Circadian rhythms in Limulus photoreceptors. II. Quantum bumps

The light response of the lateral eye of the horseshoe crab, Limulus polyphemus, increases at night, while the frequency of spontaneous discrete fluctuations of its photoreceptor membrane potential (quantum bumps) decreases. These changes are controlled by a circadian clock in the brain, which transmits activity to the eye via efferent optic nerve fibers (Barlow, R. B., S. J. Bolanski, and M. L Brachman. 1977. Science. 197:86-89). Here we report the results of experiments in which we recorded from single Limulus photoreceptors in vivo for several days and studied in detail changes in their physiological and membrane properties. We found that: (a) The shape of (voltage) quantum bumps changes with the time of day. At night, spontaneous bumps and bumps evoked by dim light are prolonged. The return of the membrane potential to its resting level is delayed, but the rise time of the bump is unaffected. On average, the area under a bump is 2.4 times greater at night than during the day. (b) The rate of spontaneous bumps decreases at night by roughly a factor of 3, but their amplitude distribution remains unchanged. (c) The resting potential and resistance of the photoreceptor membrane do not change with the time of day. (d) the relationship between injected current and impulse rate of the second order neuron, the eccentric cell, also remains unchanged with the time of day. Thus the efferent input from the brain to the retina modulates some of the membrane properties of photoreceptor cells. Our findings suggest that the efferent input acts on ionic channels in the membrane to increase the sensitivity of the photoreceptor to light.     

Pineal photoreceptor cells are required for maintaining the circadian rhythms of behavioral visual sensitivity in zebrafish

In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry)] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity.     

Cellular analysis of ocular circadian pacemaker coupling in Bulla: role of efferent impulses in phase shifting

The eyes of Bulla gouldiana, a marine snail, contain circadian oscillators that are coupled to each other. Obvious candidates for the coupling signals are the optic nerve compound action potentials (CAPs) that express the circadian rhythm and lead to efferent impulses in the contralateral optic nerve. In the present experiments, the role of the CAPs as coupling signals was evaluated. We found that, following desynchronization of the two ocular oscillators by phase-delaying one eye with manganese, subsequent phase shifts in the initially unshifted ocular rhythm only occurred during the time that efferent optic nerve signals were present. In addition, in the absence of ocular desynchrony, phase shifts of the ocular rhythm could still be effected by activation of the efferent pathway. The influence of efferent impulses on identified retinal cells was also evaluated. No effect of efferent signals on receptor layer cells was detected, while it was found that efferent impulses generated depolarizations in basal retinal neurons (BRNs), the putative circadian oscillator cells. Depolarization of the BRNs has been shown previously to be involved in the light entrainment pathway. Depolarization appears to be similarly involved in the coupling pathway, since membrane depolarizations that mimicked the efferent-induced postsynaptic potentials likewise generated phase shifts of the ocular rhythm.     

The eccentric cell of theLimulus lateral eye: encoder of circadian changes in visual responses

Efferent fibers from a central circadian clock innervate both photoreceptor cells and second-order neurons (eccentric cells) in the lateral compound eye ofLimulus, and release octopamine when activated. We have used intracellular microelectrodes to study the modulation of eccentric cell function by efferent optic-nerve activity, octopamine agonists, and a K⁺-channel blocker, TEA.

Efferent control of temporal response properties of the Limulus lateral eye

The sensitivity of the Limulus lateral eye exhibits a pronounced circadian rhythm. At night a circadian oscillator in the brain activates efferent fibers in the optic nerve, inducing multiple changes in the physiological and anatomical characteristics of retinal cells. These changes increase the sensitivity of the retina by about five orders of magnitude. We investigated whether this increase in retinal sensitivity is accompanied by changes in the ability of the retina to process temporal information. We measured the frequency transfer characteristic (FTC) of single receptors (ommatidia) by recording the response of their optic nerve fibers to sinusoidally modulated light. We first measured the FTC in the less sensitive daytime state and then after converting the retina to the more sensitive nighttime state by electrical stimulation of the efferent fibers. The activation of these fibers shifted the peak of the FTC to lower frequencies and reduced the slope of the low-frequency limb. These changes reduce the eye's ability to detect rapid changes in light intensity but enhance its ability to detect dim flashes of light. Apparently Limulus sacrifices temporal resolution for increased visual sensitivity at night.     

