A circadian oscillator in cultured cells of chicken pineal gland

The activity of serotonin N-acetyltransferase, the key enzyme of melatonin synthesis, shows a marked circadian rhythm in the pineal glands of various animal species. The regulation mechanism of the N-acetyltransferse rhythm in birds is different from that in mammals. N-Acetyltransferase activity in rat pineal gland is controlled by the central nervous system through the sympathetic nerves from the superior cervical ganglion, while in chicken the endogenous oscillator for N-acetyltransferase rhythm is presumably located in the pineal gland. Recently it has been shown that N-acetyltransferase activity oscillates in a circadian manner in the organ culture of chicken pineal glands. When chicken pineal glands were organ-cultured under continuous illumination, the nocturnal increase of enzyme activity was suppressed. These observations suggested that chicken pineal gland contains a circadian oscillator, a photoreceptor and melatonin-synthesising machinery. A central question arises whether the circadian oscillation of N-acetyltransferase activity and its response to environmental lighting are generated within the cell or are emergent properties of interaction between different types of pineal cells. I report here that in the dispersed cell culture of chicken pineal gland, N-acetyltransferase activity exhibits a circadian rhythm and responds to environmental lighting in the same manner as in the organ culture.     

Induction of photosensitivity by heterologous expression of melanopsin

Melanopsin has been proposed to be the photopigment of the intrinsically photosensitive retinal ganglion cells (ipRGCs); these photoreceptors of the mammalian eye drive circadian and pupillary adjustments through direct projections to the brain. Their action spectrum (lambda(max) approximately 480 nm) implicates an opsin and melanopsin is the only opsin known to exist in these cells. Melanopsin is required for ipRGC photosensitivity and for behavioural photoresponses that survive disrupted rod and cone function. Heterologously expressed melanopsin apparently binds retinaldehyde and mediates photic activation of G proteins. However, its amino-acid sequence differs from vertebrate photosensory opsins and some have suggested that melanopsin may be a photoisomerase, providing retinoid chromophore to an unidentified opsin. To determine whether melanopsin is a functional sensory photopigment, here we transiently expressed it in HEK293 cells that stably expressed TRPC3 channels. Light triggered a membrane depolarization in these cells and increased intracellular calcium. The light response resembled that of ipRGCs, with almost identical spectral sensitivity (lambda(max) approximately 479 nm). The phototransduction pathway included Gq or a related G protein, phospholipase C and TRPC3 channels. We conclude that mammalian melanopsin is a functional sensory photopigment, that it is the photopigment of ganglion-cell photoreceptors, and that these photoreceptors may use an invertebrate-like phototransduction cascade.     

Diurnal cycles in serotonin acetyltransferase activity and cyclic GMP content of cultured chick pineal glands

Levels of serotonin N-acetyltransferase (NAT: acetul CoA:arylamine N-acetyltransferase; EC 2.1.1.5.) activity in the chick pineal gland exhibit a marked diurnal variation in birds kept under a diurnal cycle of ilumination. Activity begins to rise rapidly at the start of the dark phase of the cycle and reaches maximum levels at mid-dark phase about 25-fold greater than the minimum basal level at mid-light phase. Thereafter, the level of activity declines to the basal level about the start of the light phase. This diurnal cycle in chick pineal NAT activity found in vivo has recently been reproduced in vitro with intact glands incubated in organ culture. The mechanism of the 'biological clock' which regulates these variations in level of chick pineal NAT activity is unknown. However, I now report that chick pineal glands cultured under a diurnal cycle of illumination exhibit a diurnal cycle in content of cyclic GMP which roughly parallels the cycles in NAT activity. In contrast, there was no correlation between variations in pineal content of cyclic AMP and in level of NAT activity.     

Research Progress on the Relationship among Circadian Rhythm,Melatonin and Aging

It is generally believed that aging is a gradual decline in the efficiency of our biological metabolism, which eventually leads to the deterioration of individual physiological function and the development of a series of age-related degenerative diseases.The circadian clock machinery orchestrates the normal metabolism of the organism in order to assure that individual growth,development and reproduction are adapted to the changes of diurnal environmental variations. The circadian rhythm in the elderly is attenuated with age and is accompanied by the onset of metabolic syndrome, the accumulation of genomic or epigenomic instability, the decline of metabolic tissue homeostasis and the change of natural feeding behavior. Existing results corroborate that light at night(LAN) and melatonin inhibition affect genomic integrity and normal metabolic function. In several animal models,LAN accelerated aging by inhibiting melatonin production in the pineal gland and promoting age-related carcinogenesis. This paper reviews the effects of the circadian rhythm on aging and discusses the complex relationship among circadian rhythms, melatonin and aging in different models of organisms, which may provide clues for prolonging human life and maintaining health.     

