Barbara-Anne Battelle

The morphology of the dorsal eye of the hydrothermal vent shrimp, Rimicaris exoculata

The bresiliid shrimp, Rimicaris exoculata, lives in large masses on the sides of hydrothermal vent chimneys at two sites on the Mid-Atlantic Ridge. Although essentially no daylight penetrates to depths of 3500 m, very dim light is emitted from the hydrothermal vents themselves. To exploit this light, R. exoculata has evolved a modified compound eye on its dorsal surface that occupies about 0.5% of the animals body volume. The eyes morphology suggests that it is extremely sensitive to light. The cornea of the dorsal eye is smooth with no dioptric apparatus. The retina consists of two wing-shaped lobes that are fused across the midline anteriorly. The rhabdomeral segments of the 7000 ommatidia form a compact layer of photosensitive membrane with an entrance aperture of more than 26 mm2. Within this layer, the volume density of rhabdom is more than 70%. Below the rhabdomeral segments, a thick layer of white diffusing cells scatters light upward into the photoreceptors. The arhabdomeral segments of the five to seven photoreceptors of each ommatidium are mere strands of cytoplasm that expand to accommodate the photoreceptor nuclei. The rhabdom is comprised of well-organized arrays of microvilli, each with a cytoskeletal core. The rhabdomeral segment cytoplasm contains mitochondria, but little else. The perikaryon contains a band of mitochondria, but has only small amounts of endoplasmic reticulum. There is no ultrastructural indication of photosensitive membrane cycling in these photoreceptors. Vestigial screening pigment cells and screening pigment granules within the photoreceptors are both restricted to the inner surface of the layer of the white diffusing cells. Below the retina, photoreceptor axons converge in a fanshaped array to enter the dorsal surface of the brain. The eyes size and structure are consistent with a role for vision in shrimp living at abyssal hydrothermal vents.

Central origin of the efferent neurons projecting to the eyes of Limulus polyphemus

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 microns. 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.

Isolation and expression of an arrestin cDNA from the horseshoe crab lateral eye

Abstract: Electrophysiological studies of photoreceptors from the horseshoe crab Limulus polyphemus continue to provide fundamental new knowledge of the photoresponse in invertebrates. Therefore, it is of particular interest to characterize the molecular components of the photoresponse in this system. Here we describe an arrestin cloned from a cDNA library constructed using poly(A)+ RNA isolated from Limulus lateral eyes. The protein, deduced from the arrestin cDNA, is most similar to arrestin from locust antennae (56% identity) and Drosophila phosrestin I (53% identity). Limulus arrestin was expressed in a heterologous system, and its properties were compared with those of a 46‐kDa light‐regulated phosprotein (pp46A) in Limulus photoreceptors described in previous studies from this laboratory. Arrestin and pp46A (a) have the same apparent molecular weight on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, (b) have an isoelectric point in the basic pH range, (c) require calmodulin and elevated Ca2+ levels for phosphorylation, (d) are immunoreactive with monoclonal antibody C10C10 directed against a sequence in bovine arrestin (S‐antigen) that is perfectly conserved in the deduced arrestin protein, and (e) are associated with photoreceptors. We conclude that the arrestin described here and pp46A are the same protein. The results of this and previous studies show that in Limulus photoreceptors, light regulates the phosphorylation of arrestin in complex ways.

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

SUMMARY 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 animals 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.

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. J. Comp. Neurol. 435:211–225, 2001.

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. Microsc. Res. Tech. 44:70–80, 1999.

Multiple mechanisms of rhabdom shedding in the lateral eye of Limulus polyphemus

Rhabdom shedding in horseshoe crab lateral eye photoreceptors was studied with anti‐opsin and anti‐arrestin immunocytochemistry. Two, possibly three, distinct shedding mechanisms were revealed in animals maintained in natural lighting. Transient rhabdom shedding, triggered by dawn, is a brief, synchronous event that removes up to 10% of the rhabdom membrane. Whorls of rhabdomeral membrane break into vesicles and form compact multivesicular bodies. These debris particles are immunoreactive for opsin and are of a relatively uniform size, averaging approximately 2 μm2 in area. Transient shedding requires that input from circadian efferent fibers to the retina precedes the light trigger, and cutting the optic nerve blocks efferent input and transient shedding. Light‐driven rhabdom shedding is a progressive process. Rhabdomeral membrane is removed by coated vesicles that accumulate into loosely packed multivesicular bodies. These debris particles label with antibodies directed against opsin, arrestin, and adaptin, and they have a large distribution of sizes, averaging almost 6 μm2 in area and ranging up to 25 μm2 or more. The amount of rhabdomeral membrane removed by light‐driven shedding has seasonal variation and depends on latitude. Light‐driven shedding does not require circadian efferent input. A possible third shedding mechanism, light‐independent shedding, is observed when transient shedding is blocked either by 48 hours of darkness or by cutting the optic nerve. Small particles, averaging 1.8 μm2 in area, exhibiting opsin but not arrestin immunoreactivity can then be found in the cytoplasm surrounding the rhabdom. The nature of light‐independent shedding is not yet clear. J. Comp. Neurol. 449:26–42, 2002.

Light-regulated proteins in Limulus ventral photoreceptor cells

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.

Identification and Function of Octopamine and Tyramine Conjugates in the Limulus Visual System

Abstract: Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as γ‐glutamyl octopamine and γ‐glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine‐rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of γ‐glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that γ‐glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that γ‐glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.

Histogenesis of dopamine-containing neurons in the rat retina.

The point during development when a neuron in the central nervous system ceases to divide is a crucial early step in differentiation which may be a prerequisite for phenotypic expression. In this double-label study, which combines [3H]thymidine autoradiography with histochemical detection of endogenous dopamine, we examined the histogenesis of dopamine-containing cells (DA-cells) in the amacrine layer of the rat retina. We found that most DA-cells present in the adult rat retina stop dividing between embryonic days 16 and 20. These results show that most cells destined to become dopaminergic neurons are generated during a discrete interval within the extended period of amacrine-cell production, and that these cells are produced in the embryonic retina.