Limulus Lateral Eye Research Articles

Modulation of function in Limulus compound eye photoreceptors by octopamine enantiomers

Octopamine has been identified as a circadian neutrotransmitter in Limulus photoreceptor organs, which can increase photoreceptor response to light. Here we report that the naturally occurring d(−)-octopamine is much more effective in increasing the response to light in Limulus lateral eye photoreceptor cells in vitro than its l(+) enantiomer, which appears to act as an antagonist of d(−)-octopamine. The effect of both enantiomers on the response to light is slow to develop, and slow to disappear following their removal. Such behavior is appropriate for a system which produces a long-term, steady change of state in the photoreceptor organs.

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.

Isolation and Expression of an Arrestin cDNA from the Horseshoe Crab Lateral Eye

Electrophysiological studies of photoreceptors from the horseshoe crab Limulus polyphemus continue to provide fundamental new knowledge of the photoresponse in invertebrates. Therefore, it is of particular interest to characterize the molecular components of the photoresponse in this system. Here we describe an arrestin cloned from a cDNA library constructed using poly(A)+ RNA isolated from Limulus lateral eyes. The protein, deduced from the arrestin cDNA, is most similar to arrestin from locust antennae (56% identity) and Drosophila phosrestin I (53% identity). Limulus arrestin was expressed in a heterologous system, and its properties were compared with those of a 46-kDa light-regulated phosphoprotein (pp46A) in Limulus photoreceptors described in previous studies from this laboratory. Arrestin and pp46A (a) have the same apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) have an isoelectric point in the basic pH range, (c) require calmodulin and elevated Ca2+ levels for phosphorylation, (d) are immunoreactive with monoclonal antibody C10C10 directed against a sequence in bovine arrestin (S-antigen) that is perfectly conserved in the deduced arrestin protein, and (e) are associated with photoreceptors. We conclude that the arrestin described here and pp46A are the same protein. The results of this and previous studies show that in Limulus photoreceptors, light regulates the phosphorylation of arrestin in complex ways.

Characterization of a calcium/calmodulin-dependent protein phosphatase in the Limulus nervous tissue and its light regulation in the lateral eye.

Calcium (Ca2+) plays an integral role in the light response of the photoreceptors in both vertebrate and invertebrate organisms. In the ventral eye of the horseshoe crab, Limulus polyphemus, a flash of light delivered to a dark-adapted photoreceptor stimulates a rapid rise in intracellular free calcium concentration ([Ca2+]i), which in turn mediates light adaptation. It has previously been demonstrated that in Limulus photoreceptors light, via Ca2+, activates a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase which increases the phosphorylation of arrestin. We now have identified biochemically, a calcium/calmodulin-dependent protein phosphatase (Ca2+/CaM PP) in homogenates of the Limulus lateral and ventral eye, brain, and lateral optic nerve using as a substrate, a 32P-labeled peptide fragment of the regulatory subunit of cAMP-dependent protein kinase (RII). This protein phosphatase shares biochemical properties with calcineurin, a Ca2+/CaM-dependent protein phosphatase (type-2B). Its activity is enhanced by Ca2+, calmodulin and Mn2+; and is inhibited by mastoparan, a calmodulin antagonist, and a synthetic peptide corresponding to the autoinhibitory domain of mammalian calcineurin. Most importantly, light regulates the Ca2+/CaM PP activity in the lateral eye. While there is no difference in basal activity in long-term dark- or light-adapted preparations, Ca2+ enhances Ca2+/CaM PP activity only in long-term light-adapted eyes.

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.

Efferent neurotransmission of circadian rhythms inLimulus lateral eye

We investigated efferent neurotransmission in the Limulus 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 microM) 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 microM, potent agonist of octopamine), forskolin (8 to 400 microM, activator of adenylate cyclase), IBMX (1 mM, inhibitor of phosphodiesterase), and 8-bromo-cAMP (500 microM, analogue of cAMP). 8-bromo-cGMP (500 microM, 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 the Limulus 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.

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 circadian rhythms in the Limulus lateral eye.

Efferent control of circadian rhythms in the Limulus lateral eye.

