Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light-dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P-phosphate and 3H-glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24-h period. Our findings showed that in DD there was a daily oscillation in 32P-labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10-12). PRCs also exhibited an in vivo rhythm of 32P-phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H-glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H-glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H-glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.     

Circadian effects of light no brighter than moonlight

In mammals, light entrains endogenous circadian pacemakers by inducing daily phase shifts via a photoreceptor mechanism recently discovered in retinal ganglion cells. Light that is comparable in intensity to moonlight is generally ineffective at inducing phase shifts or suppressing melatonin secretion, which has prompted the view that circadian photic sensitivity has been titrated so that the central pacemaker is unaffected by natural nighttime illumination. However, the authors have shown in several different entrainment paradigms that completely dark nights are not functionally equivalent to dimly lit nights, even when nighttime illumination is below putative thresholds for the circadian visual system. The present studies extend these findings. Dim illumination is shown here to be neither a strong zeitgeber, consistent with published fluence response curves, nor a potentiator of other zeitgebers. Nevertheless, dim light markedly alters the behavior of the free-running circadian pacemaker. Syrian hamsters were released from entrained conditions into constant darkness or dim narrowband green illumination (~0.01 lx, 1.3 x 10(-9) W/cm(2), peak lambda = 560 nm). Relative to complete darkness, constant dim light lengthened the period by ~0.3 h and altered the waveform of circadian rhythmicity. Among animals transferred from long day lengths (14 L:10 D) into constant conditions, dim illumination increased the duration of the active phase (alpha) by ~3 h relative to complete darkness. Short day entrainment (8 L:16 D) produced initially long alpha that increased further under constant dim light but decreased under complete darkness. In contrast, dim light pulses 2 h or longer produced effects on circadian phase and melatonin secretion that were small in magnitude. Furthermore, the amplitude of phase resetting to bright light and nonphotic stimuli was similar against dimly lit and dark backgrounds, indicating that the former does not directly amplify circadian inputs. Dim illumination markedly alters circadian waveform through effects on alpha, suggesting that dim light influences the coupling between oscillators theorized to program the beginning and end of subjective night. Physiological mechanisms responsible for conveying dim light stimuli to the pacemaker and implications for chronotherapeutics warrant further study.     

Light-dependent Structural Change of Chicken Retinal Cryptochrome4

Animals have several classes of cryptochromes (CRYs), some of which function as core elements of circadian clockwork, circadian photoreceptors, and/or light-dependent magnetoreceptors. In addition to the circadian clock genes Cry1 and Cry2, nonmammalian vertebrates have the Cry4 gene, the molecular function of which remains unknown. Here we analyzed chicken CRY4 (cCRY4) expression in the retina with in situ hybridization and found that cCRY4 was likely transcribed in the visual pigment cells, cells in the inner nuclear layer, and retinal ganglion cells. We further developed several monoclonal antibodies to the carboxyl-terminal extension of cCRY4 and localized cCRY4 protein with immunohistochemistry. Consistent with the results of in situ hybridization, cCRY4 immunoreactivity was found in visual pigment cells and cells located at the inner nuclear layer and the retinal ganglion cell layer. Among the antibodies, one termed C1-mAb had its epitope within the carboxyl-terminal 14-amino acid sequence (QLTRDDADDPMEMK) and associated with cCRY4 in the retinal soluble fraction more strongly in the dark than under blue light conditions. Immunoprecipitation experiments under various light conditions indicated that cCRY4 from the immunocomplex formed in the dark dissociated from C1-mAb during blue light illumination as weak as 25 μW/cm² and that the release occurred with not only blue but also near UV light. These results suggest that cCRY4 reversibly changes its structure within the carboxyl-terminal region in a light-dependent manner and operates as a photoreceptor or magnetoreceptor with short wavelength sensitivity in the retina.     

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.     

强光照和全黑暗条件下荒漠沙蜥视网膜内GAP-43表达的免疫组织化学

采用免疫组织化学技术研究了在强光照和全黑暗条件下荒漠沙蜥(Phrynocephalus prezewalskic)视网膜内生长相关蛋白GAP-43的表达变化。结果表明,在正常光照条件下,视网膜内GAP-43阳性表达部位主要存在于内网层;强光照条件下,GAP-43免疫染色部位主要出现在内网层、节细胞层和内核层的部分细胞核。在全黑暗条件下,在视纤维层和内网层呈阳性染色;提示视网膜在不同环境条件下GAP-43的不同定位,可能与其在相应的环境下参与不同的视觉功能有关。     

