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.

Opsin1-2, G(q)α and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock.

Dark and light adaptation in photoreceptors involve multiple processes including those that change protein concentrations at photosensitive membranes. Light- and dark-adaptive changes in protein levels at rhabdoms have been described in detail in white-eyed Drosophila maintained under artificial light. Here we tested whether protein levels at rhabdoms change significantly in the highly pigmented lateral eyes of wild-caught Limulus polyphemus maintained in natural diurnal illumination and whether these changes are under circadian control. We found that rhabdomeral levels of opsins (Ops1-2), the G protein activated by rhodopsin (G(q)α) and arrestin change significantly from day to night and that nighttime levels of each protein at rhabdoms are significantly influenced by signals from the animal's central circadian clock. Clock input at night increases Ops1-2 and G(q)α and decreases arrestin levels at rhabdoms. Clock input is also required for a rapid decrease in rhabdomeral Ops1-2 beginning at sunrise. We found further that dark adaptation during the day and the night are not equivalent. During daytime dark adaptation, when clock input is silent, the increase of Ops1-2 at rhabdoms is small and G(q)α levels do not increase. However, increases in Ops1-2 and G(q)α at rhabdoms are enhanced during daytime dark adaptation by treatments that elevate cAMP in photoreceptors, suggesting that the clock influences dark-adaptive increases in Ops1-2 and G(q)α at Limulus rhabdoms by activating cAMP-dependent processes. The circadian regulation of Ops1-2 and G(q)α levels at rhabdoms probably has a dual role: to increase retinal sensitivity at night and to protect photoreceptors from light damage during the day.

Opsin co-expression in Limulus photoreceptors: differential regulation by light and a circadian clock.

A long-standing concept in vision science has held that a single photoreceptor expresses a single type of opsin, the protein component of visual pigment. However, the number of examples in the literature of photoreceptors from vertebrates and invertebrates that break this rule is increasing. Here, we describe a newly discovered Limulus opsin, Limulus opsin5, which is significantly different from previously characterized Limulus opsins, opsins1 and 2. We show that opsin5 is co-expressed with opsins1 and 2 in Limulus lateral and ventral eye photoreceptors and provide the first evidence that the expression of co-expressed opsins can be differentially regulated. We show that the relative levels of opsin5 and opsin1 and 2 in the rhabdom change with a diurnal rhythm and that their relative levels are also influenced by the animal's central circadian clock. An analysis of the sequence of opsin5 suggests it is sensitive to visible light (400-700 nm) but that its spectral properties may be different from that of opsins1 and 2. Changes in the relative levels of these opsins may underlie some of the dramatic day-night changes in Limulus photoreceptor function and may produce a diurnal change in their spectral sensitivity.

Do weak adapting backgrounds uncover multiple components in the electroretinogram of the horseshoe crab?

The lateral eye of the horseshoe crab, Limulus polyphemus, has been used as a model system for over a century to study visual and circadian processes. One advantage of this system is the relative simplicity of the retina. The input pathway of the retina consists of photoreceptor cells that are electrically coupled to the dendrite of a second-order cell, which sends action potentials to the brain. Electroretinograms (ERGs) recorded from the lateral eye show a biphasic shape, with a leading negative wave and a later positive peak. The purpose of these experiments was to determine whether adapting backgrounds could be used to uncover multiple adaptation mechanisms within the ERG. To test this idea, ERGs were elicited using variable intensity flashes presented under dark-adapted conditions, as well as in the presence of weak adapting backgrounds. Flashes and backgrounds were generated using green LEDs (lambda max = 525 nm) under software control. ERGs were recorded using a corneal wick electrode placed on the lateral eye of the horseshoe crab. Preliminary results suggest that ERGs recorded in the presence of adapting backgrounds are linearly scaled versions of dark-adapted FRGs. This suggests that there is a single adaptation stage in the Limulus retina. This is in contrast with analogous results from mammals, including mouse, cat and monkey, which show multiple stages of adaptation within their more complex retinas.

Categorical and prolonged potentials are evoked when brief, intermediate-intensity flashes stimulate horseshoe crab lateral eye photoreceptors during octopamine neuromodulation.

