The Visual System of the Horseshoe Crab Limulus polyphemus

The Visual System of the Horseshoe Crab Limulus polyphemus WOLF H . FAHRENBACH hboratory of Electron Microscopy. Oregon Regional Primate Research Center. Beaverton. Oregon

I . Introduction . . I1. Dioptric Structures

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. . . . A . Cuticular Cones and Lenses . B. Lens Epidermis . . . . C . ConeCells . . . . . 111. Pigment Cells . . . . .

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IV . V.

VI . VII .

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IX. X.

XI.

A Guanophores . . . . B. Distal Pigment Cells . . C . Ommatidial Pigment Cells Neuroglial Cells . . . . Receptor Cells . . . . A . Retinula Cells . . . B. Eccentric Cells . . . C Arhabdomeric Cells . . Basal Lamina and Hemocoel Axons and Plexus . . . A . Afferent Axons . . . B. Efferent Axons . . . C Plexus . . . . . OpticNerves . . . . . Optic Centers . . . . Miscellaneous Aspects . . A . Abnormalities . . . . B. Pathology . . . . . Vision and Behavior . . . Addendum . . . . . References . . . . .

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Introduction

Two noteworthy events were recorded for the year 1782. to wit. the last execution of a witch and the first microscopic study of the lateral eye of Limulus (AndrC. 1782). This auspicious start of a rational age was followed after about a century by two histological studies (Grenacher. 1879; Lankester and Bourne. 1883). which to this day are a pleasure to read . Later. WatasC (1890).by painstaking maceration and teasing of eyes. provided insight into the cellular composition of the ommatidium. including the presence of a ganglion (i.e., eccentric) cell . An analysis of the cuticular dioptrics of the compound 285

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eye (Exner, 1891) and a description of the ventral eye (Patten, 1893) laid the foundation for the only previous review of eye structure in the horseshoe crab (Demoll, 1914). [The correct name of the American horseshoe crab is Limulus polyphemus. The name Xiphosura pertains to its order within the Merostomata and has been suppressed as a generic name by the International Commission on Zoological Nomenclature (Stpjrmer, 1952; Levi, 1968).] The continuing interest in Limulus eyes arises principally from their neurophysiological accessibility. Studies of visual physiology in Limulus, complete with a Nobel prize and a voluminous bibliography, have contributed considerably to our understanding of the function of photoreceptors but are treated in this article only in relation to definitive structural elements. Two recent physiological reviews, one by Wolbarsht and Yeandle (1967),which also covers some pertinent systematics and zoogeography of the xiphosurans, and the other, a comprehensive study of ommatidial electrophysiology by Smith and Baumann (1969),furnish the reader ready access to the literature on the function of the Limulus eye. To a biologist accustomed to vertebrate tissues, the fine structure of the Limulus visual system is a somewhat bewildering and alien landscape. Consequently, the primary aim of this article is to supply a diagnostic guide to the various cells and tissues of the eyes and to review the current status of information on their structural-functional correlations. The anatomical orientation of most previous publications is acknowledged by means of diagrams and survey illustrations, but a cytological emphasis is considered more useful for future experimental work with Limulus. As far as is practical in the context of a review, my own work has been updated by new observations, and various small informational gaps in the literature have been closed. The most prominent eyes of the horseshoe crab are the lateral compound eyes, bean-shaped, convex structures set into the cuticle under the lateral spines of the prosoma. The two ocelli are centrally placed on the prosoma on either side of the median dorsal spine. The lateral rudimentary eyes, rather unorganized masses of photoreceptor cells and guanophores, lie at the posterior border of each compound eye. A similar mass, the median rudimentary (endoparietal) eye, accompanies the two ocelli. It is bipartite but fused into a Y-shaped organ, An additional photoreceptor, the ventral eye, consists of two groups of cells at the base of the so-called olfactory tubercle, a small protuberance anterior to the mouth. The two nerves from this eye contain additional visual cells scattered along their length. A few

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receptor cells, associated with the ventral eye nerve but positioned at the surface of the second optic ganglion, complete the inventory of the Lirnulus photoreceptors. The phylogenetic relationship between these eyes and those of other arthropods is best appreciated by reference to Eakin’s (1970) comprehensive review of invertebrate eyes. 11. Dioptric Structures

A.

CUTICULAR CONES AND

LENSES

Only the compound and ocellar eyes contain sculptural specializations of the overlying cuticle, which is composed of tanned proteins and about 25% chitin (Hock, 1940) and is densely lamellated (Richter, 1969). The lenses of the ocelli, quite similar to those of spider ocelli (Carricaburu, 1970) and first described by Demo11 (1914), are each formed by a subspherical, internal projection of the transparent cuticle over the eye, with a diameter of about 0.5 mm in an animal of 5-cm width (Figs. 1 and 2). [Data relating molting stage, age, size, and eye size of Lirnulus have been compiled by Waterman (1954a) and Shuster (1955).]The cuticular lamellae dip into the lens

FIG. 1. Internal view of cleaned cuticle with the two ocellar lenses. The deep recess is the median frontal spine of the animal. x60.

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FIG. 2. Vertical section through a median ocellus. The spherical lens (L) is lined by lens epidermis, which changes to a pigmented epithelium laterally (P). Receptor cells (R)with contorted rhabdomeres sandwich guanophores (C).Optic fibers (A) and vascular sinusoids (S) border the base of the micrograph. x 150.

as they do into the cuticular cones of the compound eye. The lens proper has a refractive index gradient of 1.591at its proximal periphery to a lower one (1.555) at its center; but the overlying cuticle, although heterogeneous in its refractive properties, incorporates a weak converging lens (center refractive index 1.567; periphery 1.553) above the ocellar sphere (Carricaburu, 1968). However, the lens was tested at a wavelength of 585 nm, at which the ocellus is not a particularly sensitive photoreceptor. Although not image-forming,

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the shape of the lens and the immediately subjacent array of retinulae suggest that the ocellus can at least convey directional information. Its large aperture in relation to that of an ommatidium makes it particularly adapted to seeing at low light levels. The cuticle of the compound eye is smoothly curved with hardly any sculptural indication of facets. However, its internal surface is covered with blunt-tipped cuticular cones (Figs. 4 and 5), first and excellently illustrated by And& (1782). Despite the initial impression of regularity, the precision of rows is maintained only over short stretches, and the “squeezing out” of lattice rows is common (these features are best observed by viewing Fig. 4 at a glancing angle). The array of cones gains its adult number of about 850 rapidly (Wa-

FIG.3. Detail of the relationship between ocellar lens and lens epidermis (Le), retinula cells (R) and their rhabdomeres, guanophores (G), optic fibers (A), and adjacent pigmented epidermis (P). X330.

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terman, 1954a)and is practically complete in an animal 5-10 cm wide. The cones are added peripherally and grow both in size and spacing with successive molts. Only a few at the center of the eye are at right angles to the surface; the remainder deviate from normal orientation to a maximal inclination of 55" at the periphery of the eye. Discounting the acceptance angle of individual ommatidia, this slanted insertion of the cones (k55") added to the curvature of the cuticle provides a 180" horizontal field of vision for each over the eye (70") eye, The vertical field of view is about 90". Any experimentation that involves the directional sensitivity of ommatidia (Waterman, 195413) or of individual retinula cells (Ratliff, 1966) is profoundly affected by the slanted insertion of most of the cones. The system of Gemperlein (1969) for encoding the location of specific facets, although potentially an ideal method for locating the few vertical cones, may be difficult to apply to the irregular array of the Limulus compound eye. Exner (1891) originally interpreted the cones as cylinder lenses, that is, structures that bend the light toward the optical axis by a gradient of refractive indices. Such cases have since been observed in fireflies, and their properties have been discussed (Seitz, 1969; Horridge, 1969), but the Limulus cuticular cone appears to have a

FIG.4. Internal view of the compound eye cuticle cleaned of adhering tissue. The surrounding cuticle is perforated by gland ducts. X60.

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rather homogeneous refractive index (Carricaburu, 1967). Hence the function of the cone can best be understood as that of a wave guide. Light entering the cone at an angle to its axis undergoes total internal reflection, the limiting angle depending on the refractive index of the cone, the surrounding epidermal cells, and the concentration of pigment near the cone. This concentration would be expected to increase the angle of total reflection during light adaptation and would therefore allow more light to be absorbed by the pigment than in a dark-adapted eye (Seitz, 1969). Ray tracing on the basis of estimated refractive indices yields an acceptance angle of 70" for the Limulus ommatidium (Fahrenbach, 1968), which is reasonably close to the neurophysiologically derived result of 80"(Waterman, 1954b). This unusually high angle is probably not caused by leakage from

FIG.5. Vertical section through a cuticular cone and apical ommatidium. Vascular spaces (S) are bordered by guanophores (G) and distal pigment cells (P). C, Cone cells; R, retinula cells E, eccentric cell dendrite. The cuticle and subcuticular space have been invaded by fungus (F). X350.

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adjacent ommatidia because (1)the pigment surrounding the cones never leaves the region between the cones, and (2) little optical interaction between ommatidia could be detected by physiological means (Scholes and Hartline, in Shaw, 1969). Lateral inhibition probably provides a compensating mechanism to correct the overlap between the visual fields of adjacent ommatidia (Reichardt, 1961).A combination of eye curvature, angle of cone insertion, and angle of ommatidial acceptance provides for virtually omnidirectional vision in Limulus. The compound eye does not discriminate polarized light at normal incidence to the surface, but differentiates to an increasing degree with oblique illumination (Waterman, 1954~).However, the numerous concentric cuticular lamellae of the cone, first noted by Grenacher (1879),are made up of 150-A, swirling filaments which, like those of the ocellar lens, have enough form birefringence to effect complete depolarization of light transmitted through the cone (Fahrenbach, 1968). Therefore sensitivity to polarized light at slanting incidence must be attributed to spurious effects of reflection at the cuticular surface, a process that yields different intensities for different vectors of polarization. Furthermore, the cytological prerequisites for polarized light perception are lacking because of irregular rhabdomeres in the ocelli and electrotonic coupling of rhabdomeres in the ommatidia, a feature that effectively abolishes polarization sensitivity (Snyder et al., 1973).

B. LENS EPIDERMIS The epidermis underlying the nonocular cuticle is usually lightly pigmented in Limulus. The epidermis of the eyes, however, although retaining distinctive characteristics and general functions associated with molting, becomes variously modified into lens epidermis, cone cells, guanophores, and distal pigment cells. Since it is the most generalized type, the epidermis of the ocellar lens can serve here as the standard of comparison for the more deviant cell types (Figs. 2 and 3). This internal lining of the lens consists of highly interlaced, unpigmented cells with a plethora of junctions which emphasize the mechanical role of the tissue. At the apical surface, dense plaques resembling hemidesmosomes mediate the epitheliocuticular bond in conjunction with bundles of microtubules inserted on the plaques. This distinctive arthropodan junction (Bassot and Martoja, 1965, 1966; Noirot-TimothCe and Noirot, 1966) is often associated with an anchoring filament deeply embedded in the cuticle, as it is in other

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FIG. 6. Cross section of dark-adapted ommatidia. The stellate rhabdornes display a few branched or looped fins. The vascular interstices contain hemocytes. ~ 1 0 0 .

arthropods (Bouligand, 1966). During the intermolt stage the epidermal surface forms a flush apposition to the cuticle. The predominantly microvillous surface frequently found in young animals and illustrated by Whitehead et al. (1969) presumably indicates an early molting stage, when a molting space is formed (Locke, 1969). Laterally, the cells are attached by an apical macula adhaerens and by extensive septate desmosomes. The basal surface is profusely fluted and covered by a thick basal lamina. Large quantities of glycogen are a distinguishing feature of these cells, but probably not to the extent of affecting the dioptrics of the system as in some other invertebrate eyes (Wolken and Florida, 1969; Eakin and Kuda, 1972). Longitudinal bundles of microtubules bypass the small basal nucleus and are inserted on dense plaques adjacent to the basal lamina. C. CONECELLS The cone cells of the compound eye, equivalent to the Semper cells of insects, were mentioned by Grenacher (1879) and Exner (1891)and have been described in detail by Fahrenbach (1968,1969) and by Whitehead et al. (1969) (Fig. 5). About 100 of these underlie the flat tip of the cuticular cone. A dozen of the most peripheral cells, their exact number corresponding to the rhabdomal fins, have long

CuticIe

Guanophore

Distal pigment cell

Cuticular cone

Basal lamina

. Basal infolding5 . Cone cells

Cone cell process

. Eccentric

cell dendrite

. Pigment cell partition Palisade

. Rhabdome . Relinula cell

Rhabdome

- Eccentric cell - Proximal pigment cells

- Neuroglial sheath

- Retinula cell axon

-

Eccentric cell axon

FIG.7. Semidiagrammaticview of a cutaway ommatidium. (Redrawn after Fahrenbach, 1969.)

