Circadian organization in the lizardSceloporus occidentalis: The effects of pinealectomy, blinding, and melatonin

Summary Pinealectomy of the iguanid lizardSceloporus occidentalis freerunning in either continuous illumination or continuous darkness typically causes changes in the period of the activity rhythm as well as changes in the amount of daily activity (). Blinding also alters the period of the freerunning activity rhythm. Continuous long term administration of melatonin via subcutaneous capsules causes a significant lengthening of the period of the activity rhythm (as well as a decrease in ) of pinealectomized and/or blinded lizards showing that melatonin exerts its action at extrapineal and extraocular sites. However, the amount of lengthening induced by melatonin is significantly greater in pinealectomized lizards than in intact lizards. The results indicate that the pineal (and possibly the eyes) act as coupling devices or as the loci of circadian pacemakers within a multioscillator system. Melatonin may function as a chemical messenger between the pineal (or eyes) and the rest of the circadian system.     

Circadian locomotor rhythms in the desert iguana I. The role of the eyes and the pineal

Summary The pineal and the eyes are known to be important components in the circadian system of some species of lizards; their effects may be mediated by the hormone melatonin. We examined the role played by these structures in the desert iguana (Dipsosaurus dorsalis). Surgical removal of the pineal had no effect on circadian locomotor rhythms, even though this procedure abolished the circadian rhythm of melatonin in the blood. Furthermore, when the isolated pineal of Dipsosaurus was studied in organ culture, it showed no circadian rhythm of melatonin secretion, as do pineals of some other lizard species, although it did produce large quantities of this hormone. Bilateral ocular enucleation had only small effects on the freerunning period of locomotor rhythms, without affecting melatonin levels in the blood. Behavioral circadian rhythms persisted in desert iguanas subjected to both enucleation and pinealectomy. These data suggest that neither the pineal nor the eyes are central components of the circadian pacemaking system in Dipsosaurus, nor is melatonin critically involved in maintaining its organization.Abbreviations CT circadian time - ZT zeitgeber time - LL constant light - LD light-dark cycle - DD constant darkness - freerunning circadian period     

Complex circadian regulation of pineal melatonin and wheel-running in Syrian hamsters

Circadian regulation of pineal melatonin content was studied in Syrian hamsters (Mesocricetus auratus), especially melatonin peak width and the temporal correlation to wheel-running activity. Melatonin was measured by radioimmunoassay in glands removed at different circadian times with respect to activity onset (= CT 12). Pineal melatonin peak width (h; for mean 125 pg/gland) and activity duration () were both 4–5 h longer after 12 or 27 weeks than after 5 or 6 days in continuous darkness (DD). Increased peak width was associated with a delay in the morning decline (M) of melatonin to baseline, correlated with a similar delay in wheel-running offset. In contrast, the evening rise (E) in melatonin occurred at approximately the same circadian phase regardless of the length of DD. Fifteen min light pulses produced similar phase-shifts in melatonin and activity. In a phase advance shift, M advanced at once, while E advanced only after several days of adjustment. Independent timing of shifts in the E and M components of the melatonin rhythm suggest that these events are controlled separately by at least two circadian oscillators whose mutual phase relationship determines melatonin peak width. This two-oscillator control of melatonin peak width is integral to the circadian mechanism of hamster photoperiodic time measurement.Abbreviations CT circadian time - DD continuous dark - L: D light: dark cycle - PMEL pineal melatonin - PRC phase response curve - RIA radioimmunoassay; , duration (h) of the active phase of the circadian wheel-running rhythm; , free-running period     

Pineal melatonin rhythms in the lizardAnolis carolinensis: effects of light and temperature cycles

