Calcium (Ca2+) is thought to play a major role in the photoresponse of both vertebrates and invertebrates, but the mechanisms through which Ca2+ exerts its effects are unclear. In many systems, some effects of Ca2+ on cellular processes are thought to be mediated via activation of calcium/calmodulin protein kinase (CaCAM-PK) and the phosphorylation of specific proteins. Thus, protein substrates for CaCAM-PK in photoreceptor cells may be important in mediating the effects of Ca2+ on the photoresponse.

In this study, we identify eight substrates for CaCAM-PK found in both the ventral and lateral eyes of Limulus. We focus on a characterization of one of these, a 46-kD substrate. We show that its subcellular distribution in ventral photoreceptors and its isoelectric forms are identical to the 46-kD light-stimulated phosphoprotein (46A) described by Edwards et al. (1989). Furthermore, we present evidence that 46A is unique to photoreceptor cells, and that it is present throughout the cell. Based on the results of this study, and the previous study by Edwards et al. (1989), we propose that 46A is involved in mediating the effects of Ca2+ on Limulus photoreceptor cell function, and that it may be involved in dark adaptation.

The activities of both protein phosphatases and protein kinases are responsible for the transient changes in the levels of phosphorylation and probably the functions of protein intermediates involved in the biochemical and physiological mechanisms underlying the photoresponse in photoreceptor cells from both vertebrate and invertebrate organisms. Of the known protein serine/threonine phosphatases, various forms of type I (PP 1) and type 2A (PP 2A) protein phosphatases are responsible for dephosphorylating many of the known phosphoproteins including those involved in photoreceptor cell function. In this report, we provide biochemical evidence for both PP 1– and PP 2A-like activities in the visual and nonvisual tissue of the horseshoe crab, Limulus polyphemus, that membrane and soluble forms of both enzymes are present, and that the activities of both enzymes are greater in light- than in dark-adapted lateral eyes. These activities were characterized using glycogen phosphorylase a, a substrate for both PP 1 and PP 2A, and various protein phosphatase inhibitors, including okadaic acid. We also report that okadaic acid, at concentrations required to inhibit PP 1, inhibited physiological functions of photoreceptor cells from the ventral eye, causing a delayed reduction of the resting membrane, and slowing and reducing light responses.

We used a precision two-circle goniometer mounted to the stage of a compound microscope to determine the optical alignment and to measure the entrance aperture diameter of individual cuticular cones in the dioptric array of the lateral eye of juvenile horseshoe crabs in order to learn about the development of the visual field. Our results show that the extent of the visual field of juvenile horseshoe crabs with prosomal lengths about 20% ofadult size (14–21 mm) is about 70% that of the visual field of adult horseshoe crabs (prosomal lengths: 100+ mm). The visual field of such juvenile animals covers between 77 and 85 deg vertically and 140 and 145 deg horizontally. Assuming that the dioptric array is uniform and square packed, the average interommatidial angle of the juvenile animals is between 5.6 and 6.0 deg as compared to 4.6 deg for an adult animal. The diameter of the entrance aperture of individual cuticular cones increases markedly with increasing animal size. In addition, we noted a statistically significant trend for entrance aperture diameters to increase from anterior to posterior within the eye for animals of all sizes. There may be a slight trend for entrance aperture diameters to increase from dorsal to ventral within the eye. Our results indicate that the extent of the visual field and the resolution of the lateral eye approach adult values in advance of animals' reaching sexual maturity.

Efferent fibers from a central circadian clock innervate photoreceptors along the ventral nerve of Limulus and release octopamine when active. We have recorded ERG-like responses from the ventral eye in vivo over several day periods. We have also used intracellular microelectrodes to study changes in ventral photoreceptor function during exogenous applications of octopamine (the putative efferent neurotransmitter), IBMX (a phosphodiesterase inhibitor), and forskolin (an adenylate cyclase activator): (1) Responses to light measured at night from ventral photoreceptors in vivo are greater in amplitude than those recorded during the day; (2) Octopamine and agents that increase intracellular levels of cAMP in ventral photoreceptors decrease the rate of spontaneous (dark) bumps, increase photoreceptor response to light without changing threshold, and often increase the bump duration; and (3) These changes in function of ventral photoreceptors are similar to those that have been observed in the photoreceptor of the lateral eye during circadian clock activity at night, and in vitro in the presence of those same pharmacological agents.

A circadian clock modulates the structure and function of the lateral eyes of Limulus polyphemus, greatly increasing their sensitivity at night. During the mating season, male Limulus are visually attracted both day and night to females and objects that resemble females. This paper asks how well Limulus can see day and night, and whether the circadian changes in retinal sensitivity might influence the ability of these animals to find mates. We recorded the visual behavior of male and female horseshoe crabs in the vicinity of an object – a cement hemisphere (29.5 cm diameter) similar in size and shape to a female horseshoe crab – placed in a mating area near Mashnee Dike, Bourne, Massachusetts. Males oriented toward this target from an average distance of 0.94 m during the day and 0.88 m at night; and females appeared to avoid the target. We conclude that males can see potential mates at night almost as well as they can during the day. Apparently the circadian changes in the retina help compensate for the daily changes in illumination in the animal's normal environment. This study provides the first evidence for a role of visual circadian rhythms in an animal's natural behavior.

