Limulus Eye Research Articles

Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes.

The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.

Opsin expression in Limulus eyes: a UV opsin is expressed in each eye type and co-expressed with a visible light-sensitive opsin in ventral larval eyes.

The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors.

Circadian regulation of Limulus visual functions: A role for octopamine and cAMP

Abstract The purpose of this contribution is to review our current understanding of the source and biochemistry of the circadian efferent input to the eyes of the American horseshoe crab Limulus polyphemus and the impact of this input on the structure, physiology and biochemistry of Limulus eyes. Special emphasis is given to the role of the biogenic amine octopamine and biochemical cascades it activates in the eyes. In addition to reviewing published data, we present new data showing that octopamine elevates cAMP levels in Limulus lateral eyes, and we partially characterize the pharmacology of the receptors involved in this response. We also present new data showing that octopamine regulates gene expression in Limulus lateral eyes by activating a cAMP cascade.

In memoriam Robert B. Barlow, Jr

It is with great sadness that we mark the loss of Dr Robert (Bob) Barlow, a pioneering investigator into the neural basis of visual behavior. Bob had a passion for vision research that was vibrant and engaging. He would surely have remained scientifically active and productive for years to come,

Isolation and expression of Pax6 and atonal homologues in the American horseshoe crab, Limulus polyphemus

Pax6 regulates eye development in many animals. In addition, Pax6 activates atonal transcription factors in both invertebrate and vertebrate eyes. Here, we investigate the roles of Pax6 and atonal during embryonic development of Limulus polyphemus rudimentary lateral, medial and ventral eyes, and the initiation of lateral ommatidial eye and medial ocelli formation. Limulus eye development is of particular interest because these animals hold a unique position in arthropod phylogeny and possess multiple eye types. Furthermore, the molecular underpinnings of eye development have yet to be investigated in chelicerates. We characterized a Limulus Pax6 gene, with multiple splice products and predicted protein isoforms, and one atonal homologue. Unexpectedly, neither gene is expressed in the developing eye types examined, although both genes are present in the lateral sense organ, a structure of unknown function.

Circadian efferent input to Limulus eyes: anatomy, circuitry, and impact.

Much is known about the anatomy of Limulus retinal efferent neurons and the structural and functional consequences of their activation. Retinal efferent axons arise from cell bodies located in the cheliceral ganglia of the brain, and they project out all of the optic nerves. Their unique neurosecretory-like terminals contact all cell types in lateral eye ommatidia, the retinular cells of the median eye, and the internal rhabdom of ventral photoreceptors. Lateral and median rudimentary photoreceptors are also innervated. The activity of the efferents is circadian. They are active during the subjective night and inactive during the subjective day. Activation of the efferents drives dramatic and diverse changes in the structure and function of Limulus eyes and causes the sensitivity and responsiveness of the eyes to light to increase at night. Relatively little is known about the molecular mechanisms that produce these structural and functional changes, but one efferent-activated biochemical cascade has been identified. The biogenic amine octopamine is released from efferent terminals, and an octopamine-stimulated rise in cAMP in photoreceptors, with a subsequent activation of cAMP-dependent protein kinase, mediates many of the known effects of efferent input. A photoreceptor-specific protein, myosin III, is phosphorylated in response to efferent input; this protein may play a role in the efferent stimulated changes in photoreceptor structure and function. Anatomical, biophysical, biochemical, and molecular approaches are now being effectively combined in studies of Limulus eyes; thus, this preparation should be particularly useful for further detailed investigations of mechanisms underlying the modulation of primary sensory cells by efferent input.

Multiple mechanisms of rhabdom shedding in the lateral eye of Limulus polyphemus.

Rhabdom shedding in horseshoe crab lateral eye photoreceptors was studied with anti-opsin and anti-arrestin immunocytochemistry. Two, possibly three, distinct shedding mechanisms were revealed in animals maintained in natural lighting. Transient rhabdom shedding, triggered by dawn, is a brief, synchronous event that removes up to 10% of the rhabdom membrane. Whorls of rhabdomeral membrane break into vesicles and form compact multivesicular bodies. These debris particles are immunoreactive for opsin and are of a relatively uniform size, averaging approximately 2 microm(2) in area. Transient shedding requires that input from circadian efferent fibers to the retina precedes the light trigger, and cutting the optic nerve blocks efferent input and transient shedding. Light-driven rhabdom shedding is a progressive process. Rhabdomeral membrane is removed by coated vesicles that accumulate into loosely packed multivesicular bodies. These debris particles label with antibodies directed against opsin, arrestin, and adaptin, and they have a large distribution of sizes, averaging almost 6 microm(2) in area and ranging up to 25 microm(2) or more. The amount of rhabdomeral membrane removed by light-driven shedding has seasonal variation and depends on latitude. Light-driven shedding does not require circadian efferent input. A possible third shedding mechanism, light-independent shedding, is observed when transient shedding is blocked either by 48 hours of darkness or by cutting the optic nerve. Small particles, averaging 1.8 microm(2) in area, exhibiting opsin but not arrestin immunoreactivity can then be found in the cytoplasm surrounding the rhabdom. The nature of light-independent shedding is not yet clear.