Photic sensitivity ranges of hamster pupillary and circadian phase responses do not overlap

Mammalian retinal photoreceptors form an irradiance detection system that drives many nonvisual responses to light such as pupil reflex and resetting of the circadian clock. To understand the role of pupil size in circadian light responses, pupil diameter was pharmacologically manipulated and the effect on behavioral phase shifts at different irradiance levels was studied in the Syrian hamster. Dose-response curves for steady-state pupil size and for behavioral phase shifts were constructed for 3 pupil conditions (dilated, constricted, and control). Retinal irradiance was calculated from corneal irradiance, pupil size, retinal surface area, and absorption of ocular media. The sensitivity of photic responses to retinal irradiance is approximately 1.5 log units higher than to corneal irradiance. When plotted against corneal irradiance, pharmacological pupil constriction reduces the light sensitivity of the circadian system, but pupil dilation has no effect. As expected, when plotted against retinal irradiance all dose-response curves superimposed, confirming that the circadian system responds to photon flux on the retina. Pupil dilation does not increase the circadian response to increasing irradiance, since the response of the circadian system attains saturation at irradiance levels lower than those required to induce pupil constriction. The main finding shows that due to the different response sensitivities, the effect of pupil constriction on the light sensitivity of the circadian system in the hamster under natural conditions is virtually negligible. We further suggest the existence of distinct modulating mechanisms for the differential retinal irradiance sensitivity of the pupil system and the circadian system, which enables the different responses to be tuned to their specific tasks while using similar photoreceptive input.     

Proper function of the Drosophila trp gene product during pupal development is important for normal visual transduction in the adult

The response of invertebrate photoreceptors consists of the summation of quantum bumps, each representing the response to a single photon. The bumps adapt depending on the intensity of the stimulus: their average size is relatively large in dim light and small in bright light. The rate of occurrence of the bumps varies proportionally with light intensity. In the Drosophila mutant trp, unlike in the wild type, the rate does not increase with increasing light intensity and the bumps do not adapt. Here we report an analysis of the trp gene and its expression in normal and mutant flies. Our results suggest that the trp protein is a novel photoreceptor membrane-associated protein, that this protein is not required for the occurrence of bumps but is necessary for adaptation, and that proper function of the trp gene product during pupal development is important for normal visual transduction in the adult.     

Efferent neurotransmission of circadian rhythms inLimulus lateral eye

Summary We investigated efferent neurotransmission in theLimulus lateral eye by studying the action of pharmacological agents on responses of photoreceptor cells in vitro. We recorded transmembrane potentials from single cells in slices of retina that were excised during the day and maintained for several days in a culture medium. Potentials recorded in the absence of pharmacological agents resemble those recorded from cells in vivo during the day.Octopamine, a putative efferent neurotransmitter, induced changes in photoreceptor potentials that mimicked in part those generated at night by a circadian clock located in the brain. Specifically, octopamine (100 to 500 M) decreased the frequency of occurrence of quantum bumps in the dark and increased the amplitude of photoreceptor responses to intermediate and high light intensities. Similar actions were produced by naphazoline (25 to 100 (M, potent agonist of octopamine), forskolin (8 to 400 M, activator of adenylate cyclase), IBMX (1 mM, inhibitor of phosphodiesterase), and 8-bromo-cAMP (500 M, analogue of cAMP). 8-bromo-cGMP (500 M, analogue of cGMP) decreased the rate of spontaneous quantum bumps only.Our results support the hypothesis that (1) octopamine is an efferent neurotransmitter of circadian rhythms in theLimulus eye and that (2) it activates adenylate cyclase to increase levels of the second messenger, cAMP, in photoreceptor cells. Circadian changes in photoreceptor responses to moderate intensities may be a specific action of cAMP, since cGMP has no effect. Circadian changes in the rate of spontaneous quantum bumps may involve a less specific intermediate, since both cAMP and cGMP reduce bump rate. Characteristics of the retinal slice preparation precluded a detailed study of the effects of pharmacological agents on retinal morphology.Abbreviations cAMP adenosine 35-cyclic monophosphate - IBMX 3-isobutyl-1-methyl xanthine - LPF large potential fluctuation - SPF small potential fluctuation     