Circadian rhythms in the green sunfish retina

We investigated the occurrence of circadian rhythms in retinomotor movements and retinal sensitivity in the green sunfish, Lepomis cyanellus. When green sunfish were kept in constant darkness, cone photoreceptors exhibited circadian retinomotor movements; rod photoreceptors and retinal pigment epithelium (RPE) pigment granules did not. Cones elongated during subjective night and contracted during subjective day. These results corroborate those of Burnside and Ackland (1984. Investigative Ophthalmology and Visual Science. 25:539-545). Electroretinograms (ERGs) recorded in constant darkness in response to dim flashes (lambda = 640 nm) exhibited a greater amplitude during subjective night than during subjective day. The nighttime increase in the ERG amplitude corresponded to a 3-10-fold increase in retinal sensitivity. The rhythmic changes in the ERG amplitude continued in constant darkness with a period of approximately 24 h, which indicates that the rhythm is generated by a circadian oscillator. The spectral sensitivity of the ERG recorded in constant darkness suggests that cones contribute to retinal responses during both day and night. Thus, the elongation of cone myoids during the night does not abolish the response of the cones. To examine the role of retinal efferents in generating retinal circadian rhythms, we cut the optic nerve. This procedure did not abolish the rhythms of retinomotor movement or of the ERG amplitude, but it did reduce the magnitude of the nighttime phases of both rhythms. Our results suggest that more than one endogenous oscillator regulates the retinal circadian rhythms in green sunfish. Circadian signals controlling the rhythms may be either generated within the eye or transferred to the eye via a humoral pathway.     

Rapid kinetic labeling of Arabidopsis cell suspension cultures: implications for models of lipid export from plastids

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.     

Darkness during early postnatal development is required for normal circadian patterns in the adult rat

Early light experience influences the brain during development. Perinatal light exposure has an important effect on the development of the circadian system, although the role of quantity versus quality of light in this process is still unclear. We tested the development of the circadian rhythm of locomotor activity under constant bright light from the day of weaning, of six groups of rats raised under different light conditions during suckling. Results indicated that when rats received daily darkness during suckling (rats reared under constant darkness or light-dark cycles with dim or bright light) became arrhythmic when exposed to continuous bright light after weaning. However, those rats reared in the absence of darkness (constant dim or bright light, or alternating dim and bright light) developed a circadian rhythm, which was stronger and had a shorter period depending on the quantity of light received during suckling. Vasointestinal polypeptide immunoreactivity in the suprachiasmatic nucleus (SCN) was higher in those rats with weaker rhythms. However, no apparent differences among these groups were found in the melanopsin-expressing retinal ganglion cells, which provide the SCN with light input in the photoentrainment process. When bright light was shifted to dim light in three of the groups on day 57 after weaning, all of them generated a circadian rhythm with a longer period in those rats previously arrhythmic. Our results indicate the importance of the amount of light received at the early stages of life in the development of the circadian system and suggest that darkness is needed for the normal development of circadian behaviour.     

Circadian rhythms in screening pigment and invaginating organelles in photoreceptor terminals of the housefly's first optic neuropile

Screening pigment granules occur in the synaptic terminals of photoreceptors in the fly's (Musca domestica, L.) compound eye. The granules resemble ommochrome granules in the overlying photoreceptor cell body. There are also two types of invagination into receptor terminals: capitate projections (from glial cells) and invaginations from neighboring receptor terminals. The number of profiles of these organelles in the first optic neuropile, the lamina, have been counted using single-section quantitative electron microscopic methods. Pigment granules are concentrated proximally in the terminal, toward the brain. The numbers change, increasing during the night (1 h after lights off) up to values more than twice the number 1 h after lights on, apparently by longitudinal migration of granules from the cell body into the terminal. Flies entrained to day/night conditions and then held under constant darkness continue to exhibit changes in the numbers of profiles. Even though overall there were 80–90% fewer granule profiles than under day/night conditions, the numbers attained a peak many times higher at the end of the subjective day. Thus, the changes are endogenous, showing circadian rhythmicity. Although their significance is unknown, these changes parallel previously described circadian rhythms in the receptor terminals and their lamina monopolar-cell targets. The invaginations from receptor terminals were more numerous under day/night conditions than under constant darkness, and cycled in constant darkness, peaking at the end of subjective night. Capitate projections, by contrast, failed to change significantly under the experimental conditions analyzed, a lack of responsiveness they share with photoreceptor tetrad synapses. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 517–529, 1997     

Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina

Light-induced photoreceptor cell degeneration has been studied in several species, but not extensively in the teleost fish. Furthermore, the continual production of rods and cones throughout the teleost's life may result in regeneration of lost rods and cones. We exposed adult albino zebrafish to 7 days of constant darkness, followed by 7 days of constant 8000 lux light, followed by 28 days of recovery in a 14-h light:10-h dark cycle. We characterized the resulting photoreceptor layer cell death and subsequent regeneration using immunohistochemistry and light microscopy. Within the first 24 h of constant light, the zebrafish retina exhibited widespread rod and cone cell apoptosis. High levels of cell proliferation within the inner nuclear layer (INL) were observed within the first 3 days of constant light, as assessed by immunodetection of proliferating cell nuclear antigen and BrdU labeling. The proliferating cells within the INL were closely associated with the radial processes of Müller glia, similar to the pluripotent retinal stem cells observed during embryonic development. Using antibodies generated against the individual zebrafish opsins, we determined that rods and the green, blue, and ultraviolet cone cells were replaced within the 28 day recovery period. While both rods and cones were replaced, the well-ordered cone cell mosaic was not reestablished.     