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attenuated processes which project to the bottom of the rhabdome at its periphery. The cone cells are even more featureless than the epidermis of the lens, containing few scattered organelles and some glycogen but primarily conveying an impression of transparency. Their apical junctions are similar to those described for the epidermal cell but lack the conspicuous participation of microtubules or cuticular anchoring filaments. The interlocked cells are attached laterally by occluding junctions rather than by the septate or gap junctions that prevail in other regions of the ommatidium. Basally, the cone cells directly abut the retinula cells and eccentric cell dendrite and are attached to both by adhering junctions (Fig. 18). These, however, do not effect a complete seal, because hemocyanin from adjacent blood spaces frequently enters the seam and thereby gains access to the apical region of the rhabdome. The cone cell processes, called crystalline threads in insects, lie at the edge of each rhabdomal fin and are bonded by uninterrupted fasciae adhaerentes to the two adjacent retinula cells. They are flattened, 0.1-0.4 pm thick, up to 200 pm long, and contain only sparse inclusions (Figs. 7 and 14). Several functional possibilities have been advanced for these crystalline threads, primarily in insects. Exner (1891)suggested that they act as light guides, a possibility that has been explored by Horridge (1969) in the firefly Photuris uersicolor, in which the processes bridge a gap of more than 100 pm between the cuticular cone and the rhabdome and in fact act as wave guides. In Limulus, however, the cone cell processes are located at the periphery of the rhabdome and are therefore not in the proper location to direct light into it. The small dimension of the processes in relation to the wavelength of light in addition to the surrounding pigment would, furthermore, produce rapid attenuation of the conducted light. The overall optical contribution of the cone cell processes is probably negligible. A second suggestion concerns the influence of the extensive adhering junctions on current flow in the ommatidium during light-induced depolarization (Fahrenbach, 1969). The possibility that these junctions introduce an impediment to ionic flux along the intercellular cleft and increase the current flow through the eccentric cell has been clarified by tracer studies. Perrelet and Baumann (1969) exposed bees’ eyes to lanthanum and ferritin and found that both electron-opaque substances traversed the fasciae adhaerentes and entered the intermicrovillous spaces of the rhabdome. Parenthetically, the rhabdomes of all investigated arthropods are peripherally

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cemented by adhering junctions between the constituent retinula cells, but not necessarily with participation of the cone cell processes. Hence the last and simplest interpretation remains: In Limulus, the cone cell processes with their extensive junctions subserve the mechanical role of assuring cellular cohesion at the periphery of the rhabdome. At the base of the ommatidium, where retinula cells touch directly without the intervention of a cone cell process, they nevertheless display similar junctions around the rhabdome. 111. Pigment Cells A. GUANOPHORES

Guanophores are widely distributed throughout the visual system. They occur between cuticular cones of the compound eyes and in partitions between retinula cells in the ocelli, in large numbers in the rudimentary eyes (Figs. 5, 7, 8, and 9),and even under the cap-

FIG.8. Part of the lateral rudimentary eye. The mass consists of guanophores. Adjacent axons (A) are those of nearby rudimentary eye retinula cells. x 100.

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FIG.9. A cluster of receptor cells embedded in the guanophores (G) of the lateral rudimentary eye. Large inclusion masses are a conspicuous feature of these cells. Rh, rhabdomere; A, axons; N, small nerve. ~ 2 5 0 .

sule of the brain in association with common ectopic retinula cells near the second optic ganglion. Unlike the reflecting pigment in most arthropods, that in guanophores is in fact guanine (Kleinholz, 1959).Birefringent guanine crystals in the shape of polygonal blocks or thick platelets, up to 1.5 pm in diameter, are formed in opaque Golgi-derived droplets and maintain a limiting membrane. The crystals are resistant to sectioning and normally leave angular holes in the section (Fig. 15). The epidermal guanophores of the compound eye line the deep cuticular recesses between cones (Fig. 7) and thus form the white, refractile surround of the compound eye facets that accounts to a large degree for the pseudopupil of the Limulus eye. An overall impression of an active cell type is supported by ample endoplasmic reticulum, free ribosomes, Golgi bodies, and various lysosomal structures. Microtubules are inserted as previously described and often exceed 100 in a single bundle, In these cells, as well as in the distal pigment cells, two functions can be attributed to these fascicles, the static one of mediating cellular attachment to the cuticle, and the dynamic one of governing the rather rapid photomechanical pigment migration. After light adaptation the guanine platelets are concen-

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trated under the cuticle, but are distributed throughout the cell in the dark-adapted eye. The guanophores of the ocelli (Fig. 3) have no connection with the cuticle but clothe the sides of the receptor cells. Nevertheless, they are remarkably similar to the guanophores of the compound eye in their extensive Golgi cisternae, glycogen, and scattered microtubules running longitudinally in these slender and interlocked cells. The contained guanine platelets have not been observed to migrate; neurosecretory terminals and microtubules, similar to those in cells with mobile pigment, provide positive circumstantial evidence that migration may occur. The remaining category of guanophores, which incidentally are devoid of any innervation, consists of cells filled to capacity with guanine crystals at the expense of practically all other organelles except a small nucleus. These cells comprise virtually the entire mass of lateral and median rudimentary eyes (Fig. 9), and are associated with ectopic retinula cells, such as those in the brain. Their engorged contents, lack of requisite filaments or tubules, and random disposition around photoreceptor cells minimize any possibility of pigment migration. Guanophores of this type are also widely scattered in the connective tissue of Limulus, far from any photoreceptor site, and may function as storage cells of an excretory product which has found secondary utility because of its refractile properties. Rare guanophores in the ocelli and rudimentary eyes contain an approximately equal percentage of guanine platelets and ommochrome pigment droplets. These pigment droplets occur as an occasional admixture in mature guanophores, particularly those of the ocelli. The converse condition, that is, guanine “contamination” of ommatidial pigment cells, has not been found.

B. DISTALPIGMENTCELLS Each ocellus is surrounded by a broad zone of pigmented cells (Figs. 2, 3, and 10) that mirror the distal pigment cells of the compound eye in practically all cytological details except that they form an uncomplicated, pseudostratified epithelium. In the compound eye the stray light between cuticular cones is screened out by the heavily pigmented distal pigment cells (Fahrenbach, 1968; Whitehead et aZ., 1969)that are attached to the sides of the cone and form a cap over the apical part of the ommatidium. Although the cells are slender and up to 300 pm in length, they correspond closely to the previously described basic epidermal type in their apical and lateral junctional specializations, changed appearance of the apical surface

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FIG. 10. Pigment cells rimming the cuticle around the ocellar lens (see Fig. 2). This pseudostratified epithelium shows apical adhesive modification (J), longitudinal microtubule bundles (B), mitochondria (M), and large pigment droplets. The subjacent vascular tissue is bordered by reserve cells (Re) and contains a hemocyte (H), a discharged hemocyte (D), and two cyanoblasts (Cy). x 1500.

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during molting, deep basal flutings with an underlying basal lamina, and fascicles of microtubules which are particularly aggregated just below the cuticle. Apical and basal insertions of microtubules on hemidesmosomes are most easily demonstrated in these cells. The pigment, deep violet to black, is contained in 0.2 to 0.8-pm membrane-bounded droplets which are totally electron-opaque. Butenandt et al. (1958) identified the substance with chromatography and ultraviolet spectroscopy as an ommin, that is, a high-molecularweight ommochrome (contra Wasserman, 1967). A second ommochrome, an ommidin, which is a low-molecular-weight derivative of tryptophan and methionine, has been described in Limulus by Linzen (1966). The natural screening properties of the pigment were found by Wasserman (1967)to be those of a neutral absorber; but action spectra of retinula cells with and without shielding pigment, the latter in albino Limulus, demonstrate that the difference can be attributed to a red-transmitting pigment (Nolte and Brown, 1970). During dark adaptation (Fig. ll),the pigment is mostly retracted to

FIG.11. Diagrammatic representation of pigment distribution in the dark-adapted (left) and light-adapted (right) ommatidium in longitudinal and cross section.

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the base of the cells, that is, around the distal third of the ommatidium; however, a scattering of granules remains elsewhere. Light adaptation, which is complete in about 10 minutes, moves the pigment distad to a subcuticular position, leaving an apical zone of about l p m pigment-free. At the level of the cone cells, the ascent of pigment in the surrounding distal pigment cells forms an iridal aperture which restricts the exit pupil of the cone to a circle equal to or smaller than the diameter of the flattened tip of the cone (20-50 pm). The cone and apex of the ommatidium become shielded thereby to a degree that precludes light leakage to adjacent units. The distal pigment cells, like the adjacent guanophores with mobile pigment, are supplied with efferent neurosecretory fibers. C. OMMATIDIAL PIGMENTCELLS Two groups of these cells have been differentiated on the basis of location (Fahrenbach, 1969): intraommatidial pigment cells, whose nuclei and cell processes lie in the partitions between retinula cells of an ommatidium (Figs. 12 and 14); and about 100 proximal pigment

FIG. 12. Cross-sectional view of a dark-adapted ommatidium, illustrating the eccentric cell dendrite (E) in the center of the rhabdome, the nucleus of the unsymmetrical retinula cell (U), pigment cell partitions (P), and adjacent vascular space. The bridging tissue between the adjacent ommatidia carries efferent fibers. x 1OOO.