Evaluation of Circadian Rhythms in the Limulus Eye.

The visual system of the horseshoe crab, Limulus polyphemus, shows a remarkable circadian rhythm in sensitivity. About the time of sunset, a circadian oscillator in the brain of the animal generates efferent optic nerve signals to the lateral eye modulating both its structure and function. The overall increase in sensitivity nearly compensates for the reduction in ambient light at night (1, 2). High nighttime visual sensitivity appears to be important for mating, a visually guided behavior that the animals can accomplish as well at night as in the day (3). The mechanisms underlying the elegant adaptation of the Limulus eye to dim nighttime illumination include anatomical changes in the retina that increase photon catch at the expense of spatial resolution, and physiological changes that enhance the summation of photon events at the expense of temporal resolution. Here we evaluate further the effect of the circadian clock on two

Inhibition of the calcineurin-like protein phosphatase activity in Limulus ventral eye photoreceptor cells alters the characteristics of the spontaneous quantal bumps and the light-mediated inward currents, and enhances arrestin phosphorylation.

Changes in intracellular calcium are involved in phototransduction processes in both vertebrate and invertebrate photoreceptors. During this phototransduction process in the Limulus ventral eye, there is a biochemical change in the protein phosphatase, calcineurin, such that it becomes capable of activation by calcium and calmodulin. Here we show that the calcium/calmodulin-dependent calcineurin-like activity in light-adapted ventral eye was completely inhibited by the CaN autoinhibitory peptide, CaN A457-482 and the Merck analog of the membrane-permeable, immunosuppressant drug, FK 506, L-683, 590, but not an inactive analogue, L-685, 818. Whole-cell, voltage-clamp recordings of spontaneous quantal bump activity present in dark-adapted photoreceptors injected with either CaN A457-482 (500 microM) or superfused with L-683, 590 (20 microM) or L-685, 818 revealed that both CaN A457-482 and L-683, 590, but not L-685, 818, caused rapid decreases in quantal bump amplitude, rise time and fall time, resulting in smaller, sharper bumps. This was correlated with enhanced phosphorylation of arrestin in light-adapted ventral eye photoreceptors exposed to L-683, 590 or less reliably okadaic acid. Both CaN A457-482 and L-683, 590 markedly affected the light-stimulated inward currents recorded from light-adapted ventral photoreceptors, causing a "terracing" of the inward current, and an intensity-dependent delay in the time required to reach peak amplitude. Consequently, inhibition of calcineurin markedly affects two major rhodopsin-dependent electrophysiological processes, and implicates CaN as an integral component in the phototransduction cascade.

Cell-based model of the Limulus lateral eye.

We present a cell-based model of the Limulus lateral eye that computes the eye's input to the brain in response to any specified scene. Based on the results of extensive physiological studies, the model simulates the optical sampling of visual space by the array of retinal receptors (ommatidia), the transduction of light into receptor potentials, the integration of excitatory and inhibitory signals into generator potentials, and the conversion of generator potentials into trains of optic nerve impulses. By simulating these processes at the cellular level, model ommatidia can reproduce response variability resulting from noise inherent in the stimulus and the eye itself, and they can adapt to changes in light intensity over a wide operating range. Programmed with these realistic properties, the model eye computes the simultaneous activity of its ensemble of optic nerve fibers, allowing us to explore the retinal code that mediates the visually guided behavior of the animal in its natural habitat. We assess the accuracy of model predictions by comparing the response recorded from a single optic nerve fiber to that computed by the model for the corresponding receptor. Correlation coefficients between recorded and computed responses were typically >95% under laboratory conditions. Parametric analyses of the model together with optic nerve recordings show that animal-to-animal variation in the optical and neural properties of the eye do not alter significantly its response to objects having the size and speed of horseshoe crabs. The eye appears robustly designed for encoding behaviorally important visual stimuli. Simulations with the cell-based model provide insights about the design of the Limulus eye and its encoding of the animal's visual world.

