Limulus Eye Research Articles

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.

Anatomical circuitry of lateral inhibition in the eye of the horseshoe crab, Limulus polyphemus.

Lengthy uninterrupted series of sections of the neural plexus in the compound eye of the horseshoe crab, Limulus polyphemus, have been used to reconstruct all the arborizations and their synaptic interconnections in a neuropil knot. This one microglomerulus contains the axons of 19 retinular cells, which pass by without contacts; 13 efferent fibres with 44 synapses to and from eccentric cell collaterals; and arborizations from 54 eccentric cells with 577 synapses. Eccentric cell axons are devoid of synaptic input. Their collaterals ramify in synaptic knots and subserve both pre- and postsynaptic functions simultaneously. Arborizations near the axon of origin have a highly branched pattern (up to 20 bifurcations), a high synaptic input: output ratio (up to about 9:1), and high synaptic density (a maximum of 12 per micrometre of neurite length). The opposite extreme is represented by sparsely branched eccentric cell collaterals distant from their axons of origin with very little synaptic input and sparse output. Spatially graded lateral inhibition is the apparent outcome of a radially decreasing distribution of inhibitory synapses on the arborizations of eccentric cell collaterals combined with possible decremental signal transmission in the plexus. The synaptic analysis has a bearing on most physiological aspects of lateral inhibition that have been studied in the Limulus eye. Implied in the results is the suggestion that synapse formation is an intrinsic property of the presynaptic element, but that the connectivity is governed by the electrical activity of target neurons.

Calcium ion, an intracellular messenger of light adaptation, also participates in excitation of Limulus photoreceptors.

Photoreceptor cells of Limulus ventral eyes were bathed in artificial sea water (ASW) that contained 10 mM-EGTA and no added Ca2+ (EGTA-ASW). Test flashes elicited responses that increased to a maximum size within 10 min in EGTA-ASW but did not change further when dark-adapted cells were bathed for an additional 35 min in this solution. Light responses progressively declined from this maximum size if the cells were repetitively illuminated in EGTA-ASW. In this state of reduced responsiveness, response amplitudes were further reduced by intracellular ionophoretic injection of EGTA; response amplitudes were increased by intracellular ionophoretic injection of Ca2+. Both of these findings are opposite to what is normally observed for cells bathed in ASW. Also, after repetitive illumination in EGTA-ASW, both the slope of the response versus intensity relationship became steeper and light responses often had a delayed increase in amplitude. The light responses and the response versus intensity relation returned to normal when the bathing medium was changed back to ASW containing 10 mM-Ca2+. The light-induced rise in luminescence recorded from photoreceptors injected with the photoprotein aequorin (the 'aequorin response') declined by at most 50% after dark-adapted photoreceptors were bathed in EGTA-ASW for 45 min. However, the aequorin response progressively declined by 98% if cells were repetitively illuminated while bathed in EGTA-ASW. The total intracellular Ca content of whole end-organs was measured by atomic absorption spectroscopy. Total intracellular Ca content did not change significantly while photoreceptors were bathed in EGTA-ASW even after repetitive illumination. We suggest that cytosolic Ca2+ is required by one or more steps in the mechanisms that link rhodopsin isomerization to both (i) an increase in the conductance of the cell membrane to Na+ and (ii) a release of Ca2+ from a light-labile store.

Efferent innervation to Limulus eyes

The visual system of the horseshoe crab Limulus polyphemus has been important model for studies of basic aspects of vision. Experiments investigating phototransduction and contrast enhancement by lateral inhibition using Limulus eyes are famous. Recent studies with this animal are providing new information on still another feature of the visual system common to many species: the modulation of retinal function by efferent innervation from the brain. Retinal efferent systems have been identified in a variety of animals from invertebrates to mammals (see Ref. 1 for selected references) and of these the efferent system projecting to Limulus eyes is now the most thoroughly understood. Studies with Limulus have revealed that efferent innervation controls circadian changes in the sensitivity of the eye, and influences the eye's integration of visual information, adaptation to ambient light levels, and renewal of photosensitive membrane. Postsynaptic targets of efferent projections have been identified as has an efferent fiber neurotransmitter. The retinal efferent system in Limulus has opened the way for detailed studies of mechanisms underlying the central control of visual function.

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.