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cells which lie as a cup-shaped mass at the base of the ommatidium but extend for some distance distally. The two cell types are distinguishable only by their nuclei. Those of the intraommatidial cells are predominantly spherical and 4-5 pm in diameter, although some are pyknotic and quite small, depending perhaps on the stage of development. The nuclei of the proximal pigment cells, in contrast, are 10-12 pm long and located at or below the base of the retinula cells. The intraommatidial pigment cells, also described as pigmented glial cells by Lasansky (1967), form sheaths one to six cells deep around all the visual cells and partitions between them, and invaginate to a variable degree into the visual cells, particularly the eccentric cell, but not to tlie same degree as in invertebrate central neurons. The cells are filled with pigment droplets (ommochrome) and contain longitudinally oriented bundles of microtubules which lie for the most part in the peripheral folds of the deeply fluted cells. The cytoplasm abounds with glycogen in the rosette-shaped a form and has a normal set of organelles, but these cells do not appear to metabolize pigment with any intensity. Intraommatidial cells have very few junctions of any kind. At the periphery of the rhabdome, where the cone cell processes are bonded to adjacent retinula cells by adhering junctions, pigment cells are occasionally seen to participate. More commonly, proximal pigment cells provide such junctions at the base of the ommatidium proximal to the termination of the cone cell processes. Extensive quintuple-layered junctions which have been described between pigment and receptor cells (Lasansky, 1967) can be produced at will as an artifact of high sucrose concentration in the fixative (Fahrenbach, 1969). The periphery of ommatidia varies between a compact sheath of pigment cells with no interstices to speak of to an open cancellous architecture (Figs. 6 and 12), especially in older animals. Although fixation has an undeniable influence, the feature may be mostly a function of age, stage of nutrition, and degree of dark adaptation, the last of which involves drastic changes in the distribution of pigment cell volume. The proximal pigment cells (Fig. 24) form a compact, cup-shaped mass whose cellular details show no noteworthy differences from the intraommatidial pigment cells except for their larger nuclei. The pigment in both cell types, which is withdrawn to the base of the ommatidium during dark adaptation (Fig. ll), starts migrating upward in long, pearl necklace-like columns into the ommatidium within minutes of the onset of light. This movement, parallel and adjacent to microtubule sheaves, leads to a pronounced increase in the pig-

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FIG. 13. Detail of the center of the rhabdome. The eccentric cell dendrite (E) abounds with microtubules and agranular cisternae. It has a few peripheral microvilli and a subsurface filamentous zone. Rhabdomeral microvilli from two adjacent retinula cells abut along the line indicated by the arrow. The fully formed palisade (Pa) indicates the dark-adapted state. X15,OOO.

mentation and thickness of the partitions between retinula cells, particularly at the edge of the rhabdomal fins. Both cell types have a conspicuous supply of neurosecretory fibers which terminate on them. Scattered cells containing ommochrome can be found in the septa between the retinula cells of the ocelli, in the plexus, and occasionally in the ventral eye and ocellar nerves. These are usually rather compact and give no indication of pigment migration; they do not appear to receive efferent fibers. Lipetz (1960) determined that the ommatidial pigment cells have an approximately 40-fold higher electrical resistivity than the adjacent retinula cells or body fluid and suggested that they therefore

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form an insulating sleeve to confine light-induced current flow to the center of the ommatidium, that is, to the eccentric cell dendrite. Given the more recent findings of patent intercellular pathways from the rhabdome to the periphery (Lasansky, 1970; Perrelet and Baumann, 1969)and the tight coupling between receptor cells (Behrens and Wulff, 1965; Borsellino et al., 1965; Smith et al., 1965),the electrical properties of the pigment cells probably have only a marginal effect on the performance of the ommatidium. IV. Neuroglial Cells The distribution of neuroglial cells in the visual system is inversely related to the abundance of pigment cells. The ommatidia contain relatively few glial cells; most of them serve as sheaths for the eccentric cells, and some are Schwann cells carrying efferent fibers. Guanophores act as glial cells in the ocelli and rudimentary eyes, but frequently a glial cell layer is inserted between the pigment and retinula cells in these receptors. The ventral eye, which is not in intimate contact with pigment cells, displays an elaborate layering of neuroglia (Clark et al., 1969b). All axons and synaptic regions are surrounded by glial sheaths of different complexities (Figs. 22, 23, and 28). Several criteria can be adduced to identify neuroglial cells. Their condensed nuclei, with average diameters of 2-6 pm, are uniformly the smallest among those of neighboring cell types. Generally, their cytoplasm is distinctly less opaque than that of other cells and contains some lipofuscin (gliosomes) but no shielding pigment, scattered minute mitochondria, and often considerable granular vesicles and tubules which should not be confused with more regular synaptic vesicles in the plexus. Often glial cytoplasm appears so vacuous as to require visual reference to rare twists of endoplasmic reticulum or seemingly free-floating Golgi bodies to ascertain its cellular nature. Since this transparency has been observed in many animals and with many fixatives, it is probably a true characteristic. Other types of neuroglial cells in the Limulus brain display a wealth of organelles and inclusions under identical conditions of fixation. Glial cells are capable of secreting basement lamina material, as is unambiguously illustrated by the external lamina of all branches of the plexus (Fig. 27) and within the optic nerve, or more strikingly by the thick, multilayered sheath which occasionally surrounds small efferent nerves (Fig. 23). Few if any junctions are found between glial cells or adjacent to

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receptor cells. Rare five-layered junctions should probably be viewed with reservations for previously mentioned reasons. In a few places, for example, in the ventral eye, glial cells directly abut the rhabdomeral microvilli, where they form gap junctions. Glial cell membranes facing adjacent hemocoel, that is, the intervening basal lamina, are decorated with abundant dense plaques, a presumptive adhesive modification vis-8vis the basal lamina. Another characteristic of neuroglial cells, shared with both pigment and reserve cells, is their extremely attenuated and filmy shape which finds its most extreme expression in the lateral rudimentary eye, where 10 to 30 complex folds occasionally produce a solid

FIG.14. The periphery of the rhabdome. Pa, Palisade; C, cone cell process; R, retinula cell; A, efferent axon. Intraommatidial pigment cells form partitions between (arrows)and infoldings (I) into retinula cells. ~ 7 0 0 0 .

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peripheral glial mass (Fahrenbach, 1970a). In the more orthodox situations, one to several layers of glial cells invest parts of the eccentric cell, the receptor cells in the ocellar, ventral, and rudimentary eyes, and all axons. Glial folds invaginate into receptor cells to some degree but not to the extent of breaking up the periphery of the neuron into the configuration associated with a trophospongium. Large axons, like those of eccentric cells, and rudimentary or ocellar receptors, receive an individual sheath of a few neuroglial cells, whereas retinula cell axons of the compound eye travel in fascicles of two to six fibers (the number decreasing with age) in juxtaposition to one another and are usually enveloped by a single glial cell (Dumont et al., 1965; Nunnemacher and Davis, 1968; Fahrenbach, 1970a, 1971) (Fig. 22).

V. Receptor Cells An unusual attribute of Limulus photoreceptors is the presence of second-order visual neurons in juxtaposition to primary visual cells, that is, retinula cells. Eccentric cells play this role in the ommatidia and arhabdomeric cells in the ocelli. Much of the neurophysiological utility of the compound eye lies in the fact that the eccentric cell provides an accessible neural output in the form of modulated spike trains, which can be observed simultaneously with its analog input from the retinula cells. Conversely, recent interest in the ventral eye derives from its large, accessible retinula cells devoid of complicating secondary neurons or synaptic regions.

A. RETINULA CELLS The primary receptor neuron in the visual organs is the retinula cell, distinguished by a large soma, a proximal axon, and superficial, more-or-less continuous brush borders called rhabdomeres. Despite assorted structural differences in retinula cells from various locales, they are similar enough in their cytology to obviate the need for separate descriptions. Details on these cells have been published in conjunction with reports on the ventral eye (Clark et al., 1969b), the ocelli (Jones et al., 1971), the lateral rudimentary eye (Fahrenbach, 1970a), and the compound eye (Miller, 1957; Lasansky, 1967; Fahrenbach, 1969). In the simplest case (in rudimentary eyes), retinula cells are rounded, 30- to 50-pm cells indented into recesses in the accompanying mass of guanophores (Figs. 9 and 15). They assume an elongate shape in the ocelli and ventral eyes (up to 200 pm), and a

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quite precise trilateral, orange-segment form in the compound eye, where a cell can also reach a length of 200 pm (Figs. 7 and 12).

1. Rhabdomeres and Rhabdome Rhabdomeral microvilli, whose orderly disposition largely hinges on fixation, are 1-2.5 pm long and reach their greatest number in the ommatidial retinula cells which have been estimated to bear about 5 x lo5microvilli per cell. Hence an ommatidium with an average of 12 retinula cells contains about 4 mm2 in microvillous membrane, equivalent to 30-40 cm2 in one compound eye (Fahrenbach, 1969). Although the ommatidial rhabdomeres are rather precisely oriented, those on other retinula cells are highly irregular and curvilinear. The beaded appearance of microvillous membranes both in Limulus (Fahrenbach, 1969) and in other invertebrates and vertebrates (Fernandez-Morin, 1962; White, 1967) may indicate visual pigment molecules intimately incorporated into the membrane. The integrity of an ordered array of rhodopsin molecules in the photoreceptor membrane is thought to be essential in the production of an early receptor potential (Brown et al., 1967). Rhodopsin, which constitutes about 10% of the structural protein of the membrane (Hubbard and Wald, 1960), is extractable from rhabdomeres of Limulus; incorporation of the vitamin-A moiety of rhodopsin into Helix rhabdomeres has been demonstrated with autoradiography

FIG.15. Irregular rhabdome in the lateral rudimentary eye with five retinula cells, a guanophore (lower right), a possible neuroglial cell process (N), and a profile that is suggestive of an arhabdomeric cell dendrite (D). X4800.

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(Brandenburger and Eakin, 1970). Electrophysiological evidence to support the functional role of the rhabdomeral membrane has been obtained in Bombyx mod, whose retinula cells develop an electrical response to light only when the rhabdome becomes differentiated (Eguchi et al., 1962). Other evidence of the localization of photopigments in rhabdomeres has been discussed by Eakin (1972). A small amount of extracellular space is contained in the prismatic interstices between and at the bases of microvilli, a volume that has been estimated at a minimum of 7OOO-10,OOO pm3 per rhabdome (Fahrenbach, 1969). This space has free access to the hemocoel at the apex of the ommatidium-between retinula and cone cells-and probably only slightly restricted pathways through the adhering junctions that surround the sides and base of the ommatidium. Shunt pathways, which would ordinarily be of physiological interest in the context of electrotonic transmission between retinula and eccentric cells, probably play a minor role in view of the tight electrical coupling between these cells. Retinula cells contact one another only at their rhabdomeres; all other surfaces are almost invariably sheathed by cone or pigment cells (Figs. 13 and 14). Rhabdomeral microvilli form quintuplelayered gap junctions with all adjacent surfaces, a feature that is not affected by fixation (Lasansky, 1967; Fahrenbach, 1969). In ventral, ocellar, and rudimentary eyes, where rhabdomeres are commonly folded upon themselves (“self-rhabdome” of Jones et al., 1971), the tips of microvilli make contact with those of the same cell or, rarely, abut the glial cells. Microvilli in all photoreceptors are attached by gap junctions side by side or tip to tip. They form the sole contacting elements between retinula and eccentric cells. The function of these junctions between the surfaces of the same cell is admittedly enigmatic, particularly since all other arthropod rhabdomes, except those of the locust (Shaw, 1967a) and the drone honeybee (Shaw, 1967b), manage to function and cohere without additional junctions. In the compound eye, however, the gap junctions provide for electrotonic coupling in basically the same way as they do in various epithelia and neurons (Bennett et al., 1963, 1967; Loewenstein and Kanno, 1967; Pappas et al., 1971; Hudspeth and Yee, 1973).The ommatidial receptor cells can be represented as an electrotonic syncytium with all retinula cells connected to their nearest neighbors as well as to the eccentric cell (Behrens and Wulff, 1965; Smith et al., 1965; Smith and Baumann, 1969). The physiological effects are a faithful transmission of light-induced depolarization from the retinula cells to the eccentric cell without the delays or potential fatigue encountered in a chemical synapse. The complex elec-

