The distribution of acetylcholinesterase (AChE) in the lateral eye and brain of the horseshoe crab was investigated with histochemical means using standard controls to eliminate butyrylcholinesterase and nonspecific staining. Intense staining was observed in the neural plexus of the lateral compound eye, in the lateral optic nerve, and in various neuropils of the brain. Nerve fibers with moderate to weak staining were widespread in the brain. No sornata were stained in either the lateral eye or the brain. The distribution of acetylcholinesterase in the supraesophageal ganglia and nerves of the giant barnacle was also investigated for comparison. Although both the median optic nerve of the barnacle and the lateral optic nerve of the horseshoe crab appear to contain the fibers of histaminergic neurons, only the lateral optic nerve of the horseshoe crab shows AChE staining. Other parts of the barnacle nervous system, however, showed intense AChE staining. These results along with the histochemical controls eliminate the possibility that some molecule found in histaminergic neurons accounted for the AChE staining but support the possibility that acetylcholine might be involved as a neurotransmitter in lateral inhibition in the horseshoe crab retina. Two reasonable neurotransmitter candidates for lateral inhibition, histamine and acetylcholine, must now be investigated.

Enzymatic treatments that facilitated whole-cell electrophysiological recordings were used on Limulus ventral photoreceptor cells. Ventral optic nerves were treated with either collagenase or collagenase, papain, and trypsin. Either treatment greatly increased the ease of making whole-cell recordings of transmembrane potentials. Light responses obtained from enzyme-treated photoreceptor cells were nearly identical to results obtained without enzyme treatment and compared favorably to in vivo recordings of light responses from the compound lateral eye. Enzyme-treated cells also responded to applied octopamine, as do untreated cells, with an increased phosphorylation of a 122-kD protein. This suggests that the external receptors and internal biochemical machinery required for at least one second-messenger cascade are present after enzyme treatment. The morphological integrity of enzyme-treated photoreceptor cells was examined with light microscopy as well as with scanning and transmission electron microscopy. In general, we found that each enzyme treatment greatly reduced the integrity of the layers of glial cells that surround the photoreceptor cells thereby making these cells easily accessible for whole-cell recordings of transmembrane potentials. The morphology of the rhabdomere was normal after enzymatic degradation of the adjacent glial covering.

We examined inward rectification in Limulus ventral photoreceptors using the two-microelectrode voltage clamp. Hyperpolarization in the dark induced an inward current whose magnitude was distinctly dependent on extracellular K+ concentration, [K+0]. The [K+0] dependence resembled the characteristic [K+0] dependence of other inward rectifiers. The inward current was not dependent on extracellular Ca2+ or Na+, and it was unaffected by intracellular injection of Cl−. The hyperpolarization induced currents had two phases, an early nearly instantaneous phase and a slowly developing late phase. The currents were sensitive to extracellular barium and cesium. In voltage-pulse experiments, the magnitudes of the inwardly rectifying currents were variable from cell to cell, with some cells exhibiting negligible inward currents. Large hyperpolarizations (to membrane potentials more negative than about – 140 mV) caused unstable inward current recordings, irreversible desensitization, and irreversible elevation of intracellular Ca2+ concentration. The inward rectifier provides negative feedback by tending to depolarize the cell (with inward current) in response to hyperpolarization. We suggest that the inward rectifier reduces the amount of hyperpolarization that would otherwise be generated by electrogenic processes. This feature would restrict the dynamic voltage range of the photoreceptors at very hyperpolarized potentials.

The radial and longitudinal distribution of retinular screening pigment in the lateral eye of the horseshoe crab Limulus polyphemus was quantified under a variety of experimental conditions. Pigment position was characterized by the center and width of the radial distribution at four levels in the ommatidium.

Under diurnal lighting, intact animals show movement of pigment granules from the periphery of the retinular cell at night towards the junction of the arhabdomeral and rhabdomeral segments of the retinular cell in the day. In constant darkness, intact animals exhibit the same circadian rhythm in pigment migration. Animals with bilaterally cut optic nerves do not receive circadian efferent input from the brain and show little pigment movement in diurnal lighting. In all of these cases, pigment was either aggregated in a band just peripheral to the rays of the rhabdom or dispersed to the periphery of the retinular cell.

