Rhodopsin 5- and Rhodopsin 6-mediated clock synchronization in Drosophila melanogaster is independent of retinal phospholipase C-β signaling

Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.     

Modeling the role of mid-wavelength cones in circadian responses to light

Nonvisual responses to light, such as photic entrainment of the circadian clock, involve intrinsically light-sensitive melanopsin-expressing ganglion cells as well as rod and cone photoreceptors. However, previous studies have been unable to demonstrate a specific contribution of cones in the photic control of circadian responses to light. Using a mouse model that specifically lacks mid-wavelength (MW) cones we show that these photoreceptors play a significant role in light entrainment and in phase shifting of the circadian oscillator. The contribution of MW cones is mainly observed for light exposures of short duration and toward the longer wavelength region of the spectrum, consistent with the known properties of this opsin. Modeling the contributions of the various photoreceptors stresses the importance of considering the particular spectral, temporal, and irradiance response domains of the photopigments when assessing their role and contribution in circadian responses to light.     

Photoperiod perception in the blind mole rat (Spalax ehrenbergi, Nehring): involvement of the Harderian gland, atrophied eyes, and melatonin

The involvement of the Harderian gland, atrophied eyes, and melatonin in the perception of photoperiodic changes has been studied in the mole rat, a fossorial blind mammal the thermoregulatory capacity of which is photoperiod-dependent. When transferred from a long photoperiod to a short one, mole rats increase their resistance to cold, a perfectly reversible phenomenon. After 2 weeks under short photoperiod the thermoregulatory capacities of animals without Harderian glands are less than those of the controls. The Harderian gland appears thus to be implicated in the detection of photoperiodic changes. After 5 weeks, however, the Harderianectomized animals had perfectly integrated the photoperiodic change, so demonstrating that other photoreceptor organs exist. The atrophied eyes, which, under these conditions, do not seem to play an important role, are involved when the animals are transferred from short photoperiod to long photoperiod. Melatonin, but not 5-methoxytryptamine, appears to be a crucial compound in such a phenomenon. These results, which demonstrate that in mammals (at least in the mole rat, as in nonmammalian vertebrates), nonocular photoreceptors exist, suggest that the mechanism by which mammals integrate photoperiodic changes is not the same when the animals are transferred from long to short photoperiod as when transferred from short photoperiod to long photoperiod.     

What makes the Arabidopsis clock tick on time? A review on entrainment

Entrainment, the synchronization of a circadian clock with the external environment, is a crucial step in daily life. Although many signals contribute to entrainment, light and temperature are typically the strongest resetting cues. Much progress has been made concerning light resetting in the model plant Arabidopsis thaliana. Multiple photoreceptors (phytochromes, cryptochromes, LOV-domain proteins) are involved in light perception. The clock genes CCA1, LHY and TOC1 are all probable targets of light signalling, although the details of these pathways are not completely established. Temperature can entrain the clock, but little is known about the mechanism underlying this resetting; no obvious clock gene candidate for temperature resetting has been identified. Although circadian research has emphasized oscillations in free-running conditions, in the real world the circadian clock is entrained. During entrainment, short or long period mutants exhibit a 24-h period, but a mutant phenotype is often manifested as an altered phase relationship with the entraining cycle; short and long period mutants show leading and lagging phases, respectively, and this may be detrimental under some conditions. Arrhythmic CCA1-overexpressing plants display increased lethality under very short photoperiods, consistent with the circadian clock being of adaptive significance to life on a rotating world.     

Induction of reproductive diapause via perception of photoperiod through the compound eyes in the adult blow fly, Protophormia terraenovae

Covering and surgical removal of the compound eyes were performed to localize photoreceptors for photoperiodic responses in the blow fly Protophormia terraenovae (Diptera, Calliphoridae). Adult females showed a long-day photoperiodic response to control reproductive diapause. When the compound eyes were bilaterally covered with silver paint, diapause incidence increased under diapause-preventing conditions, i.e., a long-day photoperiod and constant light, as though flies were kept under constant darkness. Neither silver painting on a medial region of the head capsule nor control painting in which both compound eyes were painted in a clear solvent caused significant effects on diapause incidence. Unilateral painting of the compound eye caused an increment of diapause incidence under constant light but no effects under a long-day photoperiod. When the compound eyes were bilaterally removed, all the flies developed their ovaries both under a long-day and a short-day photoperiod. Unilateral removal of the compound eye also caused ovarian development under a short-day photoperiod, whereas removal of one antennal lobe or all ocelli caused no effects on diapause incidence. Since P. terraenovae completely lost responsiveness to photoperiod after blinding of the compound eyes, it is likely that this fly perceives photoperiod through its compound eyes. Accepted: 18 February 1997     

The regulation of circadian clocks by light in fruitflies and mice.