Summary Pineal and ocular melatonin was assessed, over 24 h periods, in male lizards (Anolis carolinensis) entrained to 24 h light-dark (LD) cycles and a constant 32 C, and in lizards entrained to both 24 h LD cycles and 24 h temperature cycles (32 C/20 C). At a constant temperature, the duration of the photoperiod has a profound effect on the duration, amplitude, and phase of the pineal melatonin rhythm (Fig. 1). The pineal melatonin rhythm under cyclic temperature peaks during the cool (20 C) phase of the cycle regardless of whether or not the cool phase occurs during the light or dark phase of a LD 1212 cycle (Fig. 3). Under a temperature cycle and constant dim illumination, a pineal melatonin rhythm is observed which peaks during the cool phase of the temperature cycle, but the amplitude of the rhythm is depressed relative to that observed under LD (Fig. 2). Illumination up to 2 h in duration does not suppress the nocturnal melatonin peak in theAnolis pineal (Fig. 4). No melatonin rhythm was observed in the eyes ofAnolis under either 24 h LD cycles and a constant temperature (Fig. 1), or under simultaneous light and temperature cycles (Fig. 3). Ocular melatonin content was, in all cases, either very low or non-detectable.Abbreviations HIOMT hydroxyindole-O-methyltransferase - NAT N-acetyltransferase     

The Circadian System of Ruin Lizards: A Seasonally Changing Neuroendocrine Loop?

Previous studies have shown that the amplitude of daily melatonin production in cultured ruin lizard pineal organs explanted in the summer is significantly higher than that from organs explanted in the winter. To test whether seasonal photoperiodic changes are decoded autonomously by the pineal gland, pineals explanted in summer were cultured in vitro and exposed to changes between winter and summer photoperiods. The changes in photoperiod duration did not affect the daily profiles of in vitro melatonin production. The discrepancy between the present in vitro results and those from lizards exposed to winter or summer photoperiods before pineal explantation supports the view that circadian information entering the pineal gland via its innervation is involved in determining seasonal changes of melatonin production in ruin lizards. We further examined whether a central component of the circadian system of ruin lizards, specifically the retinae of the lateral eyes, expresses similar seasonal changes in function as does the pineal gland. We did not find any difference between summer and autumn‐winter in the effectiveness of either bilateral retinalectomy or optic nerve lesion—at the level of the optic chiasm—in altering circadian locomotor behavior in constant conditions. Both surgical procedures mostly induced a shortening of the free‐running period of the locomotor rhythm of similar magnitude in all seasons. Thus, the retinae do not appear to participate in the seasonal reorganization of the circadian system in ruin lizards.     

Retinally perceived light is not essential for photic regulation of pineal melatonin rhythms in the pigeon: studies with microdialysis

Using in vivo microdialysis, effects of retinally perceived light on pineal melatonin release and its rhythmicity was examined in the pigeon. In the first experiment, light-induced suppression of pineal melatonin release was studied. Although light given to the whole body during the dark strongly suppressed pineal melatonin release to a daytime level, light exclusively delivered to the eyes did not remarkably inhibit melatonin release. In the second experiment, in order to determine whether retinally perceived light has phase-shifting effects on pineal melatonin rhythms, pigeons were given a single light pulse of 2 h at circadian time (CT) 18 and the phases of the second cycle after the light pulse were compared with those of control pigeons without the light pulse. In this experiment, phase advances of pineal melatonin rhythms were observed when the light was given to the whole body but not when only the eyes were illuminated. In a third experiment, after entrainment to light-dark 12:12 (LD 12:12) cycles, birds whose heads were covered with black tapes were transferred into constant light (LL) conditions and only the eyes were exposed to new LD cycles for 7 days (the phase was advanced by 6 h from the previous cycles) using a patching protocol. This procedure, however, could not entrain pineal melatonin rhythms to the retinal LD cycles. These results indicate that the eyes are not essential for photic regulation of pineal melatonin release and its rhythmicity in the pigeon.Abbreviations CT circadian time - LD light-dark - LL constant light - SCN suprachiasmatic nucleus - LLdim constant dim light - NE norepinephrine - SCG superior cervical ganglia - WB whole body - E eye - EX extraretina - C control     

Differential contribution of rod and cone circadian clocks in driving retinal melatonin rhythms in Xenopus

Background

Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release.

Methodology/Principal Findings

We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLΔQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLΔQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLΔQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLΔQ transgenic tadpoles compare to wild-type counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern.

