Synaptic ribbons in the pineal organ of the goldfish: Circadian rhythmicity and the effects of constant light and constant darkness

Summary Synaptic ribbons in photoreceptor cells of the goldfish pineal organ undergo significant daily changes in their length, distance from the plasma membrane, and number per unit area of pineal end-vesicle. The rhythms persist in fish exposed to constant darkness. Constant light abolishes the rhythms in length and distance of synaptic ribbons from the plasmalemma, but has little effect on numerical changes over a 24-h cycle. These findings suggest that synaptic ribbons in the pineal organ of lower vertebrates might be useful as indicators of metabolic activity.     

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     

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.     

Daily and circadian variation in the electroretinogram of the domestic fowl: effects of melatonin

Visual and circadian function are integrally related in birds, but the precise nature of their interaction is unknown. The present study determined whether visual sensitivity measured electroretinographically (ERG) in 7-week-old cockerels varies over the time of day, whether this rhythm persists in constant darkness (DD) and whether exogenous melatonin affects this ERG rhythmicity. ERG b-wave amplitude was rhythmic in LD and persisted in DD with peak amplitude during mid- to late afternoon in LD and mid-subjective day in DD, indicating that the ERG rhythm is endogenously generated. No daily or circadian variation in a-wave amplitude was observed, and ERG component latency and durations were not rhythmic. Intramuscular injection of 10 g/kg melatonin at ZT10 in LD significantly decreased b-wave amplitude but had no effect on a-wave. Intraocular injection of 600 pg melatonin, however, had no effect on any aspect of the ERG. These data indicate that a circadian clock regulates ocular sensitivity to light and that melatonin may mediate some or all of this effect. The level at which melatonin modulates retinal sensitivity is not known, but the present data suggest a central site rather than a direct effect of the hormone in the eye.Abbreviations DD constant darkness - ERG electroretinography - EW Edinger-Westphal nuclei - IMEL iodomelatonin - IO isthmooptic nucleus - LD light-dark cycle - SCG superior cervical ganglion - SCN suprachiasmatic nuclei - vSCN visual suprachiasmatic nucleus     

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     

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     

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light-dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights-off and declined around lights-on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights-on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights-on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights-on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase-shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.     

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light‐dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights‐off and declined around lights‐on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights‐on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights‐on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights‐on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase‐shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.     

Influence of constant light and darkness, light intensity, and light spectrum on plasma melatonin rhythms in senegal sole

Light is the most important synchronizer of melatonin rhythms in fish. This paper studies the influence of the characteristics of light on plasma melatonin rhythms in sole. The results revealed that under long-term exposure to constant light conditions (LL or DD), the total 24 h melatonin production was significantly higher than under LD, but LL and DD conditions influenced the rhythms differently. Under LL, melatonin remained at around 224 pg/ml throughout the 24 h, while under DD a significant elevation (363.6 pg/ml) was observed around the subjective evening. Exposure to 1 h light pulses at MD (mid-dark) inhibited melatonin production depending on light intensity (3.3, 5.3, 10.3, and 51.9 microW/cm(2)). The light threshold required to reduce nocturnal plasma melatonin to ML (mid-light) values was 5.3 microW/cm(2). Melatonin inhibition by light also depended on the wavelength of the light pulses: while a deep red light (lambda>600 nm) failed to reduce plasma melatonin significantly, far violet light (lambda(max)=368 nm) decreased indoleamine's concentration to ML values. These results suggest that dim light at night (e.g., moonlight) may be perceived and hence affect melatonin rhythms, encouraging synchronization to the lunar cycle. On the other hand, deep red light does not seem to inhibit nocturnal melatonin production, and so it may be used safely during sampling at night.     

Turkey retina and pineal gland differentially respond to constant environment

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.     