A circadian clock has no survival value unless biological time is adjusted (entrained) to local time and, for most organisms, the profound changes in the light environment provide the local time signal (zeitgeber). Over 24 h, the amount of light, its spectral composition and its direction change in a systematic way. In theory, all of these features could be used for entrainment, but each would be subject to considerable variation or 'noise'. Despite this high degree of environmental noise, entrained organisms show remarkable precision in their daily activities. Thus, the photosensory task of entrainment is likely to be very complex, but fundamentally similar for all organisms. To test this hypothesis we compare the photoreceptors that mediate entrainment in both flies and mice, and assess their degree of convergence. Although superficially different, both organisms use specialized (employing novel photopigments) and complex (using multiple photopigments) photoreceptor mechanisms. We conclude that this multiplicity of photic inputs, in highly divergent organisms, must relate to the complex sensory task of using light as a zeitgeber.     

Circadian modulation of light-induced locomotion responses in Drosophila melanogaster

The relationship between light and the circadian system has long been a matter of discussion. Many studies have focused on entrainment of light with the internal biological clock. Light also functions as an environmental stimulus that affects the physiology and behaviour of animals directly. In this study, we used light as an unexpected stimulus for flies at different circadian times. We found that wildtype flies showed circadian changes in light-induced locomotion responses. Elevation of locomotor activity by light occurred during the subjective night, and performance in response to light pulses declined to trough during the subjective day. Moreover, arrhythmic mutants lost the rhythm of locomotion responses to light, with promotion of activity by light in timeless(01)mutants and inhibition of activity by light in Clock(ar)mutants. However, neither ablation of central oscillators nor disturbance of the functional clock inside compound eyes was sufficient to disrupt the rhythm of light responses. We show that, compound eyes, which have been identified as the control point for normal masking (promotion of activity by light), are sufficient but not necessary for paradoxical masking (suppression of activity by light) under high light intensity. This, taken together with the clear difference of light responses in wildtype flies, suggests that two different masking mechanisms may underlie the circadian modulation of light-induced locomotion responses.     

Light sensitivity of the photoperiodic response system in higher vertebrates: wavelength and intensity effects

Most species use daily light in one way or the other in regulation of their short and/or long term activities. Light is perceived by pigment(s) present in the retinal (RP) and/or extra-retinal photoreceptors (ERPs). ERPs may be located at various sites in the body but in non-mammalian vertebrates they are found predominantly in the pineal body and hypothalamic region of the brain, Light radiations directly penetrate brain tissues to reach and stimulate the hypothalamic (deep-brain) photoreceptors. How does light information finally reach to the clock is not fully understood in many vertebrate groups? In mammals, however, the light information from the retina to the clock (the hypothalamic suprachiasmatic nuclei, SCN) is relayed through the retino-hypothalamic tract (RHT) which originates from the retinal ganglion cells, and through the geniculo-hypothalamic tract (GHT) which originates from the photically responsive cells of a portion of the lateral geniculate nucleus (LGN), called the intergeniculate leaflet (IGL). A response to light (the photoperiodic response) is the result of the interpretation of light information by the photoperiodic system. Apart from the duration, the animals use the gradual shifts in the intensity and wavelength of daily light to regulate their photoperiodic clock system. The wavelengths to which photoreceptors are maximally sensitive or the wavelengths which have greater access to the photoreceptors can induce a maximal response. There can also be differential effects of wavelength and intensity of light on circadian process(es) involved in the entrainment and induction of the photoperiodic clock. This may have some adaptive implications. Entrainment to daily light-dark (LD) cycle may be achieved at dawn or dusk, depending whether the animal is day- or night-active, when there is relatively low intensity of light. By contrast, photoperiodic induction in many species occurs during long days of spring and summer when plenty of daylight at higher intensity is available later in the day.     