Transient membrane shedding in Limulus photoreceptors: control mechanisms under natural lighting

Photoreceptors of the Limulus lateral eye shed their light-sensitive membranes (rhabdoms) in a burst early each morning when the animal is maintained in natural lighting. This shedding burst produces a cloud of multivesicular bodies which coalesce and migrate away from the rhabdom. Within 24 hr, these gradually collapse to combination bodies and ultimately to lamellar bodies. Light initiates the burst of shedding. If animals are maintained in darkness beyond their normal dawn, the shedding burst is delayed until the first onset of light. We have not been able to produce a second burst of membrane shedding within one 24-hr period. Efferent optic nerve activity generated by a circadian clock in the brain primes the shedding burst. At least 3 hr of efferent activity in darkness must precede light onset to prime membrane shedding; however, the efferent fibers need not be active when the light-initiated burst occurs. Chronically blocking the efferent input to the retina abolishes the shedding burst. The burst of membrane shedding is robust and short-lived. Within 15 min of light onset the area of photosensitive membrane decreases by about 70%, and within an hour the rhabdom returns to essentially its preburst size. At other times in the diurnal light cycle, the size of the rhabdom undergoes significant variations which are not abolished by blocking the efferent input. Apparently the daily burst of shedding overlays a second cycle of membrane metabolism that is not controlled by efferent optic nerve activity.

Effects of current pulses on the sustained discharge of visual cells of Limulus

Current pulses were used in the eccentric and retinular cells of the Limulus lateral eye to produce changes in the interspike interval of the discharge sustained by a constant light level. The effects on the interspike interval of hyperpolarizing and depolarizing perturbations, applied at various delays from the previous spike, were measured for different intensities and durations of the current pulse. The results show that when the perturbations were applied in the first part of the interval, effects contrary to what is normal were produced (i.e, hyperpolarizing pulses decreased the interspike interval instead of increasing it and vice versa for depolarizing pulses). Here we discuss briefly the implications on neural encoding models.

Efferent neurotransmission of circadian rhythms in Limulus lateral eye. I. Octopamine-induced increases in retinal sensitivity

Octopamine increases the sensitivity of the Limulus lateral eye in situ when injected beneath the cornea during the day. The effect of octopamine is dose-dependent with a threshold concentration of about 0.1 microM injected at 1 microliter/min for 15 min. Injection of 40 microM octopamine increases lateral eye sensitivity to approximately 70% of the nighttime level normally caused by the efferent output of a circadian clock. Injections of octopamine analogues and other candidate neurotransmitters indicate that the postsynaptic receptor mediating the increase of retinal sensitivity is relatively specific for the structure of octopamine. The postsynaptic receptor is tentatively classified as a type 2B octopamine receptor (Evans, P. D. (1981) J. Physiol. (Lond.) 318: 99-122). Clozapine suppresses the effects of both exogenous octopamine and the endogenous efferent neurotransmitter. Together with the results from Barlow et al. (Barlow, R. B., Jr., S. J. Bolanowski, Jr., and M. L. Brachman (1977) Science 197: 86-89) and Battelle et al. (Batelle, B. -A., J. A. Evans, and S. C. Chamberlain (1982) Science 216: 1250-1252) our study leads to the following conclusion: retinal efferents, driven by a circadian clock in Limulus brain, release octopamine that increases visual sensitivity.

Neuropeptide modulation of photosensitivity. II. Physiological and anatomical effects of substance P on the lateral eye of Limulus

A system of efferent substance P-like immunoreactive fibers innervates the ommatidia of the Limulus lateral eye. Thus, we tested the physiological effects of substance P on the lateral eye by measuring the electroretinogram, a population potential reflecting the photoreceptors' response to light, under different experimental conditions. Substance P had no direct effect on the photoreceptors, but it induced an increase in their responsiveness to test flashes of light. The latency, magnitude, and duration of this reversible modulatory effect was dose-dependent. The lateral eye displays an endogenous circadian rhythm in its responsiveness to light. Application of exogenous substance P in the daytime causes an immediate rise as well as an increase in the nocturnal peak, while injection of one of its antagonists (D-Pro2, D-Phe7, D-Trp9 substance P) in the afternoon retards the normal rise in sensitivity and reduces the nighttime levels. Passive incubation with substance P antibodies at midnight caused a drop to diurnal levels of photosensitivity. Short-term changes in photosensitivity, similar in their nature to the substance P-induced ones, were caused by arousing the subjects. Arousal had an effect on the ongoing circadian rhythm similar to that of substance P application. Thus, the substance P efferent system may regulate neural responsiveness in both a short-term, environmentally induced manner, as well as for level setting in a circadian fashion. The mechanism for substance P-induced increases in photosensitivity involves changes in ommatidial structure: contraction of distal pigment cells, resulting in an increased aperture, and contraction of the retinular cells and rhabdom, resulting in a wider diameter of the latter. These structural modifications result in a greater angle of acceptance and increased light quantum catch.

