Daily Torpor Alters Multiple Gene Expression in the Suprachiasmatic Nucleus and Pineal Gland of the Djungarian Hamster (Phodopus sungorus)

Circadian rhythms are still expressed in animals that display daily torpor, implying a temperature compensation of the pacemaker. Nevertheless, it remains unclear how the clock works in hypothermic states and whether torpor itself, as a temperature pulse, affects the circadian system. To reveal changes in the clockwork during torpor, we compared clock gene and neuropeptide expression by in situ hybridization in the suprachiasmatic nucleus (SCN) and pineal gland of normothermic and torpid Djungarian hamsters (Phodopus sungorus). Animals from light‐dark (LD) 8∶16 were sacrificed at 8 time points throughout 24 h. To investigate the effect of a previous torpor episode on the clock, we sacrificed a group of normothermic hamsters 1 day after torpor. In normothermic animals, Per1 peaked at zeitgeber time (ZT)4; whereas, Bmal1 reached maximal expression between ZT16 and ZT19. AVP mRNA in the SCN showed highest levels at ZT7. On the day of torpor, the levels of all mRNAs investigated, except for AVP mRNA, were increased during the torpor bout. Moreover, the Bmal1 rhythm was advanced. On the day after the hypothermia, Bmal1 and AVP rhythms showed severely depressed amplitude. Those distinct amplitude changes of Bmal1 and AVP on the day after a torpor episode expression suggests that torpor affects the circadian system, probably by altered translational processes that might lead to a modified protein feedback on gene expression. In the pineal gland, an important clock output, Aanat expression, peaked between ZT16 and ZT22 in normothermic animals. Aanat levels were significantly advanced on the day of hypothermia, an effect which was still visible 1 day afterward. In summary, this study showed that daily torpor affects the phase and amplitude of rhythmic clock gene and clock‐controlled gene expression in the SCN. Furthermore, the rhythmic gene expression in a peripheral oscillator, the pineal gland, is also affected.     

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.     

Effect of pinealectomy on the circadian clock of the chick retina under different monochromatic lights

The avian circadian rhythm pacemaker is composed of the retina, pineal gland and suprachiasmatic nucleus. As an intact input-pacemaker-output system, each of these structures is linked within a neuroendocrine loop to influence downstream processes and peripheral oscillations. While our previous study found that monochromatic light affected the circadian rhythms of clock genes in the chick retina, the effect of the pineal gland on the response of the retinal circadian clock under monochromatic light still remains unclear. In this study, a total of 144 chicks, including sham-operated and pinealectomized groups, were exposed to white, red, green or blue light. After 2 weeks of light illumination, the circadian expression of six core clock genes (cClock, cBmal1, cCry1, cCry2, cPer2 and cPer3), melanopsin (cOpn4-1, cOpn4-2), Arylalkylamine N-acetyltransferase (cAanat) and melatonin was examined in the retina. The cBmal1, cCry1, cPer2, cPer3, cOpn4-1, cOpn4-2 and cAanat genes as well as melatonin had circadian rhythmic expression in both the sham-operated and pinealectomized groups under different monochromatic lights, while cClock and cCry2 had arrhythmic 24 h profiles in all of the light-treated groups. After pinealectomy, the rhythmicity of the clock genes, melanopsins, cAanat and melatonin in the chick retina did not change, especially the mesors, amplitudes and phases of cBmal1, cOpn4-1, cOpn4-2, cAanat and melatonin. Compared to the white light group, however, green light increased the mRNA expression of the positive-regulating clock genes cBmal1, cAanat, cOpn4-1 and cOpn4-2 as well as the melatonin content in pinealectomized chicks, whereas red light decreased their expression. These results suggest that the chick retina is a relatively independent circadian oscillator from the pineal gland, whose circadian rhythmicity (including photoreception, molecular clock and melatonin output) is not altered after pinealectomization. Moreover, green light increases ocular cAanat expression and melatonin synthesis by accelerating the expression of melanopsin and positive-regulating clock genes cBmal1 and cClock.     

Photic resetting of the circadian clock is correlated with photic habitat in <Emphasis Type="Italic">Anolis</Emphasis> lizards

Circadian rhythms are regulated by an internal clock, which is itself synchronized to environmental cues such as light and temperature. It is widely assumed that the circadian system is adapted to local cues, which vary enormously across habitats, yet the comparative data necessary for testing this idea are lacking. We examined photic and thermal resetting of the circadian clock in five species of Anolis lizards whose microhabitats differ in the amounts of sun and shade. The primary circadian oscillator in Anolis is the pineal gland, which produces the hormone melatonin. A flow-through culture system was employed to measure rhythmic melatonin output from individually cultured pineal glands. All species showed temperature-compensated circadian rhythms of pineal melatonin. Light caused significant phase delays of the melatonin rhythm, and this effect varied among species. Controlling for phylogenetic differences, the results indicate that the pineal glands of shade-dwelling species are more sensitive to photic resetting than species living in more brightly illuminated habitats. The differences were not due to variation in free-running period, but may be due to variation in oscillator phase and/or robustness. Surprisingly, thermal resetting was not statistically significant. Overall, the results suggest that the Anolis circadian system is adapted to photic habitat.     