When dark-adapted animals are exposed to a sudden large light increment, pigment moves inward between the rays of the rhabdom. During the day, this inward response begins immediately and reverses as the ommatidial aperture begins to close. At night, the onset of the inward movement is delayed, but then occurs more rapidly than during the day. No significant longitudinal movement of photoreceptor screening pigment was detected under any of these experimental conditions.

Two opposing mechanisms control the movement of screening pigment in these cells. Release of neurotransmitters from the circadian efferents causes outward movement; large increments of light cause inward movement. In the absence of sudden changes in light intensity, circadian efferent input, not cyclic lighting, appears to be the major determinant of screening pigment position. A sudden and large increment of light triggers the rapid inward movement which appears to be a protective mechanism optimized for daytime performance.

The ommatidia of the lateral eye of the horseshoe crab, Limulus polyphemus, undergo rhythmic changes in structure that are driven by diurnal lighting and efferent neural activity from a circadian clock in the brain. This study uses cytochemical probes to investigate the cytoskeletal elements mediating these responses and to develop models for their control. Antibodies to actin and phalloidin, a specific F-actin probe, label the rhabdom of lateral eye ommatidia, the cone cells of the ommatidial aperture, the ommatidial sheath, and the peripheral regions of the photoreceptor (retinular cell) cytoplasm. These probes also label the rhabdomere of ventral photoreceptors. Antibodies to tubulin label the eccentric cell dendrite and soma in each lateral eye ommatidium, the cone cells of the aperture, and the peripheral retinular cell cytoplasm. Models are proposed for the cytoskeletal mechanisms involved in controlling aperture and rhabdom shape, pigment movement, and shedding of rhabdomeral membrane.

The lateral eyes of the horseshoe crab Limulus contain about 80 pmoles of retinal, 30 pmoles of retinol, and 4 pmoles of retinyl esters. More all-trans than 11-cis isomer was found in each category of retinoid. No consistent changes were observed in the amounts of retinal, retinol, or retinyl esters as a function of time of day. No 3,4-dehydro- nor hydroxyretinoids were found.

Aqueous extracts of the eye support the stereospecific formation of 11 -cis retinal from a11-trans retinal when irradiated with light. The reaction requires a protein that is apparently recognized by polyclonal antibodies raised against the retinal photoisomerase extracted from honeybee eyes. The isomerase is able to use as substrate either endogenous all-trans retinal in the extract or retinal supplied in vesicles of phospholipid. The spectral efficiency of this isomerization has λmax at 550 nm, but the spectrum appears too narrow compared with the absorbance spectrum of retinoid-binding proteins, probably because of inadequate correction for nonspecific isomerization at short wavelengths.

Physiological studies of intracellular messengers frequently employ intracellular injections of large molecules that either monitor or modulate the metabolism of the messenger cascade. Injected molecules have unknown mobility in the cytosol and unknown accessibility to various cytosolic compartments, including those postulated to be traversed by intracellular messenger molecules. In order to determine whether injected molecules have access to the confined spaces through which messenger molecules must diffuse, we injected 5-nm colloidal gold or horseradish peroxidase, or both, into Limulus ventral photoreceptors. Injections were made by applying pressure pulses to the back of an intracellular micropipette that also monitored membrane voltage. The tissue was fixed at varying times after injection and processed for electron microscopy by conventional techniques. Cells fixed 1–3 min after injection contained HRP reaction product only in the cell body. HRP reaction product was found at varying distances down axons in direct relation to the interval between injection and fixation. Colloidal gold particles were found throughout the cell body but not in axons of tissue fixed 1–3 min after injection. Both HRP reaction product and 5-nm colloidal gold particles were observed within the microvillar projections of internal and external rhabdomere, as well as within the extracisternal spaces of endoplasmic reticulum. We conclude that large molecules injected from an intracellular micropipette into an arbitrary locus of ventral photoreceptor cells have access to all of the presumed sites of the phototransduction cascade.

Circadian rhythms affect the anatomy, physiology, and biochemistry of the visual cells in the eyes of the horseshoe crab (Limulus polyphemus). These rhythms are mediated by the activity of efferent neurons that project from the central nervous system to all of the eyes. In this study, the optic nerves of Limulus were backfilled with Neurobiotin revealing the location of efferent cell bodies and their projections through the central nervous system. We propose that this efferent system mediates the circadian changes in visual functions in Limulus. Whether these cells are the circadian pacemaker neurons is unknown.