Octopamine release from centrifugal fibers of the Limulus peripheral visual system.

Octopamine, a biogenic amine, is synthesized and stored within centrifugal (efferent) fibers that project from the brain to the lateral and ventral eyes of the horseshoe crab, Limulus polyphemus. The experiments described here show that depolarization of Limulus lateral and ventral eyes, produced by elevating the concentration of extracellular K+, causes the selective release of newly synthesized octopamine from centrifugal fibers in a manner that requires the influx of extracellular Ca2+. Conjugates of octopamine and tyramine that are also stored within centrifugal fibers are not released in response to K+-induced depolarization. These findings add further support to the hypothesis that octopamine is a neurotransmitter synthesized by and released from centrifugal fibers in Limulus eyes. This amine may be responsible for many of the alterations in lateral eye structure and function that are mediated by centrifugal innervation.

Synchronization of impulse generation by lateral inhibition in theLimulus compound eye

Under uniform illumination the optic nerve discharge from the lateral compound eye ofLimulus polyphemus can become oscillatory. This synchronization of eccentric cell impulse generation requires strong lateral inhibition (Barlow and Fraioli 1978). The simultaneous intra- and extracellular observations of eccentric cell responses described here allow examination of the process of synchronization. They also permit measurements of the phase relationship between the oscillatory hyperpolarization of the eccentrical cell membrane potential and the periodic bursts of optic nerve activity which cause the hyperpolarization through the lateral inhibitory network of the eye. On the basis of these observations, the following conclusions can be drawn.

Neuropeptide immunoreactivity in Limulus. I. Substance P-like immunoreactivity in the lateral eye and photocerebrum.

Substance P-like immunoreactivity was examined in the Limulus lateral eye and photocerebrum by the unlabeled antibody peroxidase-antiperoxidase method. Small-diameter immunoreactive fibers innervate the corneal epidermis of the lateral eye and appear to be part of a generalized epidermal innervation. No immunoreactive neurons or fibers were found in the ventral, median, or lateral optic nerves nor in the lamina or chiasma. The optic medulla contains neurons with immunoreactive perinuclear caps in the ganglion cell layer and fibers of undetermined origin in the posterolateral regions of the neuropil. The central body contains many immunoreactive fibers in its neuropil, some or all of which arise from neurons adjacent to its anteromedial tips. Immunoreactive neurons within the curve of the central body give rise to processes which join the central neuropil of the photocerebrum. The immunoreactive innervation of the corpora pedunculata includes processes which terminate amont Kenyon cell somata, and processes in the peduncular neuropil, all of which arise from a bilateral cluster of neurons along the midline. Attempts to investigate the source of the immunoreactive fibers using cobalt impregnation of the severed circumesophageal connective have revealed two additional innervations of the corpora pedunculata, one from raphe neurons along the midline and one from lateral portions of the midline ganglion adjacent to but separate from that portion containing the immunoreactive neurons. The results demonstrate that immunocytochemical methods are another powerful tool for the study of neuronal pathways in invertebrates.

Efferent Fibers to Limulus Eyes Synthesize and Release Octopamine

Octopamine synthesized in vitro from tyramine by Limulus lateral and ventral eyes was located by light microscopic and electron microscopic autoradiography in efferent fibers which innervate ventral photoreceptors and lateral eye ommatidia. Newly synthesized octopamine was released from efferent fibers in response to depolarization in high concentrations of potassium. We propose that octopamine is a neurotransmitter of efferent fibers that may modulate basic retinal processes such as photoreceptor sensitivity, photomechanical movements, and photoreceptive membrane turnover.

Circadian clock in Limulus brain increases response and decreases noise of retinal photoreceptors.