Conclusions/Significance

These results suggest that although the Xenopus retina is made up of approximately equal numbers of rods and cones, the circadian clocks in the rod cells play a dominant role in driving circadian melatonin rhythmicity in the Xenopus retina, although some contribution of the clock in cone cells cannot be excluded.     

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     

Circadian organization in the pigeon,Columba livia: the role of the pineal organ and the eye

Summary The roles of the pineal organ and the eye in the control of circadian locomotor rhythmicity were studied in the pigeon (Columba livia). Neither pinealectomy nor blinding abolished the circadian rhythms in constant dim light conditions (LLdim). All the pinealectomized birds and the blinded birds entrained to light-dark (LD) cycles with no discernible anticipatory activity. However, the birds which had been both pinealectomized and blinded showed no circadian rhythms in prolonged LLdim. These birds entrained to LD cycles with anticipatory activity and showed residual rhythmicity for a while after transfer from LD cycles to LLdim. Continuous administration of melatonin induced suppression of the circadian rhythms and reduced total amount of locomotor activity in LLdim. These results suggest that not only the pineal organ but also the eye (perhaps the retina) is involved in the pigeon's circadian system.Abbreviations NAT N-acetyltransferase - LLdim constant dim light - cadian period - SCN suprachiasmatic nucleus - circadian activity time - LD light-dark     

Melatonin and the circadian rhythms of feeding and perch-hopping in the European starling,Sturnus vulgaris

Summary The role of melatonin plasma titers in the control of free-running circadian rhythms was investigated in European starlings,Sturnus vulgaris, held in continuous dim light. Simultaneous recordings of plasma melatonin, perch-hopping and feeding activity revealed synchronous circadian variations in all three functions with high melatonin titers during resting and low titers during activity periods. Implanting birds with melatonin silastic capsules resulted in a 100-fold increase in plasma concentrations and an abolition of the endogenous melatonin rhythm. Feeding rhythms persisted during this treatment, although the activity time () was lengthened and the circadian period () shortened. Similar changes were found in perchhopping activity, but in some birds, this activity was more or less suppressed. These data demonstrate that although melatonin plasma titers freerun synchronously with other circadian parameters, fluctuations in peripheral melatonin are not essential for the expression of circadian activity.     

Pineal-dependent locomotor activity of lamprey,Lampetra japonica,measured in relation to LD cycle and circadian rhythmicity

Summary Locomotor activity of the river lamprey, Lampetra japonica, was investigated under a light-dark (LD 1212) cycle and under continuous dark conditions. Intact lampreys were entrained to the light:dark cycle. They were active mainly in the early half of the dark period and inactive in light period. The light:dark entrainment continued in 72.7% of lampreys after the removal of bilateral eyes, but additional pinealectomy made the entrainment disappear in all lampreys. When lampreys were pinealectomized with their eyes intact, light: dark entrainment was abolished in most cases. The results indicate that the pineal organ of the lamprey is a photoreceptive organ responsible for synchronizing locomotor activity to LD cycle. Under continuous dark conditions, the locomotor activity began to free-run with a period of 21.3 ± 0.9 h (mean ± SD, n = 53). This circadian rhythmicity was not affected by the removal of lateral eyes but was abolished by pinealectomy. The pineal organ appears to function as an oscillator, or as one of the oscillators, for the circadian locomotor rhythm of lampreys.Abbreviations DD continuous dark - LD light:dark     

Static and extremely low frequency electromagnetic field exposure: Reported effects on the circadian production of melatonin