Regulation of melatonin production by light,darkness, and temperature in the trout pineal

Summary The pineal gland of the rainbow trout, Salmo gairdneri, when kept under in vitro perifusion culture conditions, displays a consistently elevated level of melatonin production in darkness (Gern and Greenhouse 1988). Upon light exposure melatonin production falls and stabilizes at a new lower level that is dependent upon the irradiance of the stimulus. To achieve the maximal response for each irradiance, the duration of the stimulus must exceed 30 min. The response amplitude is maximally sensitive to photons presented over durations of 30–45 min; is very insensitive to shorter light exposures; and is maintained with no evidence of adaptation over longer exposures. Temperature plays a role in regulation of melatonin production both in darkness and during light exposure; increased temperature increases melatonin production in darkness and also increases the sensitivity of the response to light. The action spectrum for the response is best fit by the Dartnall nomogram for a vitamin A₁ based rhodopsin with peak sensitivity near 500 nm. The possible adaptive significance of control of melatonin synthesis by light and temperature is considered.Abbreviations LD lightdark cycle - RIA radioimmunoassay - I ¹²⁵ Iodine - HIOMT hydroxyindole-O-methyltransferase     

The role of the pineal and the retinae in the expression of circadian locomotor rhythmicity in the ruin lizard,Podarcis sicula

Summary A marked interspecific variability in the role played by the pineal and the retinae characterizes the circadian system of lizards. I examined the role played by these structures in a new model species, the ruin lizard, Podarcis sicula. In constant temperature and darkness pinealectomy as well as bilateral removal of the retinae produced significant changes (both lengthening and shortening) in the freerunning period of locomotor rhythms. Circadian activity time was also affected by pinealectomy. Circadian locomotor rhythmicity persisted in all cases even when both operations were combined in the same individuals. This demonstrates in Podarcis sicula the existence of an oscillatory system outside the pineal and the retinae which can drive locomotor rhythms. The period changes recorded after pinealectomy as well as after bilateral removal of the retinae specifically suggest that both the pineal and the retinae play a modulating role on circadian oscillators located elsewhere in the system, with the final effect of stabilizing the overt rhythms.Abbreviations DD constant darkness - LL constant light - PIN-X pinealectomy - RET-X bilateral removal of the retinae - SHAM sham pinealectomy - circadian activity time - freerunning circadian period     

Presence of vasopressin, oxytocin and neurophysin in the retina of mammals, effect of light and darkness, comparison with the neuropeptide content of the neurohypophysis and the pineal gland

Arginine vasopressin (AVP) and oxytocin (OT) as well as their CNS carrier neurophysins (Np) have been found in the pineal gland. In view of the analogy between the pineal gland and the retina, the contents of these neuropeptides in rat, human and bovine retinae were determined. AVP, OT and Np were detected by specific radioimmunoassay (RIA) and their presence confirmed by RIA measurements (1) in rat and human retinae on HPLC fractions and (2) by the detection of the C-terminal portion of the precursor to AVP and its associated Np = propressophysin (CPP). The AVP and OT content in the retina of the rat was modified by light: AVP and OT content was smaller at 2 a.m. than at 2 p.m., but was increased by a 7 day constant exposure to darkness. In contrast, pituitary content was decreased after 7 days of constant darkness. If one optic nerve was cut we observed a decrease in retinal AVP content compared to the contralateral side and a decrease in pituitary AVP content. Our data clearly demonstrated the presence of AVP, OT and Np in the retina and their variation induced by light. It is probable that these peptides are of central origin.     

Circadian rhythms in the blowfly, Phormia terraenovae: the period in constant light

ABSTRACT. Locomotor activity of individual blowflies, Phormia (=Protophormia) terraenovae R.D. (Diptera, Calliphoridae), was recorded by means of running wheels made of Perspex (plexiglass). At various intervals after hatching, flies were placed in the wheel in continuous light (120 lx). The period of the free-running rhythm was often shorter than 24 h initially, but lengthened later and stabilized after 15–20 days at values around or above 25 h. The mean period was independent of age in both sexes. Within a range from 2 to 2000lx the period did not depend in a systematic way on intensity of illumination; no activity could be recorded below 1 lx.     