A Myosin III fromLimulusEyes Is a Clock-Regulated Phosphoprotein

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca2+; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.

Histamine: putative transmitter for lateral inhibition in Limulus eye.

Previous articleNext article No AccessVISIONHistamine: Putative Transmitter for Lateral Inhibition in Limulus EyeK. M. Stewart, D. M. Porcello, M. E. McSweeney, T. Saito, C. L. Passaglia, F. A. Dodge, and R. B. BarlowK. M. Stewart Search for more articles by this author , D. M. Porcello Search for more articles by this author , M. E. McSweeney Search for more articles by this author , T. Saito Search for more articles by this author , C. L. Passaglia Search for more articles by this author , F. A. Dodge Search for more articles by this author , and R. B. Barlow Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 193, Number 2October 1997 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv193n2p203 Views: 12Total views on this site Copyright © 1997 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.

Dawn, diacylglycerol, calcium, and protein kinase C — the retinal wrecking crew. A signal transduction cascade for rhabdom shedding in the Limulus eye

Vertebrate and invertebrate photoreceptors shed their photosensitive membrane on a daily basis. Although we have detailed knowledge of the morphology of the disc shedding and renewal process in vertebrate photoreceptors, and of the turnover of rhabdom in invertebrate photoreceptors, we know relatively little about the molecular mechanisms whereby these processes are triggered by light and/or by circadian efferent input to the retina. We have used the horseshoe crab, Limulus polyphemus, as a model system to unravel the molecular means by which the trigger light is communicated to the intracellular machinery responsible for the daily breakdown of the photosensitive membrane. Phorbol esters, potent and specific activators of protein kinase C (PKC), induce a robust burst of rhabdom shedding when injected subretinally into the compound lateral eye of Limulus. This occurs in the absence of the light trigger normally required to initiate shedding in the lateral eye at dawn, suggesting that PKC may play a role in the light triggering of rhabdom shedding. Diacylglycerol (DAG) analogf were also found to elicit rhabdom shedding in the lateral eye without a light trigger, but an uncharacteristically high concentrations. However, injecting inositol trisphosphate (InsP 3) and DAG analog simultaneously results in a tenfold decrease in the concentration of DAG analog required to initiate a shedding event. Immunohistochemical screening for PKC in the lateral eye shows that two isozymes (PKC βII and PKC ξ) are co-localized to the retinular cell rhabdom. Taken together, these data suggest that light triggers rhabdom shedding at dawn via a classical Ca 2+-sensitive PKC, similar to PKC βII, which is activated synergistically by the light-evoked production of DAG and InsP 3.

Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes

In rhabdomeral photoreceptors, light stimulates the phosphorylation of arrestin, a protein critical for quenching the photoresponse, by activating a calcium/calmodulin-dependent protein kinase (CaM PK). Here we present biochemical evidence that a CaM PK that phosphorylates arrestin in Limulus eyes is structurally similar to mammalian CaM PK II. In addition, cDNAs encoding proteins homologous to mammalian and Drosophila CaM PK II in the catalytic and regulatory domains were cloned and sequenced from a Limulus lateral eye cDNA library. The Limulus sequences are unique, however, in that they lack most of the association domain. The proteins encoded by these sequences may phosphorylate arrestin.

Flutter-like response in visual cortex of the semi-isolated turtle brain.

activity of nearby receptors in the eye that see an object becomes correlated (5). The lateral eye is highly sensitive to the flickering light from the overhead waves. A large component of the light reflected from grey targets centers at -2-4 Hz which is the peak of the temporal transfer function of the eye (Fig. 1B; 6, 7, 8). Phototransduction mechanisms set the underlying shape of the transfer function, which two inhibitory processes then sharpen and amplify (6, 7). As a result, the power spectrum of the spike train recorded from receptors viewing the grey target grows considerably in the range of 2-4 Hz (Fig. 1 B). In this paper we show that a consideration of the natural environment of an animal can lead to a better understanding of its visual system. The Limulus eye appears to be adapted to a particular feature of its environment-the flickering light reflected off the carapace of a potential mate. These light signals help males detect a female irrespective of the contrast of her carapace. Frequency tuning of vision is not unique to horseshoe crabs and has been observed in many animals, such as cats (9) and humans (10). Perhaps their lighting environment also deserves a closer look. Supported by NSF grant BNS9309539 and NIH grants MH4974 1 and EY00667.