Existence of an extra-retinal and extra-pineal photoreceptive organ that regulates photoperiodism in gonadal development of an Osmerid teleost, ayu (Plecoglossus altivelis)

Ayu (Plecoglossus altivelis) is an Osmerid teleost whose gonadal development exhibits clear photoperiodism: it is stimulated and prevented under short and long photoperiod, respectively. However, the photoreceptor organ involved in this process remains to be identified. In the present study, we examined whether gonads of ophthalmectomized (Ex) and pinealectomized (Px) ayu respond to short photoperiod to test whether photoreceptors other than lateral eyes and pineal complex are involved in the photoperiodic response of gonadal development. Gonadosomatic index (GSI) and plasma levels of sex steroids (testosterone and 11-ketotestosterone for males and testosterone and estradiol 17-beta for females) were significantly increased in the Ex+Px ayu kept under short photoperiod in both males and females as compared with the initial control. On the other hand, there were no significant increases in GSI and sex steroids in the Ex+Px ayu kept under long photoperiod. Histological observation of gonads in the Ex+Px ayu revealed that oocytes undergoing final maturation in females and proliferation of germ cells in males were observed under short photoperiod but not under long photoperiod. These results indicate that extra-retinal and extra-pineal photoreceptive organ regulates photoperiodic gonadal development in this species.     

BRAIN PHOTORECEPTOR PATHWAYS CONTRIBUTING TO CIRCADIAN RHYTHMICITY IN CRAYFISH

Freshwater crayfish have three known photoreceptive systems: the compound eyes, extraretinal brain photoreceptors, and caudal photoreceptors. The primary goal of the work described here was to explore the contribution of the brain photoreceptors to circadian locomotory activity and define some of the underlying neural pathways. Immunocytochemical studies of the brain photoreceptors in the parastacid (southern hemisphere) crayfish Cherax destructor reveal their expression of the blue light-sensitive photopigment cryptochrome and the neurotransmitter histamine. The brain photoreceptors project to two small protocerebral neuropils, the brain photoreceptor neuropils (BPNs), where they terminate among fibers expressing the neuropeptide pigment-dispersing hormone (PDH), a signaling molecule in arthropod circadian systems. Comparable pathways are also described in the astacid (northern hemisphere) crayfish Procambarus clarkii. Despite exhibiting markedly different diurnal locomotor activity rhythms, removal of the compound eyes and caudal photoreceptors in both C. destructor and P. clarkii (leaving the brain photoreceptors intact) does not abolish the normal light/dark activity cycle in either species, nor prevent the entrainment of their activity cycles to phase shifts of the light/dark period. These results suggest, therefore, that crayfish brain photoreceptors are sufficient for the entrainment of locomotor activity rhythms to photic stimuli, and that they can act in the absence of the compound eyes and caudal photoreceptors. We also demonstrate that the intensity of PDH expression in the BPNs varies in phase with the locomotor activity rhythm of both crayfish species. Together, these findings suggest that the brain photoreceptor cells can function as extraretinal circadian photoreceptors and that the BPN represents part of an entrainment pathway synchronizing locomotor activity to environmental light/dark cycles, and implicating the neuropeptide PDH in these functions. (Author correspondence: bbeltz@wellesley.edu)     

Tau differences between short-day responsive and short-day nonresponsive white-footed mice (Peromyscus leucopus) do not affect reproductive photoresponsiveness

In laboratory-bred rodent populations, intraspecific variation in circadian system organization is a known cause of individual variation in reproductive photoresponsiveness. The authors sought to determine whether circadian system variation accounted for individual variation in reproductive photoresponsiveness in a single, highly genetically variable population of Peromyscus leucopus recently derived from the wild. Running-wheel activity patterns of male and female mice, aged 70 to 90 days, from artificially selected lines of reproductively photoresponsive (R) and nonresponsive (NR) lines were monitored under short-day photoperiod (8 h light, 16 h dark), long-day photoperiod (16 h light, 8 h dark), and constant darkness (DD). NR mice displayed a significantly longer mean free-running period (24.08 h) in DD compared with R mice (23.75 h), due in large part to a difference between NR and R females (24.25 h vs. 23.74 h, respectively). All other entrainment characteristics (alpha, phase angle of activity) under short days, long days, and DD were similar between R and NR mice. Variation in free-running period and entrainment characteristics has been shown to affect photoresponsiveness in other rodent species by altering the manner in which the circadian system interprets short days. To determine whether variation in photoresponsiveness in P. leucopus is due to differences in free-running period instead of variation downstream from the central circadian clock in the pathway controlling photoresponsiveness, the authors exposed young R and NR mice to DD and measured the effect on reproductive organ development. If variation in free-running period affected how the circadian system of mice interpreted short days, then both R and NR mice exposed to DD should have exhibited a delay in gonadal development. Only R mice exhibited pubertal delay in DD. NR mice exhibited large paired testes, paired seminal vesicles, paired ovaries, and uterine weight typical of mice nonresponsive to short days, whereas R mice exhibited reproductive organ weight typical of mice responsive to short days. These data suggest that despite significant differences in free-running period between R and NR mice, individual variation in photoresponsiveness is not due to differences in how the circadian systems of R and NR mice interpret the LD cycle.     

