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.

Serotonin and dopamine in the retina of a lizard.

The objective of this paper is to report the presence and localization of serotonin and dopamine in the retina of the lizard Uta stansburiana. High performance liquid chromatography and electrochemical detection were used to identify and quantitate the two amines. Both compounds are present as endogenous molecules in this retina and are found in concentrations similar to those reported in other non-mammalian retinas. The same methods were employed to confirm, in the isolated retina, the synthesis of serotonin from precursor, tryptophan. Immunocytochemical methods were used to localize, in the neural retina, serotonin and the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase. Serotonin immunoreactivity was observed in bistratified amacrine cells (ca. 7 micron dia.) with processes ramifying in sublayers 1, 4, and 5 of the inner plexiform layer. Immunoreactivity to tyrosine hydroxylase was observed in a different population of bistratified amacrine cells (ca. 11 micron dia.) that had processes ramifying in sublayers 1 and 5 (and perhaps 3) of the inner plexiform layer. The enzymes for further metabolism of dopamine were not found in the retina of this lizard by immunocytochemical methods. The results of this research suggest that only single classes of serotoninergic and dopaminergic neurons are present in the retina of U. stansburiana. This retina might, therefore be an appropriate place in which to investigate the functioning of these amines in visual information processing.

Immunohistochemical and biochemical evidence for the putative inhibitory neurotransmitters histamine and GABA in lobster olfactory lobes.

As an initial effort to investigate possible inhibitory interactions in the olfactory system of the spiny lobster, studies were conducted to identify and localize the putative inhibitory neurotransmitters histamine and GABA in the olfactory lobe. Biochemical studies demonstrated that olfactory lobe tissue was capable of synthesizing histamine from radioactive histidine and GABA from glutamic acid. Immunohistochemistry was used to localize histamine and GABA in brain sections, by using either avidin-biotin conjugated peroxidase or fluorescein conjugated secondary antibody. Specific histamine-like and GABA-like immunoreactivity was found in soma clusters of olfactory interneurons, adjacent to the olfactory lobe. Small, putative glial cells displaying intense histamine-like immunoreactivity were found interspersed among the glomeruli of the lobe. The accessory lobe exhibited moderate immunostaining for both histamine and GABA. Positive immunostaining for histamine and GABA was also found in the olfactory lobes, with a predominance of staining in the outer caps of the glomeruli, which are thought to be the regions where the primary afferent terminals contact the processes of second-order olfactory neurons. These findings collectively implicate inhibition at the first synaptic level of the olfactory pathway in the spiny lobster.

Autoradiographic localization of newly synthesized octopamine to retinal efferents in the Limulus visual system.

The biogenic amine octopamine is synthesized from both tyrosine and tyramine in the lateral, median, and ventral eyes of Limulus. The autoradiographic studies presented here were designed to locate the sites of octopamine synthesis in the ventral and lateral eyes. We found that efferent fibers, which project to ventral and lateral eyes from the central nervous system, became intensely and selectively labeled during in vitro incubations with 3H-tyramine. In the ventral eye, more than 95% of the efferent fibers were labeled. Results of biochemical analyses suggested that most of the radioactive substance within these efferent fibers was newly synthesized octopamine. The selective labeling of efferent fibers during incubation with 3H-tyramine was used as an anatomical tool to study the number and distribution of efferent fibers within the ventral eye. Light microscopic (LM) reconstructions of the distribution of label in serial longitudinal sections through ventral optic nerves together with electron microscopic (EM) autoradiographic analyses revealed between 70 and 200 efferent axons. The results of these studies and of reconstructions of efferent innervation to photoreceptor somata suggest that each ventral photoreceptor cell or each small cluster of cells is innervated by a separate efferent fiber. Both LM reconstructions and EM analyses showed that efferent fibers ramify extensively and specifically in and near the internal rhabdom of ventral photoreceptor cells. In EM autoradiographs of lateral eyes incubated with 3H- tyramine, the silver grains that were located over ommatidia were concentrated exclusively over efferent fibers. All of these efferent fibers, which lay near rhabdoms and in partitions between retinular cells, were labeled. The results of our present studies support our hypothesis that octopamine is a neurotransmitter in Limulus retinal efferent fibers. This amine may modulate the biochemistry and physiology of ventral photoreceptor cells and may mediate many of the known effects of circadian efferent innervation to the lateral eye.