The Neural Network of the Limulus Retina: From Computer to Behavior

SYNOPSIS. The visual system of the horseshoe crab, Limulus polyphemus, provides an excellent opportunity for studying the neural basis of behavior. Quantitative analysis of the animal's visual behavior is now possible as is theoretical analysis of information processing in its retina. We combine these theoretical and behavioral approaches to investigate the nature of the signals the eye transmits to the brain for the animal to see. Over the years theoretical studies of the Limulus eye were restricted by the limited capabilities of single processor digital computers. However, a breakthrough in technology with the advent of parallel computers greatly enhances the analysis of large neural networks such as that of the retina. We have developed a time-dependent model of the Limulus retina on the Connection Machine (Model CM-2), which is a massively parallel computer containing 32,768 processors. The model represents a matrix of 64 × 128 receptors and simulates interactions among receptors with digital filters and transduction and adaptation within a receptor by a multistage cascade. Neural response patterns computed with the Connection Machine model replicate to a first approximation the patterns of neural activity recorded in the laboratory. Behavioral studies of Limulus vision carried out in the field can be simulated on the Connection Machine. Neural responses recorded from behaving animals serve to test the accuracy of the model. Thus far we have developed just one model of the retina, but it eventually will have two forms, “daytime” and “nighttime,” to account for the known circadian rhythms in retinal function. With a combination of field, physiological, and theoretical studies, we hope to gain a better understanding of the neural mechanisms that underlie the animal's visually-guided behavior.

Visual cells in excised Limulus eyes: 2. Excision does not reduce sensitivity to light

Excision of the lateral eye of Limulus is believed to compromise it by greatly reducing its sensitivity to light stimuli. This belief derives from an experiment in which excised eyes were studied in the daytime and intact eyes were studied at night. But since a circadian elevation of nighttime sensitivity occurs in this eye, the belief is based on a confounded experiment in which like was not compared with like. In the present paper, like is compared with like; light sensitivity is assessed by comparing the intensity-response curves of receptor potentials that were recorded intracellularly from excised and from intact eyes. These data clearly indicate that the excised eye is not less sensitive than the intact eye. They further indicate that this situation exists for periods of time that exceed 5 h after excision, which means that this preparation provides ample time to replicate behaviorally interesting experiments while recording from a single neuron.

Visual cells in excised Limulus eyes: Dark adaptation reveals evidence of response duality

The role of light adaptation on response duality was examined in excised eyes taken from mature Limulus and maintained in ordinary seawater for up to 10 h. Several manifestations of duality were found. Two types of quantum fluctuations exist in dark adaptation. Two segments were found in the intensity-response function characterizing the plateau phase of responses in the dark-adapted state. And this intensity-response function covered a wide range of intensities in dark adaptation. Light adaptation reversibly abolished all three of these expressions of response duality. Thus, despite repeated claims that response duality is a property that degenerates after excision, these data show that it is an adaptation-dependent property that can readily be seen when care is taken to allow enough time for dark adaptation.

Identification and function of octopamine and tyramine conjugates in the Limulus visual system.

Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as gamma-glutamyl octopamine and gamma-glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine-rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of gamma-glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that gamma-glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that gamma-glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.

Dynamic relationship between the slow potential and spikes in cockroach ocellar neurons.

The relationship between the slow potential and spikes of second-order ocellar neurons of the cockroach, Periplaneta americana, was studied. The stimulus was a sinusoidally modulated light with various mean illuminances. A solitary spike was generated at the depolarizing phase of the modulation response. Analysis of the relationship between the amplitude/frequency of voltage modulation and the rate of spike generation showed that (a) the spike initiation process was bandpass at approximately 0.5-5 Hz, (b) the process contained a dynamic linearity and a static nonlinearity, and (c) the spike threshold at optimal frequencies (0.5-5 Hz) remained unchanged over a mean illuminance range of 3.6 log units, whereas (d) the spike threshold at frequencies of less than 0.5 Hz was lower at a dimmer mean illuminance. The voltage noise in the response was larger and the mean membrane potential level was more positive at a dimmer mean illuminance. Steady or noise current injection during sinusoidal light stimulation showed that (a) the decrease in the spike threshold at a dimmer mean illuminance was due to the increase in the noise variance: the noise had facilitatory effects on the spike initiation; and (b) the change in the mean potential level had little effect on the spike threshold. We conclude that fundamental signal modifications occur during the spike initiation in the cockroach ocellar neuron, a finding that differs from the spike initiation process in other visual systems, including Limulus eye and vertebrate retina, in which it is presumed that little signal modification occurs at the analog-to-digital conversion process.