The cell bodies of the efferent neurons are ovoid and have a diameter of 40−80 μm. They lie within the cheliceral ganglion of the tritocerebrum, just posterior to the protocerebrum. This ganglion is on the lateral edge of the circumesophageal ring, near the middle of the dorsal-ventral axis of the ring. Each optic nerve contains axons from both ipsilateral and contralateral efferent cells, and some, possibly all, of them project bilaterally and to more than one type of optic nerve.

The efferent axons form a tract that projects anteriorly from the cell bodies to the protocerebrum, and bifurcates just lateral to the protocerebral bridge. One branch crosses the midline and projects anteriorly to the optic tract and medulla on the side contralateral to the cell of origin; the other branch follows a symmetric pathway on the ipsilateral side. Small branches arising from the major efferent axons in the optic tract project through the ocellar ganglia to the median optic nerves. The efferent axons branch again in the medulla, and some of these branches innervate the ventral optic nerves. The major branches of the efferent axons continue through the lamina and enter the lateral optic nerve.

A circadian clock in the brain of the horseshoe crab, Limulus polyphemus, has an important role in the function of the peripheral visual system. At night, the clock transmits neural activity to the lateral, ventral, and median eyes via efferent optic nerve fibers. The activity occurs in synchronous bursts (maximum rate of 2 bursts/s) with individual efferent fibers contributing a single spike in each burst. The circadian efferent activity originates in the protocerebrum. Lateral connections synchronize the efferent activity recorded from the two halves of the protocerebrum, suggesting the existence of bilateral circadian oscillators. Circadian efferent activity survives excision of the brain and isolation of the protocerebrum. We conclude that circadian clock and its complex neural circuitry are fundamental components of the Limulus visual system.

The optical alignment of individual cuticular cones in the dioptric array of the lateral eye of Limulus polyphemus was determined with a precision two-circle goniometer constructed and mounted to the stage of a compound microscope and using a new formaldehyde-induced fluorescence procedure. All measurements were made from the corneal surface of the excised eye mounted in seawater through an air/water interface perpendicular to the optic axis of the microscope. Our results revealed two variants of visual field and eye curvature which can actually be discriminated in casual examination of adult animals. We call animals possessing these two variants “morlocks” and “eloi.” Adult male and female morlocks about 25 cm across the carapace have eyes which are relatively elongated, often darker in pigmentation, smaller, and relatively flatter in curvature. Morlocks have a monocular field of view of about 3.13 steradians or 50% of a hemisphere. The coverage averages 115 deg along the vertical axis and 168 deg along the horizontal axis of the eye, with maximum resolution in the anteroventral quadrant. Adult male and female eloi of comparable size have eyes which are relatively more round, often lighter in pigmentation, larger with more ommatidia, and relatively more bulged. Eloi have a monocular field of view of approximately 3.83 steradians or 61% of a hemisphere that covers 145 deg vertically and 185 deg horizontally. Eloi have more uniform resolution than morlocks with best resolution in the posteroventral quadrant. All horseshoe crabs examined, whether morlocks or eloi, have an identical orientation of the margin of the eye relative to the animals’ coordinates.

Activity at night in efferent nerve fibers from a central circadian clock produces changes in photoreceptor function in the lateral compound eye of Limulus: the response to light is increased; membrane potential fluctuations (bumps) occurring in the dark are suppressed; and the duration of bumps occurring both in the dark and under dim illumination is increased (Barlow et al., 1977; Kaplan & Barlow, 1980; Barlow, 1983; Barlow et al., 1985, 1987). Efferent nerve terminals release octopamine when activated (Battelle et al., 1982; Battelle & Evans, 1984, 1986); exogenous octopamine in vitro produces some of the changes resulting from efferent nerve activity in vivo (Kass et al., 1988).

We report here that the increase in both on-transient and steady-state response to light induced by octopamine in the lateral eye in vitro are concentration dependent with threshold at or below 100 nM, saturation at or above 100 µM, and half-maximal increase in the range 1–10 µM. Octopamine also reduces bump activity in the dark in a concentration-dependent way. Unlike the increase in light response, the dependence of this effect on octopamine concentration is extremely variable from specimen to specimen. The effects of exogenous octopamine on light response and bump activity can sometimes be reversed by removing octopamine from the medium bathing the in vitro preparation. Octopamine also increases bump duration, apparently in a concentration-dependent manner. We have not succeeded in reversing this increase in bump duration.