Daily Oscillation in Melatonin Synthesis in The Turkey Pineal Gland and Retina: Diurnal and Circadian Rhythms

The aim of the present study was to examine arylalkylamine N‐acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light‐dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night‐time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high‐amplitude melatonin rhythms in the turkey.     

Deleting the Arntl clock gene in the granular layer of the mouse cerebellum: impact on the molecular circadian clockwork

The suprachiasmatic nucleus houses the central circadian clock and is characterized by the timely regulated expression of clock genes. However, neurons of the cerebellar cortex also contain a circadian oscillator with circadian expression of clock genes being controlled by the suprachiasmatic nucleus. It has been suggested that the cerebellar circadian oscillator is involved in food anticipation, but direct molecular evidence of the role of the circadian oscillator of the cerebellar cortex is currently unavailable. To investigate the hypothesis that the circadian oscillator of the cerebellum is involved in circadian physiology and food anticipation, we therefore by use of Cre‐LoxP technology generated a conditional knockout mouse with the core clock gene Arntl deleted specifically in granule cells of the cerebellum, since expression of clock genes in the cerebellar cortex is mainly located in this cell type. We here report that deletion of Arntl heavily influences the molecular clock of the cerebellar cortex with significantly altered and arrhythmic expression of other central clock and clock‐controlled genes. On the other hand, daily expression of clock genes in the suprachiasmatic nucleus was unaffected. Telemetric registrations in different light regimes did not detect significant differences in circadian rhythms of running activity and body temperature between Arntl conditional knockout mice and controls. Furthermore, food anticipatory behavior did not differ between genotypes. These data suggest that Arntl is an essential part of the cerebellar oscillator; however, the oscillator of the granular layer of the cerebellar cortex does not control traditional circadian parameters or food anticipation.     

Homeobox Genes in the Rodent Pineal Gland: Roles in Development and Phenotype Maintenance

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell.     

DIURNAL EXPRESSION OF CLOCK GENES IN PINEAL GLAND AND BRAIN AND PLASMA LEVELS OF MELATONIN AND CORTISOL IN ATLANTIC SALMON PARR AND SMOLTS

In Atlantic salmon, the preadaptation to a marine life, i.e., parr-smolt transformation, and melatonin production in the pineal gland are regulated by the photoperiod. However, the clock genes have never been studied in the pineal gland of this species. The aim of the present study was to describe the diurnal expression of clock genes (Per1-like, Cry2, and Clock) in the pineal gland and brain of Atlantic salmon parr and smolts in freshwater, as well as plasma levels of melatonin and cortisol. By employing an out-of-season smolt production model, the parr-smolt transformation was induced by subjecting triplicate groups of parr to 6 wks (wks 0 to 6) under a 12?h:12?h light-dark (LD) regime followed by 6 wks (wks 6 to 12) of continuous light (LL). The measured clock genes in both pineal gland and brain and the plasma levels of melatonin and cortisol showed significant daily variations in parr under LD in wk 6, whereas these rhythms were abolished in smolts under LL in wk 12. In parr, the pineal Per1-like and Cry2 expression peaked in the dark phase, whereas the pineal Clock expression was elevated during the light phase. Although this study presents novel findings on the clock gene system in the teleost pineal gland, the role of this system in the regulation of smoltification needs to be studied in more detail. (Author correspondence: ti.hu@hotmail.com)     

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina.     

Interaction and developmental activation of two neuroendocrine systems that regulate light‐mediated skin pigmentation

Lower vertebrates use rapid light‐regulated changes in skin colour for camouflage (background adaptation) or during circadian variation in irradiance levels. Two neuroendocrine systems, the eye/alpha‐melanocyte‐stimulating hormone (α‐MSH) and the pineal complex/melatonin circuits, regulate the process through their respective dispersion and aggregation of pigment granules (melanosomes) in skin melanophores. During development, Xenopus laevis tadpoles raised on a black background or in the dark perceive less light sensed by the eye and darken in response to increased α‐MSH secretion. As embryogenesis proceeds, the pineal complex/melatonin circuit becomes the dominant regulator in the dark and induces lightening of the skin of larvae. The eye/α‐MSH circuit continues to mediate darkening of embryos on a black background, but we propose the circuit is shut down in complete darkness in part by melatonin acting on receptors expressed by pituitary cells to inhibit the expression of pomc, the precursor of α‐MSH.