The protein intermediates of the photoresponse and the modulation of this response in invertebrate photoreceptors are largely unknown. As a first step toward identifying these proteins, we have examined light-stimulated changes in protein phosphorylation in preparations of Limulus photoreceptors. Here we show that light modulates the level of phosphorylation of three proteins associated with Limulus ventral photoreceptors: the upper band of a 46-kD protein doublet (46A) and a 122-kD protein, which become more heavily phosphorylated in response to light, and the lower component of the 46-kD doublet (46B), which is phosphorylated in dark-adapted cells, but not in cells maintained in the light. In dark-adapted preparations, 46A is phosphorylated within 30 s after a flash of light and dephosphorylates over a period of many minutes. It is also a major substrate for calcium/calmodulin-dependent protein kinase (Wiebe et al., 1989); therefore, we speculate that 46A is involved in some aspect of dark adaptation. Interestingly, the level of phosphorylation of 46A is the same when measured from preparations maintained in complete darkness or ambient light for at least 1.5 h. The 122-kD phosphoprotein is the same protein which becomes phosphorylated in response to efferent innervation to Limulus eyes (Edwards et al., 1988) and the efferent neurotransmitter, octopamine (Edwards and Battelle, 1987). It may be involved in the increase in retinal sensitivity and the enhanced response of photoreceptors to light that is initiated by efferent innervation. Its role in light-stimulated processes is not clear. The level of phosphorylation of 46B may be most relevant to the long-term state of adaptation of the photoreceptor cell to light and dark.

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+, activate s a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase which increases the phosphorylation of arrestin. We now have identifie d 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.

The phototransduction cascade in invertebrate photoreceptors has not been fully elucidated. It has been proposed that in Limulus ventral photoreceptor cGMP is the intracellular second messenger that directly controls the gating of the light-dependent channels (Johnson et al., 1986; Bacigalupo et al., 1991). Recently, a putative cGMP-gated channel cDNA, Lcng1, has been cloned from Limulus and shown to be expressed in the brain and the ventral eye (Chen et al., 1999). In this study, we sought to more specifically localize the LCNG1 transcript and protein. In situ hybridization was used to determine whether the gene is expressed in glia or photoreceptor cells in the ventral eye. The results clearly demonstrated that Lcng1 mRNA is transcribed in the ventral photoreceptors. On Western blots probed with a polyclonal antibody raised against the C-terminus of LCNG1, a 100-kDa band and an 80-kDa band was labeled in the membrane protein preparations from brain and ventral eye, respectively. The labeling of these bands was blocked by preabsorption of the antibody with the antigen, indicating the labeling specificity. Immunocytochemistry and confocal microscopy were applied to investigate the subcellular localization of this antigen. Immunolabeling was highly localized in the transducing lobes of ventral eye photoreceptors and lateral eye photoreceptors. In both cases, the labeling was associated with membrane regions specialized for phototransduction, but the exact pattern appeared to be somewhat different in the two eyes. Preabsorption of the antiserum with antigen abolished the labeling, confirming specificity. The results lend support to the hypothesis that a cGMP-gated channel is directly involved in the phototransduction process.

Most animals experience daily changes in light and darkness. The retinas of many of these animals show concomitant rhythmic changes in the levels of mRNAs that encode proteins involved in the photoresponse. These changes may be circadian and independent of light, independent of circadian clocks and regulated by light, or regulated by a circadian clock and light. We have taken advantage of the organization of the Limulus visual system to examine the separate and combined effects of signals from a circadian clock and light on arrestin mRNA levels in photoreceptors. The clock that regulates photoreceptors in the lateral eye of Limulus is in the brain, and signals from the clock reach the lateral eye via activation of a well-characterized efferent projection in the lateral optic nerve. In the experiments described, clock-driven efferent input to the lateral eye was eliminated by cutting the lateral optic nerve, and light input to the lateral eye was eliminated by placing an opaque patch over the eye. Arrestin mRNA levels were quantified relative to 18s rRNA with a ribonuclease protection assay. We observed the following. In lateral eyes exposed to natural diurnal light and endogenous efferent nerve activity, the level of arrestin mRNA was higher during the day in the light than during the night in the dark. Circadian efferent nerve activity was necessary and sufficient to produce normal daily fluctuations in the level of arrestin mRNA. Light influenced arrestin mRNA levels only in eyes with intact and active efferent projections. We conclude that arrestin mRNA levels in lateral eye photoreceptors are controlled entirely by efferent nerve activity, and that light exerts its effects by modulating this output from the circadian clock. Light-stimulated changes in arrestin mRNA in the vertebrate retina may likewise require interactions between light-driven biochemical cascades and clock output.

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 μM) or superfused with L-683, 590 (20 μM) 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.