Immunoreactivity in Limulus. II. Studies of serotoninlike immunoreactivity, endogenous serotonin, and serotonin synthesis in the brain and lateral eye.

Serotoninlike immunoreactivity was examined by the fluorescein-isothiocyanate-labeled secondary antibody technique in the lateral eye and brain of Limulus. Endogenous serotonin was measured with high-performance liquid chromatography and electrochemical detection. The synthesis of [3H]serotonin from [3H]tryptophan was measured in the presence and absence of reserpine. Fibers with serotoninlike immunoreactivity were found in the proximal stalks of the corpora pedunculata, in the neuropil of the central body, in the neuropils of the visual centers (lamina, medulla, and ocellar ganglion), in the optic tract that connects the ocellar ganglion with the posterior medial medulla, and in the central neuropil of the brain. Immunoreactive somata were found in four groups in the brain. Up to 50 somata were scattered through each side of the dorsal medial group that lies centered on the dorsal surface within the curve of the central body. These neurons innervate the central body neuropil and send processes into the central neuropil. Three or four reactive somata formed the ventral pole of each medullar group. These may provide the innervation of the proximal stalk of the corpora pedunculata. Five to ten reactive neurons were observed anteriorly in the ventral posterior lateral group #2 on each side that send processes into the central neuropil. Ten to 15 reactive somata were found on either side of the midline in the dorsal anterior part of the ventral medial group that contribute processes to the central neuropil. The remainder of the brain was not immunoreactive. No immunoreactive fibers or somata were found in the lateral eye or in the lateral optic nerve. Serotoninlike and substance P-like immunoreactivities were not found to be colocalized anywhere in the brain. Significant amounts of endogenous serotonin were detected in the lamina and medulla whose neuropils are rich in immunoreactive fibers and in the central body and dorsal medial group that are also rich in immunoreactive somata and fibers. No endogenous serotonin was detected in either the lateral eye or the lateral optic nerve. The lamina, medulla, and central body and dorsal medial group also synthesized and stored [3H]serotonin from [3H]tryptophan. It is likely that serotonin is a neurotransmitter in the brain, but not in the lateral eye of the horseshoe crab. In particular, it appears that serotoninergic neurons may play a role in central visual processing.

Central projections of Limulus photoreceptor cells revealed by a photoreceptor-specific monoclonal antibody.

Studies of lateral, median, and ventral eyes of the chelicerate arthropod Limulus polyphemus (the common American horseshoe crab) are providing important basic information about mechanisms for information processing in the peripheral visual system and for the modulation of visual responses by light and circadian rhythms. The processing of visual information in Limulus brain is less well understood in part because the specific central projections of the various classes of visual neurons are not known. This study describes a mouse monoclonal antibody, 3C6A3, which binds to Limulus photoreceptor cell bodies, their axons, and terminals, but not to any other cell type in the central nervous system. This antibody, and intracellular injection of biocytin, are used to demonstrate the central projections of each type of photoreceptor. Our main conclusions are that: 1) the photoreceptors (retinular cells) of the lateral eye project only to the lamina; 2) the photoreceptors of the lateral rudimentary eye project to both the lamina and medulla; 3) the photoreceptors of the median ocellus project only to the ocellar ganglion; and 4) the photoreceptors of the rudimentary median (endoparietal) eye project to the ocellar ganglion and also into the optic tract. These results, along with previous studies, allow us to infer the projections of the secondary cells. The eccentric cells of the lateral eye project to the lamina, medulla, optic tract, and ocellar ganglion. The arhabdomeral cells of the median ocellus project through the ocellar ganglion and to optic tract to the medulla.

Retinal anatomy of Chorocaris chacei, a deep-sea hydrothermal vent shrimp from the Mid-Atlantic Ridge.