The concentration dependence of changes in photoreceptor response described here agrees qualitatively with the dependence of cAMP levels on octopamine in Limulus photoreceptors (Kaupp et al., 1982), lending further support to the idea that cAMP acts as a second messenger in the circadian control of photoreceptor function. Our results also suggest that the changes induced in the transient and steady-state response to light by both efferent nerve activity and exogenous octopamine have a common origin, which may differ from that responsible for the modulation of bump activity.

We have examined some of the physiological effects associated with the replacement of extracellular Na+ with Li+ in nominally Ca2+-free saline in the ventral photoreceptors of the horseshoe crab Limulus polyphemus. We observed that replacement of Na+ saline with Li+ saline induced larger voltage-activated inward currents with similar voltage dependence. These currents were absent in Tris+ saline. Anode-break excitation was maintained in Li+ saline but blocked in Tris+ saline. Regenerative events associated with quantum bumps in dark-adapted cells illuminated with dim lights were maintained in Li+ saline. Regenerative events associated with responses to moderately bright illumination were also maintained in Li+ saline. The post-illumination hyperpolarization associated with the Na+/K+-exchange pump (Brown & Lisman, 1972) was present after brief exposure to Li+ saline but disappeared after longer exposure. Following return to Na+ saline, the post-illumination hyperpolarization reappeared. We conclude that (1) Li+ permeates the voltage-dependent Na+ channel, GNa(V), in the photoreceptor plasma membrane; (2) Li+ supports voltage-activated physiological events normally mediated by Na+; and (3) Li+ substitution briefly supports and later inhibits the electrogenic effects of the Na+/K+-exchange pump. The effects of external Li+ on cellular physiology have implications for the interpretation of other studies employing Li+ extracellularly.

The visual arrestins in rhabdomeral photoreceptors are multifunctional phosphoproteins. They are rapidly phosphorylated in response to light, but the functional relevance of this phosphorylation is not yet fully understood. The phosphorylation of Limulus visual arrestin is particularly complex in that it becomes phosphorylated on three sites, and one or more of these site are phosphorylated even in the dark. The purpose of this study was to examine in detail the light-stimulated phosphorylation of each of the three sites in Limulus visual arrestin in intact photoreceptors. We found that light increased the phosphorylation of all three sites (S377, S381, and S396), that S381 is a preferred phosphorylation site, and that S377 and S381 are highly phosphorylated in the dark. The major effect of light was to increase the phosphorylation of S396, the site located closest to the C-terminal and very close to the adaptin binding motif. We speculate that the phosphorylation of this site may be particularly important for regulating the light-driven endocytosis of rhabdomeral membrane.

Much has been learned from studies of Limulus photoreceptors about the role of the circadian clock and light in the removal of photosensitive membrane. However, little is known in this animal about mechanisms regulating photosensitive membrane renewal, including the synthesis of proteins in, and associated with, the photosensitive membrane. To begin to understand renewal, this study examines diurnal changes in the levels of mRNAs encoding opsin, the integral membrane protein component of visual pigment, and the relative roles of light and the circadian clock in producing these changes. We show that at least two distinct opsin genes encoding very similar proteins are expressed in both the lateral and ventral eyes, and that during the day and night in the lateral eye, the average level of mRNA encoding opsin1 is consistently higher than that encoding opsin2. Northern blot assays showed further that total opsin mRNA in the lateral eyes of animals maintained under natural illumination increases during the afternoon (9 & 12 h after sunrise) in the light and falls at night in the dark. This diurnal change occurs whether or not the eyes receive input from the circadian clock, but it is eliminated in eyes maintained in the dark. Thus, it is regulated by light and darkness, not by the circadian clock, with light stimulating an increase in opsin mRNA levels. The rise in opsin mRNA levels observed under natural illumination was seasonal; it occurred during the summer but not the spring and fall. However, a significant increase in opsin mRNA levels could be achieved in the fall by exposing lateral eyes to 3 h of natural illumination followed by 9 h of artificial light. The diurnal regulation of opsin mRNA levels contrasts sharply with the circadian regulation of visual arrestin mRNA levels (Battelle et al., 2000). Thus, in Limulus, distinctly different mechanisms regulate the levels of mRNA encoding two proteins critical for the photoresponse.