Photoperiodism and rhythmic response to light

Abstract. Seedlings of Pharhitis nil show a circadian rhythm in the capacity to flower in response to the timing of a second red light pulse given at various times after a first saturating exposure to red when this is given together with a benzyladeninc spray. There are also changes in the photon irradiance required for half maximum response to the second red pulse. The photochemical properties of phytochrome in the photoperiodically sensitive cotyledons were also shown to change rhythmically. Oscillations in both p_r→ P_fr and P_fr→ P_r photoconversion characteristics persisted over at least two circadian cycles with a periodicity of about 12 h. There were, however, no significant oscillations in either P_fr peak absorbance or in Δ(ΔA). The changes in sensitivity for the photoconversion of P_r→ P_fr did not parallel the much larger changes in sensitivity of the flowering response to red light. The amplitude of the P_r→ P_fr rhythm was at least as great as that for P_r→ P_fr, but the flowering response to far-red light was not rhythmic, nor was there any large change in sensitivity. The changes in photoconversion properties may reflect a basic biochemical oscillation which affects both photoreceptor properties and sensitivity to photoreceptor input. There was also a marked rhythm in the P_fr/P ratio that would be established by a saturating pulse of red light and this too may have affected the flowering response to such a pulse. Far-red light inhibited flowering when given at any time during the inductive night. After 14 h in darkness, P_fr could still be measured in the cotyledons and it was concluded that far-red light inhibited flowering by removing P_fr As red light also inhibited flowering at this time, there may be two pools of phytochrome with different kinetic properties.     

Properties of visual cells in the lateral eye of Limulus in situ: intracellular recordings

Two types of potential fluctuations, large and small, recorded intracellularly from photoreceptors in the dark-adapted Limulus eye in situ underlie the dual properties of the impulse discharge of the optic nerve fibers. The small potential fluctuations (SPFs)--the well-known quantum bumps--were normally less than 20 mV in amplitude. The large potential fluctuations (LPFs) were up to 80 mV in amplitude. LPFs appear to be regenerative events triggered by SPFs that enable single photon absorptions in retinular cells to fire off nerve impulses in the eccentric cell. In the dark, SPFs and LPFs occur spontaneously. At low light intensities, LPFs are the major components of the receptor potential. At high intensities, LPFs are suppressed and SPFs become the major components. SPFs and LPFs together enable single photoreceptor cells to encode approximately a 9-log unit range of light intensity. Excising the eye from the animal or cutting off its blood supply generally abolishes LPFs and thereby reduces the range of light intensity coded in the optic nerve discharge.     

Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates. It is hypothesized that the rhabdomeric photoreceptor cell had evolved to be the photosensitive retinal ganglion cell for the vertebrate circadian photoentrainment [2, 3 and 4] owing to the fact that some molecules involved in cell differentiation are common among them [5]. We focused on the cephalochordate amphioxus because it is the closest living invertebrate to the vertebrates, and interestingly, it has rhabdomeric photoreceptor cells for putative nonvisual functions [6]. Here, we show that the amphioxus homolog of melanopsin [7, 8 and 9], the circadian photopigment in the photosensitive retinal ganglion cells of vertebrates, is expressed in the rhabdomeric photoreceptor cells of the amphioxus and that its biochemical and photochemical properties, not just its primary structure, are considerably similar to those of the visual rhodopsins in the rhabdomeric photoreceptor cells of higher invertebrates. The cephalochordate rhabdomeric photoreceptor represents an evolutionary link between the invertebrate visual photoreceptor and the vertebrate circadian photoreceptor.     