A discrete theory of synchronization of lateral optic-nerve impulses in the horseshoe crab

A discrete theory of synchronization of optic-nerve responses during uniform, steady illumination of the compound eye ofLimulus is described here. The theory is a natural extension of the classical steady-state theory of Hartline and Ratliff (1957). In order to explain the asynchronous response to weak illumination, we find it necessary to take account of observed random fluctuations in the responses of ommatidia illuminated by themselves. Without this noisy component, the computed responses synchronize at very low excitation levels. Once synchronized, the response develops a non-linear dependence on excitation. This non-linearity is a consequence solely of synchronization and is distinct from observed excitation dependences of lateral inhibition between pairs of ommatidia (Barlow and Lange, 1974). Synchronization at the higher excitation levels is also found to reduce or destroy Mach bands which are present in responses to weaker excitations.

Limulus brain modulates the structure and function of the lateral eyes.

At night efferent optic nerve activity generated by a circadian clock in the Limulus brain changes the structure of the photoreceptor and surrounding pigment cells in the animal's lateral eyes. The structural changes allow each ommatidium to gather light from a wider area at night than during the day. Visual sensitivity is thereby increased, but spatial resolution is diminished. At daybreak efferent activity from the clock stops, the structural changes reverse, and the field of view of each ommatidium decreases. The cyclic changes are endogenous and continue in the dark. Thus, under the control of a circadian clock, the Limulus eye exchanges its daytime acuity for greater sensitivity at night.

The Effects of Constant Light on Visual Processes

This book presents the papers given at a symposium held at the Florida State University in Tallahassee on April 25 through 27,1979. Many of the papers have in the meantime appeared in print in various scientific journals, but it is nice to have them all together in one volume. About half of the book is concerned with the influence of light on the vertebrate eye, though the human eye is really not considered in depth. The other half deals with invertebrates (no monograph on visual processes would be complete without mentioning the limulus eye) and molecular aspects of photoreceptor physiology. Anybody interested in the effect of light on the visual system could read this book with benefit.

Neurotransmitter candidates in the visual system of Limulus polyphemus: Synthesis and distribution of octopamine

Synthesis of octopamine, acetylcholine, γ-aminobutyric acid and serotonin was examined in a number of tissues from the Limulus visual system, and the distribution of endogenous octopamine was determined. There was a selective association of octopamine synthetic activity with tissues rich in photoreceptor cell bodies compared to optic nerves and highest levels of endogenous octopamine were found in Limulus eyes and optic ganglia. The distribution of octopamine synthetic activity and endogenous octopamine suggests a role for this amine in Limulus visual function.

On oscillatory responses of the Limulus retina

Published observations of the dynamic properties of lateral and self-inhibition in the Limulus retina lead to a non-linear integral equation for the response of ommatidia located near the center of a uniformly illuminated region. Coleman and Renninger (1976, 1978) showed that when the excitation is constant in time and the sum of the inhibitory coefficients for the illuminated region exceeds a critical value, the integral equation has a stable periodic solution describing a sustained, spatially synchronized, oscillatory response in which bursts of activity alternate with silent periods. Such spatially synchronized "bursting" has been observed in the Limulus retina in situ by Barlow and Fraioli (1978), using the preparation of Barlow and Kaplan (1971). Employing experimental data on the temporal dependence of lateral and self-inhibition, which were then available only for the excised eye, Coleman and Renninger calculated a value of 0.34 s for the period p of the bursting response, which is significantly above the range, 0.11---0.20 s, of values of p observed for the Limulus eye in situ. Brodie et al. (1978) have recently published measurements of the temporal dependence of lateral and self-inhibition for the in situ preparation. Here we show that when the kernel functions in Coleman and Renninger's integral equation are chosen in accord with these new data, the periodic solutions of the equation have a period of approximately 0.13s, which is in the range (0.11---0.20 s) required for agreement with experiment. Other properties of the periodic solutions, i.e., their general form and the threshold levels of inhibition required for their existence, are also in accord with published observations of the behavior of the retina in situ.

Effect of boundaries on the response of a neural network

The effect an abrupt boundary has upon the dynamical response of a neural network is investigated. The retina of the Limulus eye is used as a model system for studying this effect. A theoretical technique is presented for the quantitative prediction of the manner in which this neural network responds in the vicinity of its boundary. Corresponding experimental measurements of the response to moving stimuli by single optic neurons located near retinal boundaries are presented. Theory and experiment show detailed quantitative agreement.