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trophysiological interactions have been discussed in detail by Smith and Baumann (1969). 2. Palisade In the dark-adapted state, the base of all rhabdomeres is occupied by a more-or-less regular system of distended, agranular cisternae (Figs. 13 and 14) that have been referred to in various arthropods as Schaltzone (Hesse, 1901), perirhabdomal vacuoles (Eguchi and Waterman, 1966), subrhabdomere cisternae (Lasansky, 1967), or the palisade (Horridge and Barnard, 1965). This palisade has no continuity with the surface membrane; in fact, its membrane is considerably thinner than that of the microvilli (Fahrenbach, 1969; Jones et d., 1971). Its degree of regularity is very easily altered by fixation, and it is structurally transient. On light adaptation, the palisade is gradually displaced by pigment (in the ommatidium), or at least dispersed in the receptor cell cytoplasm in other locations, a process first explored by Horridge and Barnard (1965) in the locust eye and illustrated in Limulus by Miller (1958). The dynamics of the palisade make the hypothesis of Horridge and Barnard (1965) an attractive one. They suggest that the palisade, by its low refractive index and its position next to the refractile rhabdome, serves to confine light to the inside of the rhabdome in the dark-adapted state. As in the cone, the angle of internal reflection in the rhabdome increases with light adaptation as a consequence of pigment aggregation and thereby increases the light loss out of the rhabdome. A second functional possibility emerges from studies with intracellularly injected aequorin (Lisman and Brown, 1972; Brown and Blinks, 1972; Brown, personal communication). Retinula cells of the ventral eye, which also have a palisade, release calcium ions from an intracellular compartment during light-induced depolarization. The calcium ions, which seem to throttle the sodium influx across the receptor membrane to a steady-state level, are rapidly cleared out of the cytoplasm when stimulation ceases. By analogy to the sarcoplasmic reticulum, calcium pumping by the palisade is a possibility favored by its opportune juxtaposition to the rhabdomere and by the absence of other capacious intracellular compartments. 3. Cytoplasm The cytoplasm of the receptor cells is replete with such diverse and abundant contents that it can support various functional claims, including neurosecretion (Waterman and Enami, 1954). In particular, the cells are filled with large amounts of endoplasmic reticulum (ER) and free ribosomes (Fig. 16). Commonly, the nucleus is surrounded by a wide band of ER (Clark et al., 1969b) in addition to the regular

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FIG.16. Retinula cell cytoplasm with extensive ER, pigment, and residual bodies. Y. 6300.

peripheral stacks of ER in the ommatidia. A second type of retinula cell in the compound eye (Fig. 12) has been distinguished on the basis of several criteria, particularly a flared rhabdomere, less ER, and more mitochondria than the other retinula cells of the same ommatidium (Fahrenbach, 1969); but no experimental evidence is available to equate this cell type with, say, the 10% of retinula cells that have a distinctive action spectrum (Wasserman, 1969). Two major synthetic processes presumably proceed at the same time: the production of rhodopsin (and rhabdomeral membrane protein) and the synthesis of ommochrome. The first has been investigated by the autoradiographic tracing of tritiated leucine incorporation into the compound eye of Limulus (Bumel et al., 1970). These investigators found a conspicuous light-dark effect. Animals kept in the light for 12 hours, but held in the dark after injection, incorporated 10 to 20 times more radioactivity into the rhabdome than animals exposed to the reverse regimen. This effect was attributed to a possible competition for available ATP between energy demands

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of the transduction process in illuminated cells on the one hand, and to metabolic claims of protein synthesis on the other. Much of the synthetic activity of the primary visual cells seems to be directed toward the metabolism of the ommochrome pigment. Membrane-bounded pigment granules are produced by the accretion or growth of small, opaque vesicles, seemingly in the same manner as has been related for type I (ommochrome) pigment in the eye of Drosophila melanogaster (Shoup, 1966; Fuge, 1967). There is a subtle distinction in size, but not in structure, between the pigment of sensory cells (ca. 0.5 pm maximum) and pigment cells (ca. 1.5 pm maximum) (however, see Section IX,A). Degradation of the pigment by polymorphic autophagosomes (type IV granule of Shoup, 1966) commences with the incorporation of one to three pigment droplets in a large vesicle with granular, opaque contents and continues through diverse and bizarre lysosomal forms with granular and myeloid content to a presumptive residual stage characterized by irregular stacks or whorls of membranes. Except for the lack of any oversized structures in this sequence, the entire course mirrors that found during granulolysis in the retinula cells of the mantis shrimp Squilla mantis (Perrelet et al., 1971).The abundance of residual bodies, like that in neurons of the central nervous system, appears to increase with the age of the animal. In the absence of direct experimental investigations, any discussion of the remaining, often copious, inclusions, leans toward educated extrapolation. Various small Golgi-derived dense bodies appear to be lysosomes and enter into the larger cytophagosomes. Coated vesicles are produced to such a degree that, particularly in the rudimentary eyes, they frequently form concentrated aggregates which simulate glycogen (Fahrenbach, 1970a). They are also frequently found in surface continuity with the base of rhabdomeral microvilli, a juxtaposition that has been interpreted as light-dependent pinocytotic uptake in the eye of the crab Libinia emarginata (Eguchi and Waterman, 1967) and, conversely, as a sign of mucopolysaccharide secretion into the periodic acid-Schiff (PAS)-positive rhabdomeral space in Limulus (Fahrenbach, 1969). Large multivesicular bodies are a common attribute of all retinula cells. Exploration of these structures in the eye of a cricket, Pteronemobius heydeni, has shown some continuity between multivesicular bodies and lamellar bodies, which supports their customary link to degradative processes (Wachmann, 1969); however, Eguchi and Waterman (1967) found a striking increase in multivesicular bodies with light adaptation and a progressive decrease in

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the dark in the crab Libinia. In addition to the generally favored lysosomal hypothesis, the apparently inverse relationship between these structures and the existence of the palisade warrants consideration. The multivesicular bodies in retinula cells could form a membrane reservoir of palisade cisternae dispersed into the cytoplasm during light adaptation, possibly in conjunction with lamellar bodies. Because of the irregularities of its adaptation processes, Limulus does not provide a promising model in which to study these phenomena. Retinula cells of the ocelli and lateral rudimentary eyes have an added cytological peculiarity in the form of large homogeneous pools of a featureless, PAS-positive, amylase-resistant substance, evidently a glycoprotein, mixed with a variable amount of glycogen (Fahrenbach, 1970a) (Fig. 19).This storage material, commonly in juxtaposition to masses of coated vesicles, also includes some lipid and lipofuscin and is transported along the large axons of these cells toward the brain. The fluctuating quantities and transport of these inclusions have given rise to two reports that mistakenly attribute neurosecretory function to the lateral rudimentary eye (Waterman and Enami, 1954; Nunnemacher and Davis, 1968). 4. Light Adaptation Only a brief resume of the structural changes during light and dark adaptation is appropriate because the subject has not been explored to any extent in Limulus, nor are its ommatidia, the most active organs in this respect, particularly predictable (Fig. 11). The usual process of aggregation and dispersion of pigment cannot truly be said to exist in Limulus ocular pigment cells and is demonstrable only in modified form in the retinula cells. Rather, pigment granules reciprocate within the attenuated cells as they move to a distal (i.e., dispersed) position in the light and collect basally in the dark. Intrinsic changes in the photosensitivity of retinula cells do not necessarily effect observable changes in their ultrastructure. Light adaptation in the compound eye is demonstrable after 5-10 minutes and consists of a general distal pigment migration which results in a subcuticular position of guanine and a dense pigmentary layer around the cuticular cone. A cufflike concentration of pigment at the level of the cone cells provides, in effect, a diaphragm at the exit pupil of the cone. Pigment and some cytoplasm of the proximal pigment cells move distad into the ommatidium and thereby produce more substantial and heavily pigmented partitions between the retinula cells. The palisade of the retinula cells is displaced by pigment which aggregates in the wedge of cytoplasm between rhabdomeres, a phe-

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nomenon illustrated by Miller (1958) and Bass and Moore (1970). The palisade forms and is dispersed in ocellar, ventral, and rudimentary eye cells, but no pigmentary changes analogous to those in the compound eye are seen. Dark adaptation is a more gradual process fully executed after an hour or more. The systematic changes that may occur in the cytological components, and ultrastructural corollaries of light-dependent protein synthesis (Burnel et al., 1970) in retinula cells, have not been explored.

5. Spectral Response Eyes of the horseshoe crab have diverse receptor cells with different action spectra, but to date no morphological distinctions have been established. Graham and Hartline (1935) were the first to record that about 90% of the lateral eye retinula cells (a cells) are maximally sensitive at 520-530 nm (Wasserman, 1969). This action spectrum coincides closely with the absorption spectrum (520 nm) of rhodopsin extracted from Limulus compound eyes (Hubbard and Wald, 1960). Both the ventral eye (Murray, 1966; Nolte and Brown, 1970) and the “visible” cells of the ocellus (Nolte and Brown, 1969, 1970) have a spectral characteristic similar to that of the a cell except for a slight deviation at long wavelengths (Wald and Krainin, 1963). The second class of ommatidial retinula cells, the /? cell (Wasserman, 1969), was also first noted by Graham and Hartline (1935). It has a broad spectral response with one peak at 525 nm and a second, slightly (1dB) lower one in the far-violet region (357-400 nm) (Wasserman, 1969).About 10%of the retinula cells in the compound e y e display this spectral characteristic, a feature that has been ascertained by single-cell recordings and, hence, is not due to overlapping responses of several cells. A third spectral type is restricted to the ocelli and is primarily an ultraviolet receptor (Wald and Krainin, 1963; Chapman and Lall, 1967). These retinula cells are most sensitive at 360 nm, their sharp peak being about 2 log units higher than that of the “visible” cells (Nolte and Brown, 1969, 1970; Lall, 1970). Visible light suppresses the response, that is, hyperpolarizes the ultraviolet-sensitive cells; the converse happens in the “visible” cells (Nolte et al., 1968). La11 and Chapman (1973) calculated that the median eye of Limulus can detect the near-ultraviolet component of moonlight at a water depth of 20 meters. The photosensitivity of intact lateral eyes extends over 10 log units (Kaplan et al., 1973). Most of this range-equal to the psychophysical perception of the human eye-can be attributed to the retinula cells themselves, since only 1-2 log units variation could be ascribed to

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pigment migration in a study of screening pigment effects in the moth Deilephila elpenor (Hoglund and Struwe, 1971).

B. ECCENTRICCELLS The eccentric cell of the ommatidium, first clearly discerned by Watas6 (1890), represents a secondary neuron of the optic pathway. Two eccentric cells per ommatidium are common in some animals, but instances of three or none are on record (Hartline and Ratliff, 1972). From its offset position at the base of the ommatidium, the soma sends a long, tapering dendrite through the center of the rhabdome (Figs. 7, 12, and 13). The bulbous tip of the dendrite abuts the cone cells, to which it is attached by adhering junctions (Fig. 18). All adjoining surfaces between the rhabdome are bounded by gap junctions (Lasansky, 1967). In addition, the surface of the dendrite is studded with an estimated 30,000 to 50,000 microvilli (Fahrenbach, 1969), which make similar contacts with the interlocking microvilli of the retinula cell and increase the surface area of the dendrites by

FIG. 17. Eccentric cell cytoplasm with abundant microtubules, clustered ER, and large Golgi bodies. x 15,000.

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FIG. 18. Tip of the eccentric cell dendrite. Its apex abuts cone cells (C) and hemocyanin-filled extracellular space (H). The cytoplasm is filled with faintly stained glycogen, mitochondria, and some ER. A thin terminal web lines the internal surface of the dendrite. x 15,000.