Distribution of photoperiodic receptors in the compound eyes of the bean bug, Riptortus clavatus

  The bean bug, Riptortus clavatus shows a long-day photoperiodic response with respect to the control of adult diapause. The location of photoreceptors for photoperiodism was examined in this species by complete or partial removal of photoreceptor organs. Even after one compound eye or both ocelli were removed, the insects were sensitive to photoperiod. After both compound eyes were removed, however, the insects became reproductive regardless of the photoperiod. Therefore, photoreceptors for photoperiodism were not in the ocelli but in the compound eyes. To clarify whether ommatidia in compound eyes have a regional difference in reception of photoperiod, sensitivity to photoperiod was examined after one compound eye and a part of the contralateral one were removed. Only when the central region of compound eyes was removed did the insects lose sensitivity to photoperiod. It is concluded that the ommatidia in the central region of compound eyes play a principal role in the reception of photoperiod. Accepted: 23 September 1996     

Influence of the corticosterone rhythm on photic entrainment of locomotor activity in rats

The question of involvement of glucocorticoid hormones as temporal signals for the synchronization of the timekeeping system was addressed in rats with different corticosterone status. The authors showed that adrenalectomy had no effects on the synchronization of wheel-running activity rhythms to a steady-state LD 12:12 cycle, regardless of whether it was compensated for by a corticosterone replacement therapy that either reinstated constant plasma concentrations of the hormone or mimicked its natural rhythm. However, after a 12-h phase shift (daylight reversal), the lack of circulating corticosterone induced a significant shortening of the resynchronization rate (less than 3 days vs. 7 days). Normalization required restoration of a rhythmic corticosterone secretion that was synchronized to the new photoperiod. Under constant darkness, the corticosterone rhythm did not show any synchronizing effect, providing evidence that it participates in entrainment of the locomotor activity rhythm through modulation of light effects. It is proposed that, under stable lighting conditions, circulating glucocorticoids contribute to stabilizing activity rhythms by reinforcing resistance of the circadian timing system to variations of the photoperiod. Experimental evidence that serotonergic neurons are involved in relaying their modulatory effects to the clock is also presented.     

Circadian and photoperiodic time measurement in male Syrian hamsters following lesions of the melatonin-binding sites of the paraventricular thalamus.

Autoradiographic studies using [125I]iodomelatonin in several species, including the Syrian hamster, have revealed that the rostral region of the anterior paraventricular nucleus of the thalamus (aPVT) contains a very high density of binding sites for melatonin. In two studies, small or large bilateral electrolytic lesions of the aPVT were made in adult male hamsters maintained on long days (LD 16:8). The hamsters were then transferred to short days (LD 8:16) to test whether testicular regression could occur in response to a decrease in photoperiod. Serum prolactin concentrations were measured as a second photoperiodic response. All unoperated control hamsters showed the typical short-day photoperiodic response: A decrease in serum luteinizing hormone (LH) and prolactin concentrations and testicular regression all occurred within 6 weeks in short days, followed by the development of scotorefractoriness. Lesions of the aPVT did not significantly affect the rate or the degree of the short-day-induced decline in serum levels of LH or prolactin, nor the pattern of testicular regression and the subsequent expression of refractoriness. To enable us to determine whether the aPVT might be involved in the entrainment or the expression of circadian rhythms, locomotor activity was monitored continuously in lesioned and control groups in Experiment 2, prior to and following the switch to short days. The reduction in photoperiod (involving an 8-hr advance in the time of lights-off and an 8-hr extension of the dark phase) caused a decompression of the nocturnal activity bout of control animals, so that after 2 weeks in short days, activity onset had also advanced to regain its phase relationship to the timing of lights-off. A similar pattern of reentrainment was observed in lesioned animals, and no differences were observed between treatment groups in the rate of entrainment and decompression. In addition, both intact controls and animals bearing large bilateral lesions of the aPVT exhibited robust free-running circadian rhythms of locomotor activity when held under constant dim red light. In summary, the integrity of the aPVT is not necessary for the seasonal response of the reproductive axis and prolactin secretion to photoperiod, nor for photic entrainment of activity rhythms, in the Syrian hamster.     