Photoperiodic modulation of circadian rhythms in the cricket Gryllus bimaculatus

The waveform and the free-running period of circadian rhythms in constant conditions are often modulated by preceding lighting conditions. We have examined the modulatory effect of variable length of light phase of a 24h light cycle on the ratio of activity (alpha) and rest phase (rho) as well as on the free-running period of the locomotor rhythm in the cricket Gryllus bimaculatus. When experienced the longer light phases, the alpha/rho-ratio was smaller and the free-running period was shorter. The magnitude of changes in alpha/rho-ratio was dependent on the number of cycles exposed, while the free-running period was changed by a single exposure, suggesting that there are separate regulatory mechanisms for the waveform and the free-running period. The neuronal activity of the optic lobe showed the alpha/rho-ratio changing with the preceding photoperiod. When different photoperiodic conditions were given to each of the two optic lobe pacemakers, the alpha/rho-ratio of a single pacemaker was rather intermediate between those of animals treated with either of the two conditions. These results suggest that the storage of the photoperiodic information occurs at least in part in the optic lobe pacemaker, and that the mutual interaction between the bilateral optic lobe pacemakers is involved in the photoperiodic modulation.     

Rhythmic melatonin secretion in different teleost species: an in vitro study

The rhythmic production of melatonin is governed by intrapineal oscillators in all fish species so far investigated except the rainbow trout. To determine whether the latter represents an exception among fish, we measured in vitro melatonin secretion in pineal organs of nine wild freshwater and six marine teleost species cultured at constant temperature and under different photic conditions. The results demonstrate that pineal organs of all species maintain a rhythmic secretion of melatonin under light:dark cycles and complete darkness, and strongly suggest that most fish possess endogenous intrapineal oscillators driving the rhythm of melatonin production, with the exception of the rainbow trout.Abbreviations LD light:dark - DD dark:dark - NAT N-acetyltransferase - RIA radioimmunoassay     

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.     

Effect of continuous darkness on circadian morphological rhythms in pinealocytes of the Chinese hamster,Cricetulus griseus

Summary Effects of a short-term exposure to continuous darkness on 24-h morphological variations in pinealocytes in the superficial pineal of the Chinese hamster (Cricetulus griseus) were examined. Pinealocytes contained type-1, -2 and -3 synaptic ribbons (SR), which had a central dense structure showing rod-like, various and ring-like profiles, respectively, and the quantity of each type of SR was expressed by SR index. 24-h changes in the type-1 and type-3 SR indices persisted in darkness and thus may be endogenous in nature. As under alternating light and dark (LD) conditions, the type-2 SR indices were almost constant over a 24-h period under continuous darkness, but the indices were larger in animals under darkness than in those under LD conditions. The 24-h variations in the nuclear and cytoplasmic volumes were abolished after exposing animals to darkness for 7 days, suggesting that these rhythms may be regulated exogenously. The amount of condensed chromatin exhibited a circadian change; this rhythm persisted under darkness. The results suggest that 24-h variations in the nuclear and cytoplasmic volumes in pinealocytes of the Chinese hamster are regulated by mechanisms different from those controlling the rhythms in SR and chromatin, and that the changes in the nuclear and cytoplasmic volumes and chromatin are related to the change in synthetic activity of pinealocytes.     

Ontogeny of the rhythmic melatonin production in a precocial and an altricial bird,the Japanese quail and the European starling

A distinct daily rhythm of melatonin production was found in the pineal gland of both precocial Japanese quail (Coturnix coturnix japonica) and altricial European starling (Sturnus vulgaris) during the first day of postembryonic life. Rhythmic melatonin production was reflected in a rhythmic profile in the general circulation. Significant day-night differences in melatonin content were also observed in the eyes of Japanese quail.The amplitude of the rhythm in the quail pineal gland increased steadily during the first two weeks of postem-bryonic life. A transient increase in maximum melatonin concentration was observed at the end of the first week of life in the plasma but not in the pineal gland of quail suggesting that a metabolizing pathway or a changed ocular contribution may influence the melatonin profile in the circulation and its availability to other tissues. There was no delay in the postembryonic development of melatonin rhythmicity in the altricial starling in comparison with the precocial quail. The amplitude of the plasma melatonin rhythm did not increase over the first week of life in starlings as it did in quail and the only significant increase was found between 6- and 17-day old starlings.In general, the development of the rhythm resulted from an increase of dark-time values. The day-time concentrations were low in all age groups of both species. A one-hour light pulse suppressed the high dark-time melatonin concentrations in 1-, 7- and 14-day old Japanese quail as well as in 7- and 14-day old European starlings. The manner in which the rhythm develops suggests that the circadian pacemaker(s) as well as the mechanisms of photoreception and entrainment are developed in hatchlings of both species in spite of their otherwise different developmental strategies.