Exploration of deep-sea hydrothermal vents over the past quarter century has revealed that they support unique and diverse biota. Despite the harsh nature of the environment, vents along the Mid-Atlantic Ridge are dominated by large masses of highly motile Bresiliid shrimp. Until 1989, when it was discovered that the vent shrimp Rimicaris exoculata possesses a hypertrophied dorsal eye, many believed that animals populating hydrothermal vents were blind. Chorocaris chacei (originally designated Rimicaris chacei) is a Bresiliid shrimp found at hydrothermal vent fields along the Mid-Atlantic Ridge. Like R. exoculata, C. chacei has a hypertrophied retina that appears to be specialized to detect the very small amount of light emitted from the orifices of black smoker hydrothermal vent chimneys. C. chacei lacks the sophisticated compound eyes common to other decapod crustaceans. Instead, it has a smooth cornea, with no dioptric apparatus, apposed by a tightly packed, massive array of photosensitive membrane. Photoreceptors in the C. chacei retina are segmented into a hypertrophied region that contains the photosensitive membrane and an atrophied cell body that is roughly ten times smaller in volume than the photosensitive segment. The microvillar photosensitive membrane is consistent in structure and ultrastructure with the rhabdoms of decapod and other invertebrate retinas. However, the volume density of photosensitive membrane (> or =60%) exceeds that typically observed in invertebrate retinas. The reflecting pigment cells commonly found in decapod retinas are represented in the form of a matrix of white diffusing cells that exhibit Tyndall scattering and form an axial sheath around the photoreceptors. All photoreceptor screening pigment granules and screening pigment cells are restricted to the region below the photoreceptor nuclei and are thereby removed from the path of incident light. No ultrastructural evidence of rhythmic cycling of photosensitive membrane was observed. The morphological adaptations observed in the C. chacei retina suggest that it is a high-sensitivity photodetector that is of functional significance to the animal.

Histamine: a putative afferent neurotransmitter in Limulus eyes.

Histamine has been proposed as a photoreceptor neurotransmitter in two major groups of arthropods, the insects and the crustacea. In this study biochemical and immunocytochemical approaches were used to examine the synthesis, endogenous content, and cellular distribution of histamine in the visual system of the horseshoe crab Limulus polyphemus, an ancient chelicerate arthropod. Studies with this animal have been critical to our understanding of the basic processes of vision. High-voltage paper electrophoresis was used to assay for histamine synthesis in Limulus tissues incubated with radiolabeled histidine; histamine synthesis was detected in the lateral, median, and ventral eyes and optic nerves and in the visual centers in the brain. Endogenous histamine, assayed as its orthophthalaldehyde derivative by high-performance liquid chromatography and electrochemical detection, was also detected in these tissues. Immunocytochemical analyses, with an antiserum directed against a protein conjugate of histamine, revealed histamine-like immunoreactivity in the somata of photoreceptors in each of the eyes and in the regions of the brain where the photoreceptors terminate. Histamine-like immunoreactivity was also intense in the cell bodies and axon collaterals of eccentric cells in the lateral eye and in eccentric cell projections in the brain. These results show that histamine is a major biogenic amine in the Limulus visual system, and they suggest that this amine is involved in transmitting visual information from the eyes to the brain and in lateral inhibition, a fundamental mechanism for processing visual information in the lateral eye.

Immunocytochemical localization of opsin, visual arrestin, myosin III, and calmodulin in Limulus lateral eye retinular cells and ventral photoreceptors.

The photoreceptors of the horseshoe crab Limulus polyphemus are classical preparations for studies of the photoresponse and its modulation by circadian clocks. An extensive literature details their physiology and ultrastructure, but relatively little is known about their biochemical organization largely because of a lack of antibodies specific for Limulus photoreceptor proteins. We developed antibodies directed against Limulus opsin, visual arrestin, and myosin III, and we have used them to examine the distributions of these proteins in the Limulus visual system. We also used a commercial antibody to examine the distribution of calmodulin in Limulus photoreceptors. Fixed frozen sections of lateral eye were examined with conventional fluorescence microscopy; ventral photoreceptors were studied with confocal microscopy. Opsin, visual arrestin, myosin III, and calmodulin are all concentrated at the photosensitive rhabdomeral membrane, which is consistent with their participation in the photoresponse. Opsin and visual arrestin, but not myosin III or calmodulin, are also concentrated in extra-rhabdomeral vesicles thought to contain internalized rhabdomeral membrane. In addition, visual arrestin and myosin III were found widely distributed in the cytosol of photoreceptors, suggesting that they have functions in addition to their roles in phototransduction. Our results both clarify and raise new questions about the functions of opsin, visual arrestin, myosin III, and calmodulin in photoreceptors and set the stage for future studies of the impact of light and clock signals on the structure and function of photoreceptors.

Dopamine regulates synaptic transmission mediated by cholinergic neurons of the rat retina.