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FIG. 19. Cytoplasm of rudimentary eye cell with residual bodies and accumulations of mixed glycogen and glycoprotein. x 13,000.

10-50% (Cohen, 1973). The physiological result of these junctions is an electrotonic, recti&ing synapse which transmits the receptor potential of the retinula cells to the dendrite (Smith et d., 1965; Smith and Baumann, 1969). Despite its microvilli, a condition akin to that found in the eccentric cell of the isopod Porcellio scaber (Eakin, 1972), the eccentric cell has no palisade and is not intrinsically photosensitive (Waterman and Wiersma, 1954; Smith and Baumann, 1969). Hence, in addition to providing an extensive junctional surface, the microvilli probably aid in anchoring the dendrite within the rhabdome, a possibility strengthened by the presence of a filamentous terminal web lining the internal surface of the dendrite (Lasansky , 1967). Neuronal attributes dominate the cytoplasmic constitution of the eccentric cell (Fig. 17). At first sight, numerous large Golgi bodies, scattered small arrays of ER cisternae, abundant mitochondria, and

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ubiquitous microtubules differentiate the cell from retinula cells. The dendrite (Figs. 13 and 18)is characterized by compact mixed accumulations of glycogen, mitochondria, and lipid droplets, mostly peripheral in position; evenly distributed microtubules; and agranular cisternae and tubules arranged in a transversely repeating pattern. Pigment is virtually absent but various residual bodies are in evidence, particularly in older animals. The eccentric cell soma is not particularly insulated by its surrounding pigment and glial cells, since hemocyanin often diffuses along intercellular clefts up to the soma in the same manner as it does at the tip of the dendrite. The receptor potential of the retinula cells is conveyed electrotonically via gap junctions to the eccentric cell dendrite. The resultant generator potential of the eccentric cell produces a propagated spike either some distance along the axon (Fuortes, 1959) or among the collaterals of the eccentric cell axon (Purple and Dodge, 1965). The spike, however, does not invade the soma and dendrite except by passive spread. Although several examples of eccentric cells or seemingly similar structures exist, namely in Bombyx mori, the silkworm (Eguchi, 1962), Macrocyclops albidus, a copepod (Fahrenbach, 1964), and Porcellio scaber, an isopod (Eakin, 1972), these cases probably represent separate evolutionary trends without relationship to the condition found in Limulus. C. ARHABDOMERICCELLS Available information on the arhabdomeric cell suggests that this cell of the ocellus is the equivalent of the eccentric cell, hence a secondary receptor neuron. It is located in the receptor layer of the ocellus at a level just below the rhabdomeral zone, or else near the base of the retinula cells. A 100- to 150-pm dendrite ascends into the region of rhabdomeres, where it branches repeatedly. Its terminals are surrounded by rhabdomeral microvilli of adjacent retinula cells. The termini, which like the eccentric cell dendrite contain microtubules, glycogen, and mitochondria, have the usual gap junctions to the adjacent microvilli. The surface of the branches also has a small number of microvilli but no hint of a palisade. The cytoplasm of the soma appears to be identical with that of the eccentric cell, that is, it contains ample glycogen, some ER, large Golgi complexes, and no pigment. Recordings from these cells (Jones et al., 1971) show that they produce spikes in response to ultraviolet light, a characteristic that sets them clearly apart from the two types of retinula cells in the ocellus.

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Some profiles, suggestive of arhabdomeric cells, are occasionally found in the rudimentary eyes (Fig. 15).Even a few such cells with their presumably spiking axons would largely negate the need for unusual length constants of retinula cell axons, a requirement without which rudimentary eyes would be incapable of transmitting any electrical message to the brain. VI. Basal Lamina and Hemocoel The 0.5- to 3-pm thick basal lamina that clothes the general epidermis follows the surface of each ommatidium and the ocellus, covers the surface of the rudimentary eyes, and is continuous with the laminae of adjacent neural and vascular tissues (Figs. 7,20, and 22). The layer, previously illustrated in various contexts of the visual system (Clark et al., 196913; Fahrenbach, 1969, 1970a,b, 1971; Whitehead et al., 1969), is partly composed of a faintl$ fibrous ground substance, presumably mucopolysaccharide, and embedded fibrils of invertebrate collagen (Harper et al., 1967) which average 100 A in diameter and have a periodicity of about 510 A. The basal lamina does not pose an impediment to the diffusion of even large molecules, since hernocyanin is found in most cellular interstices in the photoreceptors. Some small nerves, particularly efferent ones, are surrounded by 30 or more roughly concentric laminae of faintly fibrous glycocalyx without an admixture of collagen (Fig. 23). The hemolymph contains at least 1% (wlv) of the dissolved respiratory protein, hernocyanin, as toroidal granules 190 A in diameter ( F e m b d e z - M o r h et al., 1966; Fahrenbach, 1970b). In many instances the hemocyanin serves as a natural marker for patent diffusion channels in photoreceptors. Thus it enters ommatidia through the basal lamina and diffuses up to the adhering junctions at the periphery of the rhabdome and often partly into the rhabdome at the apex and base of the ommatidium. That this infiltration is not artifactitious can be ascertained by the fact that hemocyanin frequently stagnates and becomes more concentrated in the interstices than in the general circulation and thereby gives rise to the “granular ground substance” described by Lasansky (1967) (Fig. 18). Rare, free-floating crystals of hemocyanin resembling bundles of microtubules result from the normal breakdown of cyanocytes. A second large (100-A) molecular species, a hemagglutinin (Femhdez-Morhn et al., 1968), cannot be differentiated from hernocyanin in sectioned material, although it is distinct after negative staining. It constitutes about 5 % of the hemolymph protein.

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FIG.20. Small blood vessel near the ocellus. A thick lamina coats the endothelial cells, which are surrounded by several connective tissue layers. A small nerve (N; see Fig. 23) accompanies the vessel. X 1200. FIG.21. A nearly 100-pm long cyanocyte in a circulatory space. Beyond this stage the cell breaks up and liberates the contained hemocyanin. x 1200.

Two principal cell types circulate in the bloodstream and are frequently intimately associated with various parts of the visual system, particularly the lateral eye plexus, even though they remain separated from adjacent tissue by a basal lamina. Most numerous (92-99%) are the granular, ameboid hemocytes responsible for the clotting of blood by aggregation and release of their granules (Loeb, 1928; Holme and Solum, 1973; Dumont et al., 1966) (Figs. 10 and 22). Within a minute after the blood is exposed to air or seawater, the

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homogeneous content of the dense, large granules is changed to a microtubular and subsequently granular one, which is discharged from the cell. Simultaneous ameboid activity results within 3 minutes in a seemingly new cell type (type H2of Herman and Preus, 1972), devoid of specific granules but full of swollen cisternae and a variety of inclusions (Clark et al., 1969a) (Fig. 10). The mode and speed of dissection determine the abundance of degranulated hemocytes. The granules of hemocytes, surrounded by a conspicuously asymmetric membrane, have been mistaken for pigment and neurosecretory material where these cells have squeezed themselves into narrow interstices of adjacent tissues. The second blood cell type, occurring with a frequency of less than 1-8%, depending on molting stage, forms hemocyanin (Fahrenbach, 1970b). The youngest identifiable stage, the cyanoblast, is distinguishable from a degranulated hemocyte by its conspicuous content of free ribosomes. With maturation, the cell accumulates hemocyanin in crystalline form until ultimately, as a cyanocyte, it resembles a large crystal, up to 100 pm long, with an appended nucleus and minimal cytoplasm (Fig. 21). The breakup of this cell results in the previously mentioned crystalline hemocyanin fragments which gradually disperse into individual molecules. Cancellous vascular spaces with extremely thin partitions, the expression of a true open hemocoelic system (Figs. 2 and lo), surround the eyes and fill the spaces between photoreceptors and adjacent organs. The partitions are commonly composed of a thin central fibrous lamina and a cellular lining on both sides. The highly attenuated lining cells are probably identical with the tissue reserve cells (R cells) described by Schlottke (1934, 1935) and Herman and Preus (1972) in connection with the hepatopancreas in Limulus. Depending on the nutritional state of the animal, the reserve cells are more or less filled with the conspicuous rosettes of *glycogen, variable numbers of large homogeneous lipid droplets, numerous mitochondria, and an assortment of dense bodies and vacuoles (Figs. 10 and 27). Despite the hemocoelic nature of the circulatory system, progressively smaller branches of the sturdy muscular arteries can be traced between sinusoidal spaces. Only thin-walled “capillaries” are found in immediate proximity to the eyes, often in the space between adjacent ommatidia. These vessels, mentioned by Whitehead et al. (1969),have a wall one to two cells thick and a ridged internal surface covered by a deep lamina similar to the fibrous lamella lining the lumen of large arteries (Dumont et al., 1965) (Fig. 20).

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In the peculiar internal anatomy of Limulus, its optic nerves penetrate the hepatopancreas and gonad. Several articles describe the unusual histology and cytology of these organs (hepatopancreas: Schlottke, 1935; Herman and Preus, 1972; Johnson et al., 1973; ovary: Munson, 1898; Dumont and Anderson, 1967; testis: Benham, 1883; Fahrenbach, 1973a). VII. Axons and Plexus A. AFFERENT AXONS

With few exceptions the cytology of the visual axons is remarkably uniform (Nunnemacher and Davis, 1968; Clark et al., 1969b; Fahrenbach, 1970a, 1971). Their cytoplasm is filled with quite regularly spaced, longitudinal microtubules with a center-to-center spacing of 1000-1500 A. Mitochondria, mostly in a size range of less than 0.2 pm, are distributed toward the periphery of the fiber. Scarce agranular reticulum occurs in some axons (eccentric cell) in a transverse disposition of cisternae, which is also seen in the eccentric cell dendrite (Gur et al., 1972). Several of the axonal types contain further specialized contents, some of which have diagnostic value but are usually enigmatic as to function. Eccentric cell axons often carry small, compact masses of a PAS-positive, diffusely fibrillar material, apparently a glycoprotein. Chains of lipid droplets lie in an occasional retinula cell axon of the lateral optic nerve, virtually occluding the entire cross section of the fiber. An extremely rare fiber type in the lateral optic nerve, intermediate in size between eccentric and retinula cell axons, is filled with mitochondria and dense bodies and may represent the tip of a growing retinular axon (Fahrenbach, 1971). Profuse quantities of aglycogen and glycoprotein, produced in the lateral rudimentary eyes, travel along the large axons of these cells and have contributed to a mistaken diagnosis of neurosecretion (Waterman and Enami, 1954; Pannesi, 1964; Nunnemacher and Davis, 1968). The diameter of visual fibers, although typical for a given cell type of origin, is unfortunately not related to its functional properties (Fig. 22). Action potentials have been recorded from eccentric cells, arhabdomeric cells, and scattered retinula cells in the ocellar nerve (Waterman, 1953). The axons of the latter two cells have a 5-pm diameter, whereas those of eccentric cells increase from a minimum of 1 pm in a young animal to an average of 10-15 p m in an adult. A similarly large diameter has been recorded for the axons of retinula

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FIG.22. Juxtaposition of eccentric cell (E) and retinula cell (A) axons in the lateral optic nerve. Both contain microtubules and are enveloped by sheaths of glia and basal lamina. Two neuroglial nuclei are shown. Hemocytes lie in the intervening circulatory space. X 17,000.