Octopamine, a major efferent neurotransmitter in the lateral eye of the horseshoe crab (Limulus polyphemus), has previously been shown to modulate photoreceptor responses evoked by long flashes. Quantification of these data indicates that this modulation produced a genuine increase in sensitivity to light which cannot be entirely due to an increase in optical efficiency consequent on an anatomical alteration. Other previous studies demonstrated that extrinsic current can modulate Limulus lateral eye photoreceptor cells by inducing a bistable membrane potential with two distinct states. The present study was therefore undertaken to find out if octopamine could modulate visual responses by inducing prolonged and bistable polarization shifts similar to those demonstrated in several other neural systems. Intracellular microelectrodes were used to execute an electrophysiological study of the receptor potentials evoked in the lateral eye of Limulus when brief (20-ms) flashes were delivered while 50 microM octopamine perfused dark-adapted photoreceptors. The combined chemical and optical stimuli prolonged photoreceptor responses to light to the degree that they often exceeded the duration of the brief stimulus by hundreds of milliseconds. Moreover, these prolonged potentials were clearly bistable because they were categorical--either a prolongation was perceptually clear-cut and present or it was not, with no intermediate patterns being observed. During seawater control perfusions, such prolongations were absent. This appears to be the first demonstration of such categorical and prolonged potentials in a photoreceptor neuron. This finding particularly suggests that efferent-driven neuromodulation can enable the development of a persisting short-term representation of a brief stimulus, with this representation being retained at the most distal possible neural site.

Ca2+/calmodulin-binding peptides block phototransduction in Limulus ventral photoreceptors: evidence for direct inhibition of phospholipase C.

Phototransduction in Limulus photoreceptors involves a G protein-mediated activation of phospholipase C (PLC) and subsequent steps involving InsP3-mediated release of intracellular Ca2+. While exploring the role of calmodulin in this cascade, we found that intracellular injection of Ca2+/calmodulin-binding peptides (CCBPs) strongly inhibited the light response. By chemically exciting the cascade at various stages, we found the primary target of this effect was not in late stages of the cascade but rather at the level of G protein and PLC. That PLCdelta1 contains a calmodulin-like structure raised the possibility that PLC might be directly affected by CCBPs. To test this possibility, in vitro experiments were conducted on purified PLC. The activity of this enzyme was strongly inhibited by CCBPs and also inhibited by calmodulin itself. Our results suggest that the calmodulin-like region of PLC has an important role in regulating this enzyme.

Fast desensitization of the response to InsP3 in Limulus ventral photoreceptors.

In Limulus ventral photoreceptor cells the time-course of the desensitization of InsP3 response was measured by an injection-pair paradigm. Pressure pulses of InsP3 were delivered into the cell with various interpulse intervals. The desensitization of the response to the second injection of each pair approached totality at 200 ms, which is the duration of the response to a single pressure pulse of InsP3. Lowering extracellular calcium did not affect the time-course of the desensitization. Lowering the temperature slowed down both the time-course of the response to InsP3 and the time-course of the desensitization to the same extent. These findings suggest that the desensitization is powerful enough and its onset fast enough to contribute to the transience of the InsP3 response. The time-course of the desensitization suggests it may influence light adaptation.

Light-stimulated rhabdom turnover in Limulus ventral photoreceptors maintained in vitro.

The role of light in turnover of photosensitive membranes was studied in isolated photoreceptors maintained in vitro. Ventral photoreceptors of the horseshoe crab, Limulus polyphemus, were used since they have been the subjects of many in vitro physiological studies. This study shows that the two classes of ventral photoreceptors, the large and small photoreceptors (Herman: companion paper), differ in their morphological response to light. The rhabdom of small photoreceptors is remarkable for its regularity, independent of lighting condition. The photosensitive microvilli of the rhabdom of small photoreceptors are narrow and almost always tightly packed in a hexagonal arrangement. In contrast, the morphology of the rhabdom of the large ventral photoreceptors is different in the dark and in the light, and the rhabdom undergoes turnover during lighting transitions. When fully dark-adapted, the photosensitive microvilli of large photoreceptors are narrow and well organized, sometimes in a crystalline array. However, in the light-adapted state, the microvilli are much thicker and very irregular. The transitions between the dark and light-adapted states, examined at midday, are rapid. After 5 minutes light exposure, the microvilli are dilated at their bases and shed membranes are present in the cytoplasm. By 30 minutes after light onset, the appearance of the rhabdom of large photoreceptors is indistinguishable from fully light-adapted cells. The transition to the dark-adapted state is equally rapid. Even at 5 or 12 minutes after light offset, most microvilli are narrow and quite regular, and by 30 minutes, the rhabdom usually appears to be fully dark-adapted. These experiments show that both the synthetic and degradative phases of rhabdom renewal take place in isolated photoreceptors. No efferent neural activity is required to initiate turnover; rather, changes in illumination alone are sufficient to generate rhabdom turnover in large ventral photoreceptors in vitro.