Photic regulation of pineal function. Analogies between retinal and pineal photoreception

Light absorbed by a photopigment in a photoreceptor cell causes a photochemical reaction converting the 11-cis retinal chromophore into the all-trans configuration. These changes lead to a series of events that causes cGMP hydrolysis, a following decrease of cGMP in the cytoplasm of the photoreceptor outer segment and a closure of cGMP-gated cationic channels. As a consequence of these processes the membrane hyperpolarizes. In pineal photoreceptor cells of lower vertebrates these processes are only partly investigated. Molecules involved in the phototransduction process and the desensitization, like opsin, vitamin A, α-transducin and arrestin, have been immunocytochemically localized in pineal photoreceptors and also electrophysiological studies have shown that phototransduction mechanisms in pineal photoreceptors might be very similar to those found in retinal photoreceptors. This review will summarize some of the current knowledge on pineal photoreception and compare it with retinal processes.     

哺乳动物昼夜节律调节的神经基础——昼夜光感受器

哺乳动物昼夜光感受器为一组具有直接感光功能的特殊视网膜神经节细胞 ,其基本感光色素为黑视素 .昼夜光感受器具有直接、广谱和稳定感受昼夜光变化的功能特点 .昼夜光感受器的功能是通过导引作用 ,使下丘脑视交叉上核内的昼夜节律活动与外界明 暗周期变化同步     

A "melanopic" spectral efficiency function predicts the sensitivity of melanopsin photoreceptors to polychromatic lights

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A-based photopigment (nomogram) with a peak sensitivity (λ(max)) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin's sensitivity to broad-spectrum, including "white," lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity (rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term "melanopic") relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.     

The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18-30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.     

Protective gene expression changes elicited by an inherited defect in photoreceptor structure

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.     

Light adaptation in Drosophila photoreceptors: I. Response dynamics and signaling efficiency at 25 degrees C

Besides the physical limits imposed on photon absorption, the coprocessing of visual information by the phototransduction cascade and photoreceptor membrane determines the fidelity of photoreceptor signaling. We investigated the response dynamics and signaling efficiency of Drosophila photoreceptors to natural-like fluctuating light contrast stimulation and intracellular current injection when the cells were adapted over a 4-log unit light intensity range at 25 degrees C. This dual stimulation allowed us to characterize how an increase in the mean light intensity causes the phototransduction cascade and photoreceptor membrane to produce larger, faster and increasingly accurate voltage responses to a given contrast. Using signal and noise analysis, this appears to be associated with an increased summation of smaller and faster elementary responses (i.e., bumps), whose latency distribution stays relatively unchanged at different mean light intensity levels. As the phototransduction cascade increases, the size and speed of the signals (light current) at higher adapting backgrounds and, in conjunction with the photoreceptor membrane, reduces the light-induced voltage noise, and the photoreceptor signal-to-noise ratio improves and extends to a higher bandwidth. Because the voltage responses to light contrasts are much slower than those evoked by current injection, the photoreceptor membrane does not limit the speed of the phototransduction cascade, but it does filter the associated high frequency noise. The photoreceptor information capacity increases with light adaptation and starts to saturate at approximately 200 bits/s as the speed of the chemical reactions inside a fixed number of transduction units, possibly microvilli, is approaching its maximum.     

Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors

The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.     

CELL JUNCTIONS IN OMMATIDIA OF LIMULUS

The intercellular relationships in the ommatidia of the lateral eye of Limulus have been investigated. The distal process of the eccentric cell gives origin to microvilli which interdigitate with the microvilli of the retinular cells. Therefore, both types of visual cells contribute to form the rhabdom and may have an analogous photoreceptor function. Quintuple-layered junctions are found within the rhabdom at the lines of demarcation between adjoining microvilli, whether the microvilli originate from a single retinular cell, from two adjacent retinular cells, or from a retinular cell and the eccentric cell. Furthermore, quintuple-layered junctions between the eccentric cell and the tips of the microvilli of the retinular cells occur at the boundary between the distal process and the rhabdom. These findings are interpreted to indicate that the rhabdom provides an extensive electrotonic junction relating retinular cells to one another and to the eccentric cell. Quintuple-layered junctions between glial and visual cells, as well as other structural features of the ommatidial cells, are also described.