cells in the median and ventral eyes (Jones et al., 1971; Clark et al., 196913). The fibers of the lateral rudimentary eye are initially the largest in the lateral optic nerve (3-10 pm) but increase more slowly in diameter than eccentric cell axons to a diameter of 10-25 pm (Waterman and Wiersma, 1954; Fahrenbach, 1971). Since the initially segregated grouping of rudimentary eye fibers in juvenile animals is not maintained in the adult nerve, the axons can be distinguished from eccentric cell fibers only by unusually large size or by the previously mentioned inclusions. All these fibers are enveloped in one or more neuroglial cells, and

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each is surrounded by a separate thick external lamina. In contrast, the retinula cell fibers of the compound eye, measuring only 0.5-1.5 prn in diameter, are bundled into fascicles of two or three directly adjacent fibers, invested by one to several glial cells. This arrangement develops from larger bundles of 6 to 10 or more fibers in a young animal. The one-to-one correspondence between spiking axons in the lateral optic nerve and eccentric cells was established by Waterman and Wiersma (1954). However, identification of retinular fibers in the optic nerve depends on their correct numerical ratio in relation to eccentric cell fibers and their size, given the absence of detectable electrical activity in them. The small admixture of efferent and rudimentary eye axons can be singled out only by optic nerve section and pileup of secretory products in the proximal and distal stumps, respectively (Fahrenbach, 1971; Pannesi, 1964). Spiking in the nerve of the ocellus, first observed by Waterman (1953), is mostly attributable to arhabdomeric cell fibers, although retinula cells have not been ruled out (Jones et al., 1971) and ectopic retinula cells within the nerve seem to produce propagated action potentials (Waterman, 1953).Despite intensive studies on the physiology of the ventral eye (Millecchia and Mauro, 1969a,b; Lisman and Brown, 1972; Yeandle and Spiegler, 1973), no action potentials have been detected in its nerve. The ventral eye plays a definite role in visual behavior (Wasserman, 1973a) notwithstanding the 2- to 3-cm-long nerve, a length that would drastically attenuate signal transmission by electrotonic spread. Both lateral and median rudimentary eyes produce receptor potentials (Millecchia et al., 1966)but do not give rise to propagated spikes. Their functional role is unknown. Numerical axonal input to the brain increases with age but can be approximated for an animal of 10-cm prosomal width as follows (Nunnemacher and Davis, 1968; Fahrenbach, 1971) (both sides combined) : Compound eyes Eccentric cells Retinula cells Lateral rudimentary eyes Ocelli Retinula cells Arhabdomeric cells Endoparietal eye Ventral eyes

1,200 16,000 200 300 50 100 600

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B. EFFERENTAXONS All Limulus photoreceptors are supplied with efferent, neurosecretory fibers which originate in the central nervous system (FahrenThis peculiar innervation appears to be unique bach, 1970c, 1973~). to Limulus with the possible and unconfirmed exception of neurosecretory fibers seen in the eye of the honeybee by Baumann (personal communication, cited in Clark et al., 1969b). Less than 10 small (1-to 2 - p ) axons course in the lateral optic nerve to supply the compound and lateral rudimentary eyes; the numerical complement of efferent fibers in the other nerves has not been determined. Only rarely does one encounter the characteristic neurosecretory granules -opaque, cylindrical bodies with a period substructure - in the nerve, but ligature or transsection produces rapid proximal pileup of the secretory product (Fahrenbach, 1971) (Fig. 25). This procedure also causes degranulation and gradual degeneration of the terminals. In the plexus of the lateral eye, efferent fibers are conspicuous by

FIG. 23. Small nerve containing efferent axons (A). The enveloping neuroglial cells have numerous adhesion plaques which face the surrounding, lamellated sheath. x21,Ooo.

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their increased number, presumably the result of progressive branching, and by their content of elementary granules (Fig. 27). Each ommatidium receives about 70 minute efferent axons which terminate in approximately equal numbers on the proximal pigment cells, intraommatidial pigment cells, and adjacent epidermal cells, including guanophores. The fibers travel to their destination in the partitions between retinula cells and at the periphery of the ommatidium and occasionally form short commissural tracts between adjacent ommatidia. The terminals, basically identical with the “synaptoid” neurosecretory endings of insects (Scharrer, 1968),end on the surface of ommatidial pigment cells and on receptor cells of rudimentary, median, and ventral eyes; often, however, they indent or deeply invaginate into the target cell (Clark et al., 1969b; Fahrenbach, 1969, 1970a, 1973c) (Fig. 24). In all cases the basal lamina is penetrated. The synaptoid terminal is distinguished by a presynaptic, submembranous density surrounded by some clear vesicles and many elementary granules, but no postsynaptic specialization is present. In retinula cells of the ventral and, in extremely rare instances, the compound eye, efferent axons end in immediate juxtaposition to the rhabdomere. The region of the axon hillock in retinula cells of the lateral rudimentary eye is occasionally invaded by terminals. Cells that seem to be morphologically unresponsive to changes in illumination, that is, guanophores outside the compound eye, epidermal cells of the ocellus, and eccentric cells, lack an efferent supply. The origin of these fibers is uncertain, but circumstantial evidence points to a group of large neurosecretory cells in the central body, the protocerebral center on which all visual inputs converge (Fahrenbach, 1973c) (Figs. 30 and 32); however, neurosecretory cells elsewhere in the nervous system (Herman, 1970, 1972; Herman and Preus, 1973) cannot be excluded. Efferent innervation probably governs the rapid phase of pigment migration, which occurs during light adaptation. Chromatophorotropic hormones in crustaceans are generally responsible for ocular pigment migration (Kleinholz, 1966)and have, in fact, been extracted from the central nervous system of Limulus (Brown and Cunningham, 1941; Fingerman et al., 1971); however, the brain is only one-third as active as the subesophageal ganglion. In any event, by combining attributes of both neurohormonal and neurohumoral control systems, the efferent supply to the eyes is a typical example of the intermediate mode of neuroendocrine interaction (Scharrer, 1972).

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FIG.24. Several efferent fibers near their terminations in proximal pigment cells. The largest terminal illustrates several views of the granules and a synaptoid junction (arrow). x 19,OOO. FIG.25. Proximal stump of a severed optic nerve (3days after operation), showing the nerve sheath, degenerating axons and glia, and pileup of granules in an efferent fiber. C, Collagen, F, fibrocyte. X8000.

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C. PLEXUS

1. Architecture Both the median ocelli and the compound eyes have a plexus, that is, a zone of interconnecting and synapsing receptor axon collaterals below the layer of retinula cells. Since the median eye plexus has not been described, the following discussion deals exclusively with the plexus of the compound eye. The open architecture of this region combined with its volume, 75 mm3 or more in a large animal, provides a severe impediment to any structural studies in which precise fiber tracing is contemplated. The important process of lateral inhibition, discovered by Hartline (1949) and subsequently extensively explored by Hartline and his collaborators (see reviews in Ratliff, 1965; Wolbarsht and Yeandle, 1967; Hartline, 1969; Knight et aZ., 1970; Hartline and Ratliff, 1972), takes place within the plexus and involves the reciprocal inhibition of impulse rate of one ommatidium by the activity of its neighbors. Axons descend from the ommatidium singly at first and lie in a partly delimited vascular space corresponding to the periphery of the ommatidium before becoming grouped into small bundles of a few fibers (Hartline et aZ., 1961; Miller, 1965). Synaptic neuropile appears slightly below the ommatidia and is especially conspicuous at the intersections of the predominantly vertical visual fibers and the more or less horizontal fiber tracts that link neighboring ommatidia (Fig. 26). The interconnections, 10-30 pm in diameter, may lie anywhere between the base of the ommatidia to a millimeter proximal to that. Beyond that level the optic fibers aggregate into several nerves, which pass through a cribriform, cuticular plate separating the inside of the adult compound eye from the body cavity. Below the plate all fibers coalesce into the lateral optic nerve. The branching pattern of the visual axons has been investigated by Procion Yellow injection (Schwartz, 1971) and by serial sectioning for electron microscopy (Gur et aZ., 1972). The value of the former technique is limited by the uncertainty of whether the finest axonal branches are indeed labeled. Serial sectioning, although theoretically the ultimate method of ascertaining pre- and postsynaptic contributions to the plexus, is impeded by the practical barriers of continuous tracing through an uninterrupted series. For example, Gur et al. (1972) collected 2000 to 3000 sections from the plexus region underlying each of several ommatidia, lost about 20% of the series, and assembled in montage about 5%. Large collaterals can be traced by eye through incomplete stretches of the series, but the continuity of

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,

FIG. 26. Horizontal section through the plexus of the compound eye. The dark knots of neuropile (see Fig. 27) correspond approximately to the position of overlying ommatidia. The thick interconnecting tracts of axons are not necessarily as unidirectional as shown here. X280.

the smallest branches becomes ambiguous with the loss of even one or two sections. Injected preparations of the retinula cell axons show only small (ca. 5-pm) spines and no long collaterals. From serial sections it appears that a short distance below the ommatidium the retinula cell axons of that ommatidium enter into a mutual synaptic plexus by way of collaterals, without the apparent participation of the eccentric cell or contributions from adjacent ommatidia. This region has only a very limited vertical extent. The eccentric cell axons, in contrast, contribute the main substance of the plexus by long and short collaterals. The long collaterals, mainly traceable in Procion Yellow-injected preparations (Schwartz, 1971), extend through the previously mentioned interconnections and terminate in proximity to axons from adjacent ommatidia. They usually measure less than 1 pm in diameter and have been traced over a distance of 200 pm, which just about corresponds to the in-

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FIG.27. Cross section of a synaptic column in the plexus underlying an ommatidium. A, Efferent fiber; N, neuroglial cell; R, reserve cell; H, hemocytes. ~10,OOo.

terommatidial spacing in adult animals. However, much longer collaterals probably exist. In a study of inhibitory fields, which are presumably a direct reflection of long eccentric cell collaterals, Barlow (1969)found that inhibitory interaction reaches a peak at a distance of 3 to 5 ommatidia and approaches zero at about 12 ommatidia. These inhibitory fields are at their widest in a horizontal plane (anteroposterior on the eye) and are compressed vertically. No morphological information over a comparable spatial extent is available. The short eccentric cell axon collaterals (Miller, 1965; Schwartz, 1971; Gur et al., 1972) form a more-or-less continuous neuropile sleeve around the axon through the depth of the plexus (Figs. 27 and 28). This neuropile generally starts just beyond the level of the axon hillock and receives contributions from sizable branches of the eccentric cell axon. These give off clusters of smaller offshoots, which are highly irregular in course and diameter. Apparently, many collat-

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erals synapse with others of the same eccentric cell, particularly in the distal part of the plexus, that is, close to the ommatidia. Contributions of the retinula cell collaterals to the eccentric cell plexus are minimal, if present at all. The distribution of the interacting collaterals supports the contention that the inhibition is the recurrent type, that is, the inhibitory influence is exerted at or near the site of impulse generation (Ratliff, 1965). Thus impulse reduction in an inhibited ommatidium is effected by a lowered level of impulse generation rather than by the suppression of impulses in an existing spike train. The contents of the collaterals of either type consist of microtubules, abundant mitochondria, various synaptic and related vesicles and minor amounts of glycogen. Golgi bodies are found only in adjacent glial cells; hence, they cannot be presumed to play a role in the neuropharmacology of the synaptic regions (Adolph and Tuan,

1972).