CGRP modulates mucin synthesis in surface mucus cells of rat gastric oxyntic mucosa

We examined the effects of the calcitonin gene-related peptide (CGRP), including the possible participation of nitric oxide (NO), on mucin biosynthesis in the surface epithelium and remaining deep mucosa as well as the entire mucosa and compared the distribution of CGRP and NO synthase (NOS) using a combination of double immunofluorescence labeling and multiple dye filter. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing [(3)H]glucosamine and CGRP, with or without the NOS inhibitor. CGRP dose-dependently enhanced [(3)H]glucosamine incorporation into the corpus mucin but had no effect on antral mucin biosynthesis. The CGRP receptor antagonist, CGRP-(8-37), prevented the increase in (3)H-labeled corpus mucin. This stimulation of corpus mucin synthesis disappeared after removal of the surface mucus cell layer. CGRP activated the mucin biosynthesis in the surface mucus cells. In the full-thickness corpus mucosa, CGRP-induced activation was completely blocked by the NOS inhibitor. CGRP-immunoreactive fibers were intertwined within the surface mucus cell layer with type I NOS immunoreactivity. These results show that CGRP-stimulated mucin biosynthesis mediated by NO is limited to surface mucus cells of the rat gastric oxyntic mucosa.     

Diurnal and Circadian Light-Harvesting Complex and Quinone B-Binding Protein Synthesis in Leaves of Tomato (Lycopersicon esculentum)

In leaves of tomato (Lycopersicon esculentum), the synthesis of a light-harvesting complex (LHC) polypeptide of photosystem II and the quinone B (Q_B)-binding protein varies at different time points during the day. In vivo labeling with [³⁵S]methionine revealed diurnal oscillations of synthesis of these thylakoid membrane proteins. Both proteins are synthesized at elevated levels right after the transition from darkness to light, a maximum is reached around noon, and decreasing levels were measured during the afternoon and night. In addition, in constant darkness both proteins were also synthesized to varied extents at different diurnal time points. Together, these results indicate that the synthesis of a LHC II and the Q_B-binding protein is under the control of the circadian clock. This circadian oscillation of LHC II protein synthesis correlates with the very well documented circadian Lhc a/b mRNA accumulation.     

Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Spatial and temporal expression patterns of GDNF family receptor alpha4 in the developing chicken retina

GDNF family receptor alpha (GFRalpha) receptors are involved in the regulation of different aspects of embryonic development such as proliferation, migration, differentiation and survival. To determine the possible role of GFRalpha4 in retinal development, we analysed its expression in the developing chicken retina. We found that GFRalpha4 is temporally co-expressed with c-ret. Both, the temporal and spatial expression of GFRalpha4 is developmentally regulated during retinogenesis and is first detected in cells of the ganglion cell layer at E6. As development of the retina proceeds, the expression of GFRalpha4 extends to cells of the inner half of the inner nuclear layer and to cells of the outermost cell row of the inner nuclear layer. Later on, GFRalpha4 expression is also found in additional cells of the outer half of the inner nuclear layer and in a subpopulation of photoreceptors. A central-to-peripheral gradient of retinal differentiation is evident, as the onset of GFRalpha4 expression is first detectable in the central retina, while it is delayed by two days in its periphery.     

Circadian rhythm of behavioural responsiveness to carbon dioxide in the blood-sucking bug Triatoma infestans (Heteroptera: Reduviidae)

The temporal modulation of the behavioural response to carbon dioxide and its chronobiological basis were investigated in larvae of Triatoma infestans. We analysed the orientation towards CO(2) of insects kept under three different illumination regimes: (1) 12 h light/12 h darkness cycles (L/D), (2) constant darkness (D/D) and (3) constant light (L/L). When maintained under L/D conditions, insects exhibited an oriented response towards airstreams added with 1500 ppm of CO(2) during the first hours of the scotophase only. Bugs maintained under D/D also showed a positive orientation response towards CO(2) during the first hours of the subjective night, while bugs kept under L/L did not show a rhythmic oriented behaviour. Thus, T. infestans displayed a daily rhythm of orientation towards CO(2) (i.e. a potential food source) only at the beginning of the scotophase. The persistence of the rhythm under constant darkness reveals the existence of an endogenous circadian control of this behaviour.     

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. We found that flies overexpressing the pacemaker gene per or the morgue gene are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF--the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness--is dispensable for rhythmicity in constant light. Molecular circadian rhythms are only found in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light.