The circadian rhythm of melatonin production (high melatonin levels at night and low during the day) in the mammalian pineal gland is modified by visible portions of the electromagnetic spectrum, i.e., light, and reportedly by extremely low frequency (ELF) electromagnetic fields as well as by static magnetic field exposure. Both light and non-visible electromagnetic field exposure at night depress the conversion of serotonin (5HT) to melatonin within the pineal gland. Several reports over the last decade showed that the chronic exposure of rats to a 60 Hz electric field, over a range of field strengths, severely attenuated the nighttime rise in pineal melatonin production; however, more recent studies have not confirmed this initial observation. Sinusoidal magnetic field exposure also has been shown to interfere with the nocturnal melatonin forming ability of the pineal gland although the number of studies using these field exposures is small. On the other hand, static magnetic fields have been repeatedly shown to perturb the circadian melatonin rhythm. The field strengths in these studies were almost always in the geomagnetic range (0.2 to 0.7 Gauss or 20 to 70 μtesla) and most often the experimental animals were subjected either to a partial rotation or to a total inversion of the horizontal component of the geomagnetic field. These experiments showed that several parameters in the indole cascade in the pineal gland are modified by these field exposures; thus, pineal cyclic AMP levels, N-acetyltransferase (NAT) activity (the rate limiting enzyme in pineal melatonin production), hydroxyindole-O-methyltransferase (HIOMT) activity (the melatonin forming enzyme), and pineal and blood melatonin concentrations were depressed in various studies. Likewise, increases in pineal levels of 5HT and 5-hydroxyindole acetic acid (5HIAA) were also seen in these glands; these increases are consistent with a depressed melatonin synthesis. The mechanisms whereby non-visible electromagnetic fields influence the melatonin forming ability of the pineal gland remain unknown; however, the retinas in particular have been theorized to serve as magnetoreceptors with the altered melatonin cycle being a consequence of a disturbance in the neural biological clock, i.e., the suprachiasmatic nuclei (SCN) of the hypothalamus, which generates the circadian melatonin rhythm. The disturbances in pineal melatonin production induced by either light exposure or non-visible electromagnetic field exposure at night appear to be the same but whether the underlying mechanisms are similar remains unknown.     

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.     

Pineal and retinal melatonin is involved in the control of circadian locomotor activity and body temperature rhythms in the pigeon

Summary Although pinealectomy or blinding resulted in loss of the clarity of the free-running rhythm of locomotor activity and body temperature and reduced the peak level of circulating melatonin rhythms to approximately a half in intact pigeons, neither pinealectomy nor blinding abolished any of these rhythms. However, when pinealectomy and blinding were combined, the rhythms of locomotor activity and body temperature disappeared in prolonged constant dim light, and melatonin concentration was reduced to the minimum level of detection. In order to examine the role of melatonin in the pigeon's circadian system, it was administered either daily or continuously to PX + EX-pigeons in LLdim. Daily administration of melatonin restored circadian rhythms of locomotor activity which entrained to melatonin injections, but continuous administration did not induce any remarkable change of locomotor activity. These results suggest that melatonin synthesized in the pineal body and the eye contributes to circulating melatonin and its rhythmicity is important for the control of circadian rhythms of locomotor activity and body temperature in the pigeon.Abbreviations LD Light-dark - LLdim constant dim light - LLbright constant bright light - PX pinealectomy - EX blinding - SCN suprachiasmatic nucleus     

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.     

The circadian system of ruin lizards: a seasonally changing neuroendocrine loop?

Previous studies have shown that the amplitude of daily melatonin production in cultured ruin lizard pineal organs explanted in the summer is significantly higher than that from organs explanted in the winter. To test whether seasonal photoperiodic changes are decoded autonomously by the pineal gland, pineals explanted in summer were cultured in vitro and exposed to changes between winter and summer photoperiods. The changes in photoperiod duration did not affect the daily profiles of in vitro melatonin production. The discrepancy between the present in vitro results and those from lizards exposed to winter or summer photoperiods before pineal explantation supports the view that circadian information entering the pineal gland via its innervation is involved in determining seasonal changes of melatonin production in ruin lizards. We further examined whether a central component of the circadian system of ruin lizards, specifically the retinae of the lateral eyes, expresses similar seasonal changes in function as does the pineal gland. We did not find any difference between summer and autumn-winter in the effectiveness of either bilateral retinalectomy or optic nerve lesion-at the level of the optic chiasm-in altering circadian locomotor behavior in constant conditions. Both surgical procedures mostly induced a shortening of the free-running period of the locomotor rhythm of similar magnitude in all seasons. Thus, the retinae do not appear to participate in the seasonal reorganization of the circadian system in ruin lizards.