2. Synapses The synapses in the plexus were first studied by Miller (1965), who described peculiarly thickened membranes at presumptive sites of inhibitory synaptic interaction without any evidence of subsynaptic specializations. This peculiarity, although apparently a differentiated feature of limited areas, can be demonstrated only with the fixative used (10%glutaraldehyde) and has not been observed by subsequent investigators. In any event the tight junctions described by Miller (1965) as presumptive sites of excitatory interaction must be viewed with some reservation in view of the ease with which they can be produced artifactitiously. A more orthodox picture, akin to that of primary receptor synapses in insects (Trujillo-Cenbz, 1965; Trujillo-Cenbz and Melamed, 1967), emerges from the study of Whitehead and Purple (1970).An opaque hillock and a diffuse synaptic ribbon are attached to the presynaptic membrane at the trigonal point of one presynaptic and two postsynaptic profiles. A zone clear of synaptic vesicles surrounds the ribbon. Quite commonly, two reciprocal synaptic elements face each other. This particular specialization, unusual if not unique for a chemical synapse, can be construed as forming the substratum for (1) the process of self-inhibition of an ommatidium (Purple and Dodge, 1966), provided the two contributing collaterals originate from the same ommatidium, or (2) reciprocal lateral inhibition, if components from two different ommatidia are involved (Gur et al., 1972). Not only do synapses lie between small fibers, but large (2-pm) axons can

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FIG. 28. Synaptic detail of the plexus. One axon collateral (A) has conspicuously larger synaptic vesicles than average. This and the preceding figure are indicative of the relative sparsity of dense vesicles. Arrows point at synapses. N, Neuroglial cells. x 25,000.

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provide en passant synapses to adjacent small collaterals or, conversely, small branches may synapse on the eccentric cell axons, even occasionally near the axon hillock. The great majority of synaptic terminals are filled with round, clear vesicles about 400-500 A in diameter (Miller, 1965) (Fig. 28). Conspicuous efferent fibers, characterized by the typical angular granules described for efferent terminals, transverse the plexus without synapsing. In addition to these vesicles, many terminals contain a variable admixture of dense or dense-cored vesicles (Adolph and Tuan, 1972). A rather rare type of synapse is populated by large and irregular vesicles which average about 800 A in diameter, Subtle differences in synaptic morphology may exist but have not been explored. Whether the lateral excitatory interactions that have been detected in the eye (Tomita e t al., 1960; Purple and Dodge, 1965) can be attributed to some of the less common synapses is unknown.

3. Pharmacology The neuropharmacology of the plexus has been approached by micropipet injection of active substances into the lateral eye (Behrens and Wulff, 1970; Adolph, 1966), as well as by a combination of these and biochemical techniques (Adolph and Tuan, 1972). Unless certain experimental conditions are observed, for example, simultaneous recording of the output of interacting ommatidia or inhibition of a test ommatidium by antidromic stimulation of neighboring fibers, it is difficult to sort out synaptic influences from relatively unspecific membrane effects that derive from unphysiological, topical concentrations of an applied pharmacological agent. Depending on their specificity, metabolic poisons of known steps in the synthetic pathway of a putative transmitter are less likely to confuse the picture with direct neurophysiological sequelae. Both y-aminobutyric acid (GABA) and the GABA inhibitor picrotoxin affect the spike action potentials of an ommatidium (Adolph, 1966; Behrens and Wulff, 1970) by depressing or only slightly increasing frequency, respectively. However, inhibition by antidromic stimulation is blocked by the protracted action of 0.1 mM picrotoxin. Brief (30-minute) exposure of the eye to aminooxyacetic acid, an inhibitor of GABA metabolism, does not produce marked effects on lateral inhibition. Serotonin (5-HT) also depresses ommatidial spike activity (Behrens and Wulff, 1970; Adolph and Tuan, 1972). The increase in spike activity observed by Adolph (1966) has been attributed to the

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phosphate buffer vehicle (Behrens and Wulff, 1970). After the application of 5-HT, the resultant depression of lateral inhibition recovers more slowly than the firing rate of the affected ommatidia, an indication of a specific synaptic effect in addition to any nonspecific influence on the cell membrane. The eye does not respond to monoamine oxidase inhibitors nor does it contain the normal (vertebrate) 5-HT metabolite 5-hydroxyindole acetic acid, according to Adolph and Tuan (1972). These investigators, however, found a seemingly higher concentration of melatonin than 5-HT, its metabolic precursor. In physiological concentrations melatonin has no effect but may be associated with ocular pigment migration. With either GABA or 5-HT, the inhibitory effect is most pronounced if the region of application lies proximal to the test ommatidium by 0.5-1 mm. Epinephrine, norepinephrine, and various amino acids affect the spike output of ommatidia (Adolph, 1966; Behrens and Wulff, 1970), but their participation in any snyaptic mechanism in the plexus is unconfirmed. Dilute solutions of ethanol abolish lateral inhibition (MacNichol and Benolken, 1956) without depressing the spike activity, an observation that sheds little light on synaptic pharmacology, useful though it is to neurophysiological studies. For a convincing solution of this problem, ultrastructural studies will have to be combined with autoradiographic, histochemical, and biochemical investigations. VIII. Optic Nerves The lateral optic nerves, the only ones studied in any detail (Pannesi, 1964; Nunnemacher and Davis, 1968; Fahrenbach, 1971), contain fibers from the lateral rudimentary and compound eyes. They travel forward, then turn ventrad and backward to enter the optic laminae at the dorsal anterolateral bulges of the brain. The ocellar nerves loop together usually to the right side of the underlying digestive tract and enter the brain in the dorsal midline. The ventral eye nerves course directly backward and join the ganglionic mass of the lateral eyes at the level of the medulla (Fig. 29). Limulus nerves have the peculiar attribute of being enclosed by an arterial wall. Hence the nerve sheath is more complex and substantial than would be expected of an epineurium. Its structure, detailed by Dumont et al. (1965), consists of alternating layers of fibrocytes and extracellular material (Fig. 25). The interdigitated cells have conspicuous Golgi cistemae but are otherwise rather undistinguished. The intervening acellular layer, termed the fibrous la-

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mella, is made up of a medial layer of fine collagen fibrils and a peripheral thick external lamina of the adjacent fibrocytes. Scattered muscle fibers in the wall of optic nerves display the characteristics of arthropod visceral muscles (Fahrenbach, 1967; Dewey et al., 1973), that is, a sarcomere length of about 10 pm and a myofilament array of one myosin filament being surrounded by 10 to 12 actin filaments. Both outer and inner surfaces of the vessel wall are covered by a basal lamina which faces the circulatory space. A deviant nerve type is found near the lateral rudimentary and ocellar eyes, often containing only efferent axons (Fahrenbach, 1970a). Here a small number of fibers are carried in an irregular, fluted, and interlocked array of neuroglial cells (Fig. 23). These secrete a cylindrical sheath, up to 10 pm thick, composed of multiple

FIG. 29. Dissection of the brain in an oblique, ventral view. Almost the entire mass consists of corpora pedunculata. The circumesophageal ring starts at the upper left and right. A, Lateral optic nerve; B, ventral eye nerve; C, frontal organ nerve; D, ocellar nerves; E, stomodeal nerve. X40.

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external laminae without any participation of collagen. The fluted glial cells embedded in this sheath exhibit numerous conspicuous hemidesmosomes.

IX. Optic Centers The various optic ganglia have received nowhere near the same attention as the eyes. The principal publications outlining the internal anatomy of the protocerebrum, that is, the purely sensory anterior region of the brain, are those of Packard (1891), Viallanes

FIG.30. Representative neurons of the optic ganglia and central body in frontal section. 1, Ocellar nerves; 2, ventral eye nerve; 3,compound eye nerve; 4, lamina; 5, chiasma; 6, medulla; 7,corpora pedunculata; 8, optic tract; 9, central body; 10, ocellar ganglia; 11, ectopic retinula cells; 12,globuli cells; 13, neurosecretory celIs; 14,protocerebral neurons with axons into both sides of circumesophageal ring; 15, medullary axon to contralateral medulla; 16,fibers to circumesophageal ring and corpora pedunculata. [Modified after Hanstrom (1926a) and Patten (1912).]

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(1893), Patten (1893), Patten and Redenbaugh (1900), Hanstrom (1926a,b), and Johansson (1933).A detailed study has been directed at the projection of the lateral eye mosaic onto the first visual ganglion and the subsequent processing of visual information (Snodderly and Barlow, 1970; Snodderly, 1971). Only a brief coverage is given here (Fig. 30). The lateral optic nerve commonly breaks up into a number of bundles before entering into the first optic ganglion (lamina) (Fig. 31).Visual fibers, presumably of eccentric cell origin, form optic cartridges (Hanstriim, 1926a), and some continue after synapsing into the second ganglion (Patten, 1912; Snodderly, 1971). Horizontal bands of ommatidia are projected in virtually continuous fashion onto the lamina, their relative dorsoventral positions coinciding between ommatidial origin and laminar termination (Snodderly and Barlow, 1970).Anteroposterior projection may also occur but has not been explored in detail. Postsynaptic off-responses, akin to those in vertebrate ganglion cells, are generated in the optic lamina (Wilska and Hartline, 1941; Snodderly, 1971).

FIG. 31. Horizontal section through the lateral optic ganglia. Orientation of the midline is indicated by the long arrow. N, Lateral optic nerve; L, lamina neuropile; C, chiasma; M, medulla neuropile; small lobe of corpora pedunculata is at upper left. x 110.

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The lamina is connected to the medulla (second optic ganglion) by way of a chiasma. The crossing of fibers has been illustrated by Hanstrom (1926a),but occurs in a plane tilted from exact frontal; hence it is not particularly striking in most sections (Figs. 30 and 31). The neuropile of the medulla is stratified into four dense layers which alternate with three intervening less compact ones (Hanstrom, 1926a). Ganglion cells cover part of the surface of the medulla and range from the size of minute (ca. 10 pm) association neurons, called globuli cells, to a cluster of cells up to 70 pm in diameter. The ventral eye nerves enter the nervous system at the level of the medulla,

FIG.32. Horizontal section of the central body. The vascular spaces around the central body contain hemocytes and a trabecular meshwork of neuroglial cells. M, Optic medulla; N, ocellar nerve; 0, ocellar ganglion; C1, central body neurons (globuli cells); C2, protocerebral neurons; C3, neurosecretory cells. ~ 9 0 .

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which is connected to the central body by a sizable tract. Neurophysiological recordings from the medulla (Snodderly, 1971) have produced on-units which display spatial summation and a receptive field of about half the compound eye; off-units firing for at most 1-2 seconds after the cessation of a light stimulus; extended-response units which fire both during and after the stimulus; delayed-off units responding for up to a minute after termination of light; and neurons with various rhythms subject to modification by light stimulation. Fibers from the medulla penetrate into both sides of the circumesophageal ring and the contralateral medulla. The corpora pedunculata, which contain most of the neurons (5 x log in a 10-cm animal) and occupy 80% of the volume of the brain, also have connections with the second optic ganglion (Hanstrom, 1926b; Fahrenbach, 1973b). Ectopic retinula cells, associated with the ventral eye nerve, frequently lie underneath the sheath of the brain in immediate proximity to the medulla. Presumably, these cells are responsible for light responses recorded from the medulla severed from its laminar input (Snodderly, 1971). Fibers from the ocellar complex terminate in two small ganglia located between the anterior horns of the central body (Figs. 30 and 32). Small secondary neurons of these ganglia synapse within the central body. This structure is a horseshoe-shaped mass of neuropile, dorsal and superficial in location, its two free arms curving ventrad and approaching each other in the midline (Fig. 32). It is covered with globuli cells on its dorsal and lateral surfaces, receives the previously mentioned visual inputs, and has ventrally broad connections with the protocerebral neuropile, the stalks of the corpora pedunculata, and the circumesophageal ring.