The sensitivity and structure of ommatidia in the Limulus lateral eye exhibit circadian rhythms. The cyclic changes are mediated by the activity of efferent optic nerve fibres that originate in the brain and terminate in the retina. We report here that the effects of the efferent input to the retina are detectable in single photoreceptor cells. At night the efferent input decreases photoreceptor noise (discrete waves of the membrane potential in darkness) and increases the photoreceptor response to light (amplitude of the receptor potential).

Specificity of serotoninergic inhibition in Limulus lateral eye.

The receptor specificity for synaptically mediated lateral inhibition in Limulus lateral eye retina was studied by structure-activity correlations of the action of the putative indoleaminergic neurotransmitter, serotonin (5-HT), and its isomers and structural analogs, tryptamine (TRYP), 6-hydroxytryptamine (6HT), 5,6-dihydroxytryptamine (5,6-DHT), 5-hydroxydimethyltryptamine (5-HDMT), and 5-hydroxytryptophan (5-HTP). The 5-HT blockers, lysergic acid diethylamide (LSD), bromo-LSD (BOL), and cinanserin, were also tested. The inhibitory action of the indoleaminergic agonists is highly structure-specific. An hydroxyl group in the 5 position of the indole nucleus, sterically unencumbered by hydroxyls in neighboing positions, is essential. In order of decreasing potency, 5-HT, 5-HDMT, and 5-HTP are active agonists; TRYP, 6-HT, and 5,6-DHT are inactive. Configuration and mobility of the side chains of the active agonists also affect the interaction, and these side-chain characteristics correlate with agonist potency. The receptors for inhibitory action and for transmembranal transport in reuptake are different. Both active agonists and inactive analogs appear to be taken up (Adolph and Ehinger, 1975. Cell Tissue Res. 163:1-14). LSD and BOL have bimodal actions: direct inhibition and agonist blockade. These actions may be mediated via low-specificity presynaptic uptake receptor sites rather than highly specific, postsynaptic, agonist receptor sites.

Influence of calcium on the Limulus photoreceptor potential

Evidence is presented demonstrating that the magnitude of the initial component (C 1) of the Limulus photoreceptor potential elicited by moderately intense light flashes is both sodium and calcium dependent. The evidence is: (1) the reversal potential of C 1 of the lateral eye retinular cell and ventral eye cell receptor potential is appreciably more positive than the reversal potential of C 2; (2) exposure of both types of photoreceptors to environments deficient in Na + rapidly reduces and abolishes C 2 of the receptor potential whereas C 1 persists with reduced magnitude and rate of rise; (3) exposure of ventral photoreceptors to environments deficient in Ca 2+ reduces the magnitude and rate of rise of C 1 and enhances both the magnitude and duration of C 2; (4) exposure of ventral photoreceptors to calcium enriched environments rapidly increases the magnitude of C 1 and curtails the duration of C 2; (5) the increase in magnitude of C 1 for a ten-fold increase in the external CaCl 2 concentration (10 to 100 mM) is 21 mV; and (6) verapamil, an organic calcuim current blocking agent, reduces the magnitude of C 1 of the ventral photoreceptor potential and enhances the magnitude and duration of C 2. This evidence, although indirect, strongly suggests that Ca 2+ entry contributes to the production of C 1 of the Limulus ventral photoreceptor potential as well as C 1 of the Limulus lateral eye retinular cell receptor potential.

Partial voltage clamping of Limulus ventral photoreceptor potentials: Evidence of programmed conductance changes

Light-initiated currents elicited by brief light stimuli from Limulus ventral photoreceptors bathed in normal sea water generally exhibit a smooth contour, although the unclamped receptor potential elicited by an identical light stimulus usually exhibits distinct C 1 and C 2 components. However, light-initiated currents obtained from cells exposed to chlorobutanol often exhibit two components. Data from such experiments indicate that peak C 2 current is more strongly voltage dependent than peak C 1 current, as in Limulus lateral eye retinular cells. The results of partial voltage clamp experiments with ventral photoreceptors in which the clamping episode terminated at different times during the receptor potential reveal relatively minor perturbations of the rebound receptor potential when compared with the undamped control response. These findings suggest that the temporal pattern of the membrane conductance changes which underlie the receptor potential is determined prior to the occurrence of the receptor potential. It is likely that the program for these conductance changes is developed during the latent period of the receptor potential.