Circadian photoreception in Drosophila: functions of cryptochrome in peripheral and central clocks.

In Drosophila melanogaster, disruption of night by even short light exposures results in degradation of the clock protein TIMELESS (TIM), leading to shifts in the fly molecular and behavioral rhythms. Several lines of evidence indicate that light entrainment of the brain clock involves the blue-light photoreceptor cryptochrome (CRY). In cryptochrome-depleted Drosophila (cry(b)), the entrainment of the brain clock by short light pulses is impaired but the clock is still entrainable by light-dark cycles, probably due to light input from the visual system. Whether cryptochrome and visual transduction pathways play a role in entrainment of noninnervated, directly photosensitive peripheral clocks is not known and the subject of this study. The authors monitored levels of the clock protein TIM in the lateral neurons (LNs) of larval brains and in the renal Malpighian tubules (MTs) of flies mutant for the cryptochrome gene (cry(b)) and in mutants that lack signaling from the visual photopigments (norpA(P41)). In cry(b) flies, light applied during the dark period failed to induce degradation of TIM both in MTs and in LNs, yet attenuated cycling of TIM was observed in both tissues in LD. This cycling was abolished in LNs, but persisted in MTs, of norpA(P41);cry(b) double mutants. Furthermore, the activity of the tim gene in the MTs of cry(b) flies, reported by luciferase, seemed stimulated by lights-on and suppressed by lights-off, suggesting that the absence of functional cryptochrome uncovered an additional light-sensitive pathway synchronizing the expression of TIM in this tissue. In constant darkness, cycling of TIM was abolished in MTs; however, it persisted in LNs of cry(b) flies. The authors conclude that cryptochrome is involved in TIM-mediated entrainment of both central LN and peripheral MT clocks. Cryptochrome is also an indispensable component of the endogenous clock mechanism in the examined peripheral tissue, but not in the brain. Thus, although neural and epithelial cells share the core clock mechanism, some clock components and light-entrainment pathways appear to have tissue-specific roles.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Photoperiod differentially modulates photic and nonphotic phase response curves of hamsters

Circadian pacemakers respond to light pulses with phase adjustments that allow for daily synchronization to 24-h light-dark cycles. In Syrian hamsters, Mesocricetus auratus, light-induced phase shifts are larger after entrainment to short daylengths (e.g., 10 h light:14 h dark) vs. long daylengths (e.g., 14 h light:10 h dark). The present study assessed whether photoperiodic modulation of phase resetting magnitude extends to nonphotic perturbations of the circadian rhythm and, if so, whether the relationship parallels that of photic responses. Male Syrian hamsters, entrained for 31 days to either short or long daylengths, were transferred to novel wheel running cages for 2 h at times spanning the entire circadian cycle. Phase shifts induced by this stimulus varied with the circadian time of exposure, but the amplitude of the resulting phase response curve was not markedly influenced by photoperiod. Previously reported photoperiodic effects on photic phase resetting were verified under the current paradigm using 15-min light pulses. Photoperiodic modulation of phase resetting magnitude is input specific and may reflect alterations in the transmission of photic stimuli.     

Photoperiod and temperature interaction in the determination of reproduction of the edible snail, Helix pomatia

Snails were kept in self-cleaning housing chambers in an artificially controlled environment. Mating was frequent under long days (18 h light) and rare under short days (8 h light) regardless of whether the snails were kept at 15 degrees C or 20 degrees C. An interaction between photoperiod and temperature was observed for egg laying. The number of eggs laid (45-50/snail) and the frequency of egg laying (90-130%) were greater in long than in short days (16-35/snail and 27-77%) but a temperature of 20 degrees C redressed, to some extent, the inhibitory effect of short days. At both temperatures only long photoperiods brought about cyclic reproduction over a period of 16 weeks, confirming the synchronizing role of photoperiod on the neuroendocrine control of egg laying in this species of snail.