X. Miscellaneous Aspects A. ABNORMALITIES

Limulus appears to have an unusual propensity toward developing supernumerary eyes. Among 190 animals, Hanstrom (1926a) found six specimens with three ocelli. The third ocellus was always located behind the normal median eyes in the midline, had its own lens, and gave rise to two optic nerves. This condition, reminiscent of the nauplius eyes of crustaceans and the ocelli of insects, may represent a not so “rudimentary” condition of the endoparietal eye, which is normally bipartite, including its nerve. In young Tuchgpleus tridentutus, an Asiatic xiphosuran, a transparent cuticular region overlies the endoparietal eye (Waterman, 1953).Four ventral eyes in a side-

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FIG.33. Pigment cell partition in the ommatidium of an albino Limulus (compare to Fig. 14). The retinula cells are normally pigmented, but the pigment cells contain virtually no pigment and are thickened. Their content is apparently of lysosomal nature. x6500. FIG.34. An encysted metacercaria of Microphallus limuli surrounded by globuli cells of the corpora pedunculata. ~ 4 5 0 .

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by-side position with four separate nerves have been observed (J. E. Brown, personal communication). A far more exotic variant has been reported in the form of an animal with an extra pair of compound eyes, located on a single, 2cm-high solid stalk on the dorsal carapace, 3 cm off the midline (Barlow and Kaplan, 1972). These eyes, about two-thirds the size of the lateral eyes, produced pronounced activity in their optic nerves, and as in the lateral eyes, a changed level of illumination affected the heart rate. Two types of rare white-eyed mutants have been found on the western coast of Florida. The eyes of the first, totally unpigmented type have been explored in considerable physiological detail by Nolte and Brown (1970) because the absence of shielding pigment makes it possible to determine pure action spectra unaffected by the passage of light through ommochromes. The eyes of the second mutant (Fahrenbach, unpublished observations; live animal provided by courtesy of J. E. Brown) were light brown and showed normal, possibly increased pigmentation of the retinula cells, and normal guanophores, but highly aberrant ommatidial pigment cells. These had very few, generally small, ommochrome droplets but an abundance of cytophagosomes of the type described in normal eyes. Pigment cell partitions between retinula cells (Fig. 33) were much thicker than in control animals, an observation that suggests increased cytoplasmic migration of proximal pigment cells into the ommatidium in response to sustained photic stress unalleviated by the usual adaptive pigment migration. The overall architecture of the ommatidia and the efferent innervation appeared normal.

B. PATHOLOGY Most horseshoe crabs larger than 20 mm in width (about the fourth molting stage) are infested with the metacercariae of a digenetic trematode, Microphallus ZimuZi (Fig. 34). The life history of this fluke, unraveled by Stunkard (1951, 1953, 1968), starts in the snail, Hydrobia rninuta, which is invaded by the miracidium and harbors two generations of sporocysts in its hernolymphatic sinuses. The cercariae released by the snail invade young LimuZus (even animals of 3to 4-mm width in an experimental situation) and encyst as metacercariae. These ovoid bodies, 150-200 pm in length, are found mainly in interstitial connective tissue but also commonly in the lateral eye plexus and brain. Encapsulation of the cysts by host cells is minimal, and no foreign body reaction or tissue disruption except physical displacement has been observed. A foreign body reaction, however, has

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been observed in Limulus in another context (Loeb, 1902). After consuming horseshoe crabs, the herring gull Lams argentatus becomes the final, natural host to the fluke. In old, infrequently molting animals and in aquarium-stored specimens, the cuticle, particularly that over the compound eyes, is attacked by bacteria and fungi. The former generally erode small pits in the cuticle (Fig. 2), whereas a fungal infestation can penetrate the generally intact cuticle, invade the ommatidia (Fig. 5), and cause profound local destruction and possible secondary bacteremia, which would cause death by generalized intravascular clotting (Bang, 1956).Chitinolytic fungi of arthropods have been studied primarily in connection with the recent outbreak of the European crayfish plague (Unestam, 1965, 1968). A single instance of an abnormal cuticular concretion at the anterior border of the brain has been described by Hanstrom (1926~). Since injury responses can frequently be put to use in neuroanatomical tracing, several largely unpublished observations may provide some useful information. Individual ommatidia or small groups of them can be extirpated by laser irradiation through a suitable aperture. Affected ommatidia are marked by a rapid invasion of hemocytes, and subjacent synapses show degenerative changes by an increase in electron opacity. Although this approach might lend itself to the morphological tracing of the inhibitory field, it appears unsuitable to the study of projection to the optic ganglia since severed axons maintain an essentially normal appearance for many months except for an increase in subsurface cistemae and some degeneration near the cut (Fahrenbach, 1971). Synapses of severed efferent optic fibers become degranulated within a few days but involute at an extremely slow rate (at 13°C) (Fahrenbach, 1971). Similarly, a retrograde injury reaction in peripheral visual or central neurons has not been detected despite considerable effort (Fahrenbach, 1973c) and may unfortunately occur to an experimentally useful degree only in insects among the arthropods (Cohen and Jacklett, 1967; Boulton, 1969; Young et al., 1970).

XI. Vision and Behavior To prevent the further dissemination of apocryphal tales about the total or partial blindness of Limulus, its light-induced responses and behavior should be discussed here. The earliest studies were concerned with phototaxis and demonstrated, even from a retrospective standpoint, a perplexing array of factors that influence these seem-

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ingly simple reactions. Newly hatched animals are positively phototactic when swimming (ventral eye up), but negatively so when crawling or in warm water (Loeb, 1893). Older animals are positively phototactic and exhibit circus movements with unilaterally occluded eyes (Cole, 1923; Wolf and Zerrahn-Wolf, 1937), but their behavior is easily reversed by such diverse influences as hunger, being tied by the tail, protracted darkness, “fright,” or no apparent reason at all (Cole, 1924; Northrop and Loeb, 1923; von Campenhausen, 1967). Unrestrained animals moving on the ocean bottom at a shallow depth execute rapid turns toward the light when they are being shaded on one side (Adolph, 1971). The effect of ocelli versus lateral eyes in positive phototaxis was explored by La11 and Chapman (1973), who found that one ocellus or one lateral eye alone is sufficient to mediate the response. As might be expected from the neurophysiological characteristics of ocellar receptor cells, the ocelli influence the behavior most markedly in ultraviolet light but less so under full sunlight, in which the ultraviolet-sensitive cells might be partially inhibited. La11 and Chapman (1973) suggested that the extraordinary sensitivity of the ocelli to ultraviolet light and the rapid attenuation of ultraviolet light in water serve as a basis for depth detection when horseshoe crabs ascend to the beaches for breeding. A corollary observation to phototactic turning is an increase in leg movement and closing of the terminal flaps of the fifth walking leg, both primarily on the contralateral side (Corning and Von Burg, 1968; Lahue, 1973). Attempts to exploit these turning tendencies to establish an optomotor response gave results on the border of statistical significance (von Campenhausen, 1967); the principal impression generated is that of unremediable “disobedience” of the animal. A more tractable and reliable unconditioned response is that of a downward tail movement in response to illumination (Wasserman and Patton, 1970; Wasserman, 1973a,b).The discovery of this behavior was the offshoot of unsatisfactory attempts to produce conditioned responses in Limulus (Smith and Baker, 1960; Wasserman and Patton, 1969; Makous, 1969; Wasserman, 1970). The reaction has a latency of between 2.5 and 4 seconds, in keeping with the generally phlegmatic deportment of the animal, but in contrast to a low winter response reaches almost 100% in frequency in a group tested during the spring. Over a total of about 1300 trials, 33% reacted to stimulation of the lateral eyes, 30% to ocellar illumination, and an unexpected 91% to ventral eye stimulation. Although Wasserman (1973a) did not positively exclude the participation of retinula cells at the surface of the brain-the deafferented medulla responds to light

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stimulation (Snodderly, 1971)- this reliable behavioral response invites reconsideration of “the apparently senseless waste of photoreceptor cells strung out along the silent ventral eye nerve where they transduce light with great elegance to absolutely no purpose” (Clark et al., 1969b). Permanently implanted electrodes (Coming et al., 1965; Adolph, 1971) to monitor activity in the optic nerves, heart, and abdominal ganglia have revealed that the heartbeat is considerably influenced by visual input (Coming and Von Burg, 1968; Coming et al., 1971). The principal effect is a brief acceleration of heart rate at the onset of illumination and the converse at “off.” In addition, short- and long-term cardiac periodicities (3 minutes versus 15-20 minutes) are affected by illumination as well as by lesions to the visual area of the central nervous system (Coming and Von Burg, 1970). Even the ectopic compound eyes found by Barlow and Kaplan (1972)had a similar effect on the heart rate, although the relative influence of the other photoreceptors has not been explored. The potential usefulness of such investigations is enhanced by the various studies on the neurophysiology (Tanaka et al., 1966; Von Burg and Coming, 1969; Corning and Von Burg, 1970; Palese et al., 1970; Lang, 1971; Rulon et al., 1971), morphology (Bursey and Pax, 1970; Leyton and Sonnenblick, 1971; Sperelakis, 1971; Lang, 1972), and pharmacology of the Limulus heart (Pax and Sanbom, 1967a,b;Von Burg and Coming, 1971). Various other aspects of the life of Limulus are undoubtedly guided by visual stimuli but have not been explored. The animals have been dredged from depths of 20 meters several miles offshore (Shuster, 1960), yet are said to adjust their breeding time to the phase of the moon (Lockwood, 1870). In Cape Cod Bay, Massachusetts, tagged animals have been observed to travel an average of 1.1 km per day over a 2-week period and to migrate as far as 34 km in 2 months (Shuster, 1950).Given their apparent maximum speed of about 2.5 km per day (extrapolated from Cole, 1923),this means that, despite a complex and changing coastline, some animals maintain a stubborn directional orientation over a period of weeks. ACKNOWLEDGMENTS

I am grateful to Drs. N. J. Alexander, G . F. Gwilliam, J. W. Hawkes, L. H. Kleinholz, and C. J. Russell for constructive criticism of the manuscript and to Mr. J. H. It0 for his artistry in rendering the diagrams. The personal research cited in this article has received painstaking and patient technical attention from Ms. A. J. Gri5n. I am indebted to Ms. M. T. Barss and S. E. Maher for editorial and clerical assistance.

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This paper constitutes Publication No. 717 of the Oregon Regional Primate Research Center. The research of the author has been supported by Grants RR00163 and EY00392 from the National Institutes of Health and by a Bob Hope Grant in Aid by Fight-for-Sight, Inc., New York City. Figures 9, 12, 15, 18, and 32 are reproduced by permission of Springer-Verlag, Berlin and New York.

ADDENDUM Two recent articles [M. E. Behrens, J . Comp. Physiol. 89, 45 (1974);W.H.Miller and D. F. Cawthon, Znuest. Ophthulmol. 13,401 (1974)l provide new details about the process of light adaptation in the Limulus compound eye. Behrens (1974)illustrates (in material fixed in boiling water) an increase in the depth of the cone cells,

length of the eccentric cell dendrite, and radial extent of the rhabdomal rays with light adaptation. Maintenance of adaptation after short-term denervation or partial occlusion of the eye points to direct photomechanical responses of the pertinent cells. An apparent circadian rhythm of morphological changes that persists during 24 hours of constant darkness may be attributable to neurosecretory influences. Miller and Cawthon (1974)confirm the changes in the shape of the rhabdome and dendrite and show that high concentrations of topically injected colchicine, dissolved in dilute dimethyl sulfoxide, cause pigment migration in retinula cells to the light-adapted position. Both studies mention the variable response of ommatidia to light and dark adaptation. REFERENCES

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