The purpose of this study was to investigate the effect of dopamine on the function of synapses formed by cholinergic neurons derived from the rat retina. We used an experimental culture system in which rat striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. We found that dopamine could facilitate evoked transmission at retina-muscle synapses. This facilitation by dopamine was reversible and could be blocked by haloperidol, a dopamine receptor antagonist. The adenosine 3':5'-phosphate analogue, 8-bromoadenosine 3':5'-phosphate, mimicked the facilitating effect of dopamine. In addition, dopamine elevated markedly the levels of adenosine 3':5'-phosphate in cultures of rat retinal cells. The results suggest that dopamine can regulate transmission through retinal neurons. Our findings support the hypothesis that a dopamine-induced facilitation of stimulus-evoked transmission involves the activation of dopamine receptors and the intracellular accumulation of adenosine 3':5'-phosphate.

Identification of putative neurotransmitters in the lizard parietal eye.

The retina of the parietal eye of lizards is a simple vertebrate retina that may be useful in studies of basic principles of information processing in visual systems. The chemical substances which mediate the cell-to-cell communication of this eye are not known. As a step in attempting to understand the cellular basis for visual information processing the authors have studied the ability of the parietal eye to synthesize conventional neurotransmitter substances. The eyes were incubated in radiolabeled precursors, then an extract of the tissue was subjected to high-voltage paper electrophoresis. The results of this study indicate that gamma-amino butyric acid (GABA) was synthesized from glutamic acid, acetylcholine (ACh) was synthesized from choline and serotonin (5-HT) was synthesized from tryptophan. Endogenous 5-HT was localized at sites outside the sensory epithelium by immunohistochemical means. Autoradiography following an incubation with 3H-GABA revealed uptake by lens cells and probably both glia and neurons. The authors conclude that GABA and ACh may be involved in the processing of visual information within the parietal eye but that 5-HT probably is not.

Cellular distributions and functions of histamine, octopamine, and serotonin in the peripheral visual system, brain, and circumesophageal ring of the horseshoe crab Limulus polyphemus.

The data reviewed here show that histamine, octopamine, and serotonin are abundant in the visual system of the horseshoe crab Limulus polyphemus. Anatomical and biochemical evidence, including new biochemical data presented here, indicates that histamine is a neurotransmitter in primary retinal afferents, and that it may be involved in visual information processing within the lateral eye. The presence of histamine in neurons of the central nervous system outside of the visual centers suggests that this amine also has functions unrelated to vision. However, the physiological actions of histamine in the Limulus nervous system are not yet known. Octopamine is present in and released from the axons of neurons that transmit circadian information from the brain to the eyes, and octopamine mimics the actions of circadian input on many retinal functions. In addition, octopamine probably has major functions in other parts of the nervous system as octopamine immunoreactive processes are widely distributed in the central nervous system and in peripheral motor nerves. Indeed, octopamine modulates functions of the heart and exoskeletal muscles as well as the eyes. A surprising finding is that although octopamine is a circulating neurohormone in Limulus, there is no structural evidence for its release into the hemolymph from central sites. The distribution of serotonin in Limulus brain suggests this amine modulates the central processing of visual information. Serotonin modulates cholinergic synapses in the central nervous system, but nothing further is known about its physiological actions.

Maturation of neurotransmission at cholinergic synapses formed in culture by rat retinal neurons: regulation by cyclic AMP.

The objective of this study was to investigate how the maturation of neurotransmission is regulated. We used a retina-muscle cell culture system to explore the effects of cyclic AMP analogues on the developmental step in which a presynaptic neuron acquires the ability to transmit excitatory information across a synapse. Cholinergic neurons dissociated from the perinatal rat retina form synapses in culture with rat striated muscle cells. Early in the functional maturation of these retina-muscle synapses, there is a period in which the release of acetylcholine occurs spontaneously, but cannot be evoked. This stage is followed by the emergence of transmitter release that is stimulus-evoked. We report here that exposure of cultured embryonic neurons of the rat retina to 8-bromo-cyclic AMP precociously induced in these neurons the ability to release acetylcholine at synapses in response to excitatory stimulation. This effect on synaptic development could be mimicked by an inhibitor of phosphodiesterase, isobutylmethylxanthine. The results of a variety of experiments lead us to propose that 8-bromo-cyclic AMP may accelerate the development of neurotransmission by influencing presynaptic events linking neuronal depolarization with acetylcholine release. Our data support the hypothesis that cyclic AMP may be an intracellular mediator for developmental signals which regulate the emergence of effective neurotransmission across nascent synapses.