The optical mechanism of the eye of Limulus

THE compound eyes of many marine and some terrestrial arthropods have smooth surfaces with no convex curvature to the facets. This means that image formation by ordinary spherical refraction is impossible. Nevertheless, apposition eyes of this kind form images behind each surface facet1,2 (Fig. 1b) and the question has repeatedly arisen as to how these images are produced. Exner chose the king crab, Limulus, as providing the definitive example of such an eye, and from a mixture of observation and theory came up with the idea of a lens-cylinder, a flat-ended optical device in which the refractive index decreases from the central axis to the periphery in a roughly parabolic manner1. This arrangement will form an image (Fig. 2a), and devices of this kind have actually been manufactured3,4. Exner's theory has long been considered to be the correct account of image formation in this type of eye5. However, Levi-Setti, Park and Winston2 have recently produced a fundamentally different explanation of image formation in Limulus eyes (Fig. 2b). This was based on the idea that each optical element, or crystalline cone, concentrated light by reflection, not refraction, and that it was the shape of the crystalline cone's reflective surface, and not its internal refractive index gradient, that led to the image-forming properties of the structure. This principle had already been used in another optical device, the ‘ideal light collector’ (ref. 6) which was invented as a tool for concentrating faint radiation. In this report I discuss the two suggested optical mechanisms for the eye of Limulus, and present evidence from interference microscopy that Exner's theory is probably the correct one.

A Limulus eye on cognitive psychology

A Limulus eye on cognitive psychology

The response of the Limulus retina to moving stimuli: a prediction by Fourier synthesis.

The Limulus retina responds as a linear system to light stimuli which vary moderately about a mean level. The dynamics of such a system may conveniently be summarized by means of a spatiotemporal transfer function, which describes the response of the system to moving sinusoidal gratings. The response of the system to an arbitrary stimulus may then be calculated by adding together the system's responses to suitably weighted sinusoidal stimuli. We have measured such a spatiotemporal transfer function for the Limulus eye. We have then accurately predicted, in a parameter-free calculation, the eye's response to various stimulus patterns which move across it at several different velocities.

The spatial temporal response of the Limulus eye neural set

The Limulus lateral eye provides an example of a neural set showing lateral inhibition. Experimental evidence suggests the use of a linear, time-invariant, spatio-temporal, differential-integral equation as an approximation describing the set. A general solution is obtained by the Fourier and Laplace transform methods. The temporal pattern of the neural response depends both on the subset interactions and the intrinsic dynamics of component subsets. Since subset interaction strengths depend on the spatial frequencies (pattern) of the input light intensity, the output time response from the whole set depends on the input spatial frequencies. A possible temporal code for spatial patterns is discussed.

Enhancement and phototransduction in the ventral eye of limulus.

Limulus ventral photoreceptors were voltage clamped to the resting (dark) potential and stimulated by a 20-ms test flash and a 1-s conditioning flash. At a constant level of adaptation, we measured the response to the test flash given in the dark (control) and the incremental response produced when the test flash occurred within the duration of the conditioning flash. The incremental response is defined as the response to the conditioning and test flashes minus the response to the conditioning flash given alone. When the test flash was presented within 100 ms after the onset of the conditioning flash we observed that: (a) for dim conditioning flashes the incremental response equaled the control response; (b) for intermediate intensity conditioning flashes the incremental response was greater than the control response (we refer to this as enhancement); (c) for high intensity conditioning flashes the incremental response nearly equaled the control response. Using 10-mum diam spots of illumnination, we stimulated two spatially separate regions of one photoreceptor. When the test flash and the conditioning flash were presented to the same region, enhancement was present; but when the flashes were applied to separate regions, enhancement was nearly absent. This result indicates that enhancement is localized to the region of illumination. We discuss mechanisms that may account for enhancement.

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.

Photopigments of the lateral eye ofLimulus

Photoreceptor membrane fractions of the lateral eye ofLimulus were solubilized in the detergent emulphogene, and three photobleachable materials were observed with respectiveλ max values at 330nm±10nm, 450 nm±10 nm, and 530 nm±10 nm. A530 is the pigment which had been reported earlier by Hubbard and Wald (1960), and it can be separated from A330 and A450 on the basis of differential solubility in digitonin. Approximately the same number of incident quanta were required for a unit absorbance change atλ max for all three pigments, but measurable photo-products were not observed after bleaching A330 and A450.