein kinases, have been, and continue to be, examined. Moreover, several clock genes similar to those found in Drosophila and mouse are expressed, and their dynamics and interactions are being studied. Finally, the bases for acute and clock regulation of the key enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), are described. The ability to study entrainment, the oscillator itself, and a physiological output in the same tissue at the same time makes the avian pineal gland an excellent model to study the bases and regulation of circadian rhythms.

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm ref lects circadian changes in the activity of serotonin Nacetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.

The properties of the P-adrenergic receptor which regulates adenylate cyclase [ rapid decrease in the number of available receptors and in hormone-sensitive adenylate cyclase activity; conversely, lack of stimulation causes an increase in these parameters. It is suggested that these changes contribute to the phenomena of super-and subsensitivity in the pineal gland by regulating the capacity of the pineal to synthesize cyclic AMP in response to f-adrenergic stimulation.The pineal gland has proven to be a useful model in the study of the interaction between sympathetic nerves and the ,B-adrenergic receptors of responsive cells (1). Stimulation of the pineal ,B-adrenergic receptor results in increased levels of intracellular cyclic adenosine 3':5'-monophosphate (cyclic AMP) as a consequence of enhanced adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (2, 3). In addition, through its influence on cyclic AMP levels, the f0-adrenergic receptor controls both the induction and maintenance of serotonin N-acetyl-transferase activity (= arylamine acetyltransferase; acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5) (4, 5). Serotonin N-acetyltransferase forms N-acetylserotonin, a precursor for the synthesis of melatonin, the pineal hormone (1).Recent studies have shown that both the sensitivity and the magnitude of these biochemical responses of the pineal to f.-adrenergic stimulation are rapidly affected by the degree of prior stimulation of the receptor (6-8). Thus, following exposure of the pineal to either the noradrenergic neurotransmitter or to synthetic j3-adrenergic agonists, the subsequent responses are "subsensitive" in comparison with responses obtained from previously unstimulated glands. In contrast, after the reduction of sympathetic nerve firing by exposure of animals to light (9, 10), the responses are "supersensitive" (7,8). These changes in the sensitivity of the pineal to ,B-adrenergic stimulation can occur rapidly; subsensitivity has been shown to develop within hours and supersensitivity can be demonstrated within a day.Recently, competitive ,B-adrenergic antagonists of high specific activity have been used to characterize binding sites whose properties are similar to those of the f3-adrenergic receptor (11-13). In the rat pineal gland the sites which specifically bind 1-[3H]alprenololt were found to be indistinguishable from the fl-adrenergic receptor coupled to adenylate cyclase (14). The present study utilizes the binding of 1- [3H]alprenolol and the activity of hormone-sensitive adenylate cyclase to characterize changes in the properties of the ,B-adrenergic receptor which occur following stimulation of this receptor in vvo. We find that decreased ,B-adrenergic stimulation of the pineal results in both an increase in the hormone-sensitive adenylate cyclase activity and an increased number of specific l-[3H]alprenolol binding sites. In contrast, increased stimulation causes a decrease in both the amount of hormone-sensitive adenylate cyclase and the number of s...

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