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.

Retinal anatomy of a new species of bresiliid shrimp from a hydrothermal vent field on the Mid-Atlantic Ridge.

A new species of shrimp (Rimicaris sp.) was recently collected from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge. Until the discovery in 1989 that the deep-sea, hydrothermal vent species, Rimicaris exoculata, possessed a hypertrophied dorsal eye, everyone believed that animals recovered from vent environments were blind. Like R. exoculata, Rimicaris sp., a small orange bresiliid shrimp, has an enlarged dorsal eye specialized for detecting light in a very dim environment instead of the expected compound eye. The individual lenses characteristic of a compound eye adapted for imaging have been replaced in Rimicaris sp. by a smooth cornea underlain by a massive array of photosensitive membrane. The number of ommatidia in this species is about the same as in shrimp species that live at the surface; however, the photoreceptors are larger in the deep-sea species and the shape of the photoreceptors is markedly different. The light-sensitive region of the photoreceptor is much larger than those of other shrimp and the rest of the receptor is much smaller than normal. All screening pigment has moved out of the path of incident light to a position below the retina, and the reflecting pigment cells have adapted to form a bright white diffusing screen between and behind the photoreceptors. The ultrastructure of the microvillar array comprising the rhabdom is typical for decapod crustaceans; however, there is a much greater volume density of rhabdom (80% to 85%) than normal. There is no ultrastructural evidence for cyclic rhabdom shedding or renewal. Rimicaris sp. has apparently adapted its visual system to detect the very dim light emitted from the throats of the black smoker chimneys around which it lives.

Molecular and immunological characterization of a Gq protein from ventral and lateral eye of the horseshoe crab Limulus polyphemus.

GTP binding proteins of the Gq family have been implicated in phototransduction in rhabdomeral photoreceptors. In this study we used molecular and immunochemical techniques to characterize a GTP-binding protein alpha subunit of the Gq family in ventral and lateral photoreceptors of the horseshoe crab, Limulus polyphemus. Both ventral photoreceptors and lateral eye retinular cells became strongly labeled with an antibody directed against the common carboxyl tail of two Gq family proteins, G alpha q and G alpha 11. This antibody also labeled a 42 kDa band on Western blots of proteins from ventral photoreceptor cell bodies, ventral photoreceptor axons, lateral eyes and lateral optic nerves. The reverse transcription-polymerase chain reaction (RT-PCR), along with degenerate oligonucleotide primers designed against conserved regions of known G alpha q and G alpha 11 proteins, was used to isolate a cDNA from ventral eye RNA which encodes a protein with high identity to known Gq proteins. Ribonuclease protection assays showed that the corresponding message was expressed in ventral eye, but these assays, as well as Northern blots, failed to detect expression in lateral eye. Therefore, while photoreceptors of both ventral and lateral eyes contain a Gq-like protein, the mRNA encoding the Gq protein in the ventral eye may differ in nucleotide sequence from its lateral eye counterpart.

What the clock tells the eye: lessons from an ancient arthropod.

Circadian changes in visual sensitivity have been observed in a wide range of species, vertebrates, and invertebrates, but the processes impacted and the underlying mechanisms largely are unexplored. Among arthropods, effects of circadian signals on vision have been examined in most detail in the lateral compound eye (LE) of the American horseshoe crab, Limulus polyphemus, a chelicerate arthropod. As a consequence of processes influenced by a central circadian clock, Limulus can see at night nearly as well as they do during the day. The effects of the clock on horseshoe crab LE retinas are diverse and include changes in structure, gene expression, and rhabdom biochemistry. An examination of the known effects of circadian rhythms on LEs shows that the effects have three important outcomes: an increase in visual sensitivity at night, a rapid decrease in visual sensitivity at dawn, and maintenance of eyes in a relatively low state of sensitivity during the day, even in the dark. All three outcomes may be critically important for species' survival. Specific effects of circadian rhythms on vision will certainly vary with species and according to life styles. Studies of the circadian regulation of Limulus vision have revealed that these effects can be extremely diverse and profound and suggest that circadian clocks can play a critical role in the ability of animals to adapt to the dramatic daily changes in ambient illumination.