Cyclic GMP is involved in the excitation of invertebrate photoreceptors

The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by the second messenger, cyclic GMP. Injection of cGMP into rods or application of cGMP to inside-out membrane patches activates a conductance resembling that produced by light. Light produces a rapid reduction of cGMP in living rods, leading to closure of sodium channels and membrane hyperpolarization. In most invertebrate photoreceptors the response to light is depolarizing. We have investigated whether cGMP is involved in controlling the increase in sodium conductance that underlies this depolarization. We show here that injection of cGMP into Limulus photoreceptors produces a depolarization that mimics the receptor potential. We also show that the cGMP concentration of the squid retina increases rapidly during exposure to light. These results support the hypothesis that cGMP mediates the light-induced depolarization in invertebrate photoreceptors and suggests that vertebrate and invertebrate phototransduction may be more similar than previously thought.     

褪黑素在抑郁症中的研究

褪黑素(MT)是松果腺分泌的主要激素,对机体有着广泛的影响。抑郁症患者MT水平降低、生理节律紊乱。对褪黑素在抑郁症中的研究新进展进行了综述。     

4种变温动物松果腺复合体的形态结构比较

本文采用光镜和透射电镜，对日本七鳃鳗，黑斑蛙和丽斑麻蜥４种变动物的松果腺复合体，进行了观察和比较，并对其结构与功能的关系作了初步探讨，变温动物的松果腺复合体，由副松果体和松果体构成，在系统发育中，副松果体始终是光感受器，松果体则从光感觉器向着内分泌腺发展，从这４种动物松果腺复合体的变化揭示；最早脊椎动物的松果眼是２个，以后在动物从低等向高等进化中，其中一个保留在变温动物，始终是光感觉器，另一个变成     

Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes

The pineal gland has proven to be an excellent model for the study of adrenergic control systems. Noradrenaline, released from sympathetic nerve terminals in the pineal gland, regulates a large nocturnal increase in melatonin synthesis by stimulating the activity of arylalkylamine N-acetyltransferase (NAT, EC 2.3.1.87) 30-70-fold. An essential step in both the induction and maintenance of high NAT activity is an increase in intracellular cyclic AMP. Noradrenaline acts via beta-adrenoceptors to increase pineal cyclic AMP by activating adenylate cyclase, and the activation of pineal alpha 1-adrenoceptors potentiates beta-adrenergic stimulation not only of NAT but of both cyclic AMP and cyclic GMP. Here we describe investigations designed to test whether alpha 1-adrenergic potentiation of beta-adrenergic stimulation of pineal cyclic AMP involves protein kinase C. Our results suggest that kinase activation is involved and the data provide the first demonstration of a synergistic interaction between Ca2+-phospholipid-dependent protein kinase (protein kinase C) and neurotransmitter-dependent stimulation of cyclic AMP.     

Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision

Rod and cone photoreceptors detect light and relay this information through a multisynaptic pathway to the brain by means of retinal ganglion cells (RGCs). These retinal outputs support not only pattern vision but also non-image-forming (NIF) functions, which include circadian photoentrainment and pupillary light reflex (PLR). In mammals, NIF functions are mediated by rods, cones and the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs). Rod-cone photoreceptors and ipRGCs are complementary in signalling light intensity for NIF functions. The ipRGCs, in addition to being directly photosensitive, also receive synaptic input from rod-cone networks. To determine how the ipRGCs relay rod-cone light information for both image-forming and non-image-forming functions, we genetically ablated ipRGCs in mice. Here we show that animals lacking ipRGCs retain pattern vision but have deficits in both PLR and circadian photoentrainment that are more extensive than those observed in melanopsin knockouts. The defects in PLR and photoentrainment resemble those observed in animals that lack phototransduction in all three photoreceptor classes. These results indicate that light signals for irradiance detection are dissociated from pattern vision at the retinal ganglion cell level, and animals that cannot detect light for NIF functions are still capable of image formation.     

Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice

In the mammalian retina, besides the conventional rod-cone system, a melanopsin-associated photoreceptive system exists that conveys photic information for accessory visual functions such as pupillary light reflex and circadian photo-entrainment. On ablation of the melanopsin gene, retinal ganglion cells that normally express melanopsin are no longer intrinsically photosensitive. Furthermore, pupil reflex, light-induced phase delays of the circadian clock and period lengthening of the circadian rhythm in constant light are all partially impaired. Here, we investigated whether additional photoreceptive systems participate in these responses. Using mice lacking rods and cones, we measured the action spectrum for phase-shifting the circadian rhythm of locomotor behaviour. This spectrum matches that for the pupillary light reflex in mice of the same genotype, and that for the intrinsic photosensitivity of the melanopsin-expressing retinal ganglion cells. We have also generated mice lacking melanopsin coupled with disabled rod and cone phototransduction mechanisms. These animals have an intact retina but fail to show any significant pupil reflex, to entrain to light/dark cycles, and to show any masking response to light. Thus, the rod-cone and melanopsin systems together seem to provide all of the photic input for these accessory visual functions.     

Melanopsin signalling in mammalian iris and retina

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.     

持续黑暗状态下对大鼠松果体细胞分泌松果体素的昼夜节律研究

为研究体外培养的大鼠松果体细胞分泌的松果体素是否具有内在节律,收集体外培养的大鼠松果体细胞在白天6∶00,8∶00,10∶00,12∶00,15∶00,和夜晚18∶00,20∶00,22∶00,1∶00,4∶00各个时间点的培养上清,并用放射免疫方法(RIA)检测各个时间点松果体细胞培养上清中的松果体素水平.CCF(complex cosine function)统计分析结果表明,在持续黑暗的环境下,体外培养的大鼠松果体细胞分泌的松果体素在培养3,6,9天后仍然表现出内在节律.然而,这种内在节律在培养12天后消失.上述结果提示体外培养的大鼠松果体细胞可以作为一个有用的体外模型来分析节律系统的生理基础.     

Cyclic GMP increases photocurrent and light sensitivity of retinal cones

Like retinal rods, cone photoreceptors contain cyclic GMP and light-activated phosphodiesterase. The cGMP phosphodiesterase cascade is thought to mediate phototransduction in rods. Biochemical assays of nucleotide content in cone-dominant retinas, however, have failed to demonstrate light-induced changes in cGMP. Changes in cyclic AMP following light exposure have been reported, leading to the suggestion that in cone phototransduction cAMP assumes a role analogous to that played by cGMP in rods. Cyclic GMP introduced from tight-seal pipettes into isolated cones of the larval tiger salamander, Ambystoma tigrinum, rapidly increases light-modulated membrane current more than 10-fold. In cones, as in rods, cGMP also causes an approximately 10-fold increase in photocurrent duration and a 5- to 10-fold increase in light-sensitivity. Cyclic AMP has no effect on cone photocurrents under the same conditions. Because cGMP has similar effects on photocurrent magnitude and kinetics in both rods and cones, we conclude that cGMP plays corresponding roles in transduction in both vertebrate photoreceptor classes.     

The ELF3 zeitnehmer regulates light signalling to the circadian clock

The circadian system regulates 24-hour biological rhythms and seasonal rhythms, such as flowering. Long-day flowering plants like Arabidopsis thaliana, measure day length with a rhythm that is not reset at lights-off, whereas short-day plants measure night length on the basis of circadian rhythm of light sensitivity that is set from dusk, early flowering 3 (elf3) mutants of Arabidopsis are aphotoperiodic and exhibit light-conditional arrhythmias. Here we show that the elf3-7 mutant retains oscillator function in the light but blunts circadian gating of CAB gene activation, indicating that deregulated phototransduction may mask rhythmicity. Furthermore, elf3 mutations confer the resetting pattern of short-day photoperiodism, indicating that gating of phototransduction may control resetting. Temperature entrainment can bypass the requirement for normal ELF3 function for the oscillator and partially restore rhythmic CAB expression. Therefore, ELF3 specifically affects light input to the oscillator, similar to its function in gating CAB activation, allowing oscillator progression past a light-sensitive phase in the subjective evening. ELF3 provides experimental demonstration of the zeitnehmer ('time-taker') concept.     

大弹涂鱼松果体形态结构研究

鱼类松果体是传达外界信息到体内的主要传感器之一,其分泌的褪黑激素与鱼类生殖活动密切相关.为了阐明大弹涂鱼松果体形态结构与其功能的关系,运用组织细胞学方法研究了大弹涂鱼松果体的形态结构.结果表明,大弹涂鱼松果体位于端脑和中脑之间,由松果体柄和终囊两部分组成,具有背囊结构.松果体柄细长,细胞排列紧密,柄中有空腔,开口与第三脑室相通;松果体终囊膨大成梨形,且有空腔,腔内有皱褶,终囊顶部表面有黑色素细胞;背囊位于终囊下方,形状不规则,其囊壁向囊腔内反复折叠,形成许多复杂、盘旋曲折的内褶,背囊内部可见血细胞.研究结果为将来揭示大弹涂鱼半月周期产卵的机制提供了基础资料.     