The second messenger systems involved in the final stages of the phototransduction cascade in Limulus photoreceptors remain unclear. Excised patches of transducing membrane contain cGMP-gated channels, suggesting the involvement of cGMP in the excitation process. To further explore this possibility, we tested the effects of inhibitors and agonists of guanylate cyclase. The active site cyclase inhibitors guanosine 5′-tetraphosphate and adenosine 5′-tetraphosphate produced a reversible reduction of the response to light without affecting resting membrane properties. The cyclase inhibitor Rp-GTPαS produced a similar reduction, but the effect was only slightly reversible. The reduction in the response produced by these inhibitors was robust, often producing over a 95% decrease in the amplitude of the light response. Previous work had shown that an end-product cyclase inhibitor, imidodiphosphate, also inhibited the response. The consistent results with four different guanylate cyclase inhibitors strongly support the involvement of this enzyme in the phototransduction cascade. To determine whether the guanylate cyclase involved is the NO-dependent soluble form, we applied inhibitors and activators of the nitric oxide synthase/guanylate cyclase pathway such as L-N5-(1-iminoethyl) ornithine, sodium nitroprusside, and carboxy-PTIO. None of these agents had any substantial effect on phototransduction. Taken together, these results support a role for a particulate guanylate cyclase in Limulus photoreceptor excitation.

Recently, it was reported that the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA) and linolenic acid (LNA), activate the light-sensitive channels in Drosophila photoreceptors (Chyb et al. 1999). We have examined whether these PUFAs activate the light-sensitive channels in Limulus ventral photoreceptors. We find that, whether applied from the outside or injected into a Limulus ventral photoreceptor, either AA or LNA fails to activate the light-sensitive channels. Moreover, the synthetic diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, also fails to activate the light-sensitive channels. We suggest that these findings require us to rethink our view about the generality of the process of phototransduction in invertebrate microvillar photoreceptors. We propose that the photoreceptors of Drosophila and Limulus evolved to utilize different branches of the phosphoinositide pathway for phototransduction: those of Limulus evolved to utilize IP3-mediated calcium release while those of Drosophila evolved to utilize diacylglycerol and it's downstream products.

Arrestins participate in the termination of phototransduction in both vertebrates and invertebrates. However, the visual arrestins of invertebrates and vertebrates differ significantly from one another in that the invertebrate visual arrestins become phosphorylated rapidly in response to light while those in the photoreceptors of vertebrates do not. In an effort to understand the functional relevance of arrestin phosphorylation, we examined this process in the photoreceptors of the horseshoe crab Limulus polyphemus. We report that Limulus visual arrestin can be phosphorylated at three sites near its C-terminus and show that arrestin molecules phosphorylated on one, two, and three sites are normally present in both light- and dark-adapted photoreceptors. Light adaptation increases the amount of arrestin phosphorylated at three sites.

The phosphoinositide (PI) intracellular signaling pathway, which triggers Ca2+ release from intracellular stores, appears to be a central feature of phototransduction in most invertebrate species studied. Procedures designed to inhibit PI-pathway reactions cause suppression of excitation to dim lights. However, in Limulus photoreceptors, responses to bright stimuli are in fact enhanced by some of these procedures, suggesting that PI metabolism is not obligatory for light-induced excitation. Other studies, however, suggest that Ca2+ release is obligatory for excitation. We studied this issue by examining the effects of PI-pathway inhibitor, Li+, on electrophysiological responses to light in Limulus photoreceptors. Li+ is reported to cause depletion of intracellular PI-pathway intermediate, inositol; and it offers the pharmacological advantage that its block can be bypassed by introducing exogenous inositol. Introduction of Li+ caused a very slowly developing but complete suppression of responses to dim stimuli. In contrast, Li+ caused a rapidly developing but partial suppression of responses to bright stimuli. Li+-induced suppression was reversed by exogenous introduction of inositol. In addition, inositol prevented Li+-induced suppression of excitation. Li+ enhanced light adaptation (light-induced desensitization) but slowed response deactivation, indicating a difference in the processes underlying these phenomena. Li+ slowed dark adaptation, the recovery of sensitivity following light adaptation. All of these effects were prevented or rescued by extracellularly applied inositol, suggesting the presence of a transmembrane inositol transport system. The overall results suggest that PI-dependent signaling is central and obligatory for excitation in Limulus, at least for responses to dim to moderate illumination. The failure of Li+ to suppress bright light-induced excitation completely may be due to a failure of Li+ to block PI metabolism completely, as in other systems; however, it may point to a parallel, PI-independent excitation pathway possessing very low light sensitivity when PI metabolism is inhibited.