Role of visual pigment properties in rod and cone phototransduction

Retinal rods and cones share a phototransduction pathway involving cyclic GMP. Cones are typically 100 times less photosensitive than rods and their response kinetics are several times faster, but the underlying mechanisms remain largely unknown. Almost all proteins involved in phototransduction have distinct rod and cone variants. Differences in properties between rod and cone pigments have been described, such as a 10-fold shorter lifetime of the meta-II state (active conformation) of cone pigment and its higher rate of spontaneous isomerization, but their contributions to the functional differences between rods and cones remain speculative. We have addressed this question by expressing human or salamander red cone pigment in Xenopus rods, and human rod pigment in Xenopus cones. Here we show that rod and cone pigments when present in the same cell produce light responses with identical amplification and kinetics, thereby ruling out any difference in their signalling properties. However, red cone pigment isomerizes spontaneously 10,000 times more frequently than rod pigment. This high spontaneous activity adapts the native cones even in darkness, making them less sensitive and kinetically faster than rods. Nevertheless, additional factors are probably involved in these differences.     

A Drosophila mutant defective in extracellular calcium-dependent photoreceptor deactivation and rapid desensitization

CALCIUM is involved in the adaptation of vertebrate photoreceptors to light and may have a similar role in invertebrate phototransduction. But the molecular mechanisms mediating this stimulus-dependent regulation are not well understood in any G protein-coupled transduction system. We have developed a preparation of isolated Drosophila photoreceptors that has allowed us to carry out an electrophysiological characterization of the light-activated response in these sensory neurons using patch-clamp techniques. We report here that extracellular calcium entering through the light-activated conductance is a key regulator of both the activation and deactivation phases of the phototransduction cascade, and that inaC mutant photoreceptors are specifically defective in the calcium-dependent deactivation mechanism. These data suggest that the light-dependent calcium influx inactivates this cascade through a biochemical pathway that requires the inaC gene product, and that this mechanism represents a molecular basis for stimulus-dependent regulation of visual transduction in Drosophila photoreceptors.     

Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane

The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance which appears to be the light-sensitive conductance involved in phototransduction. Recently, it has been found that this conductance is partially blocked by Mg2+ and Ca2+ at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches and for the unusually small apparent unit conductance deduced from noise measurements on intact cells. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of approximately 25 pS. Both individual channel openings (mean open time approximately 1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.     

Suppression by glutamate of cGMP-activated conductance in retinal bipolar cells.

Depolarizing bipolar cells (DBCs) of the retina are the only neurons in the vertebrate central nervous system known to be hyperpolarized by the neurotransmitter glutamate. Both glutamate and its analogue L-2-amino-4-phosphonobutyrate (APB) hyperpolarize DBCs by decreasing membrane conductance. Furthermore, glutamate responses in DBCs slowly decrease during whole-cell recording, suggesting that the response involves a second messenger system. Here we report that intracellular cyclic GMP or GTP activates a membrane conductance that is suppressed by APB, resulting in an enhanced APB response. In the presence of GTP-gamma-S, APB causes an irreversible suppression of the conductance. Inhibitors of G-protein activation or phosphodiesterase activity decrease the APB response. Thus, the DBC glutamate receptor seems to close ion channels by increasing the rate of cGMP hydrolysis by a G protein-mediated process that is strikingly similar to light transduction in photoreceptors.     

Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock

The segmented aspect of the vertebrate body plan first arises through the sequential formation of somites. The periodicity of somitogenesis is thought to be regulated by a molecular oscillator, the segmentation clock, which functions in presomitic mesoderm cells. This oscillator controls the periodic expression of 'cyclic genes', which are all related to the Notch pathway. The mechanism underlying this oscillator is not understood. Here we show that the protein product of the cyclic gene lunatic fringe (Lfng), which encodes a glycosyltransferase that can modify Notch activity, oscillates in the chick presomitic mesoderm. Overexpressing Lfng in the paraxial mesoderm abolishes the expression of cyclic genes including endogenous Lfng and leads to defects in segmentation. This effect on cyclic genes phenocopies inhibition of Notch signalling in the presomitic mesoderm. We therefore propose that Lfng establishes a negative feedback loop that implements periodic inhibition of Notch, which in turn controls the rhythmic expression of cyclic genes in the chick presomitic mesoderm. This feedback loop provides a molecular basis for the oscillator underlying the avian segmentation clock.