Unorthodox pattern of microvilli and intercellular junctions in regular retinular cells of the porcellanid crab Petrolisthes.

1. Retinular fine structures in compound eyes of the porcellanid crab Petrolisthes differs significantly from two paguroid anomurans Clibanarius and Pagurus which basically conform to the usual conservative decapod crustacean retinular pattern. 2. Bidirectional orientation of microvilli has been discovered in rhabdomeres of retinular cells R1-R7 in Petrolisthes. Distally the regular rhabdom has mainly a typical banded microvillus structure (Figs. 7,8). Proximally rhabdom banding continues but uniquely all seven regular retinular cells contribute sets of alternately orthogonal microvilli to each band (Figs. 5, 6, 12). This unorthodox pattern should reduce polarization sensitivity and enhance sensitivity to unpolarized light. 3. In this special region microvillus layers are strongly elliptical in cross section with the minor axis parallel to the microvilli (Fig. 12). Hence the ends of the major axes protrude considerably from the central area of overlap (Fig. 6). 4. Retinular cell eight has bidirectional microvilli (Figs. 5-7) as usual in brachyuran crabs. Unlike the latter as well as paguroid crabs, Petrolisthes has square facets and a rectangular retinular array (Figs. 1, 3) similar to other galatheids and macruran decapods generally. It also resembles macrurans (shrimps and lobsters) in having perirhabdomal vacuoles absent or much reduced. 5. Tight junctions occur widely between adjacent retinular cells (Figs. 14, 17) especially basally immediately distal to longitudinal zonular adherentes (Figs. 6, 16) typical of compound eyes. Freeze fracture reveals in addition numerous rectangular arrays of particles on the protoplasmic face of retinular cell membrance near, but not part of, the rhabdom (Figs. 19, 20). Other authors have hypothesized polarized transfer functions for similar particle aggregates in certain vertebrate cells.

Crustacean eye fine structure seen with scanning electron microscopy.

The internal fine structure of crustacean compound eyes has been reexamined with scanning electron microscopy. Several different preparative techniques were used in a comparative study of crab, crayfish, shrimp, and stomatopod eyes. The three-dimensional pattern of photoreceptive, dioptric, and screening components of these eyes has been directly demonstrated, and new insight has been gained into their functional organization. Particularly interesting in apposition eyes is the elaborate array of boundary membranes and protoplasmic strands linking the photoreceptive microvilli to their parent cell cytoplasm across the large intracellular vacuoles surrounding the axial rhabdom. Quantitative application of scanning electron microscopy to this system promises to advance our understanding of its proven high rate of receptor membrane turnover.

Freeze-etch and histochemical evidence for cycling in crayfish photoreceptor membranes.

Freeze-etched rhabdoms and adjacent cytoplasmic cytoplasmic organelles from crayfish compound eyes have been studied for evidence of photoreceptor membrane cycling. The protoplasmic leaflet face (PF) of split photoreceptor membrane of the microvilli is richly particulate. The particles (92 +/- 16 A in diameter in surface fractures; 70 +/- 9 A in cross fractures; density about 8000/mum2) probably indicate rhodopsin molecule localization. Closely similar particles appear in membranes of pinocytotic vesicles, multivesicular bodies (MVB) and secondary lysosomes. In contrast other retinular cell membranes like plasma membrane remote from the rhabdom are quite distinct (60 +/- 23 A particle diameter, density ca 1000/mum2.) Histochemical tests for acid phosphatase demonstrate its presence in well-developed (but not early stage) MVBs, mixed lamellar vesicular bodies (LVB) and lamellar bodies. Density of PF particles decreases from 8000 in MVB to roughly 4500/mum2 in LVB indicating a degradative sequence from rhabdom to lamellar bodies. Membrane leaflet orientations show that primary endocytosis from microvilli must be followed by secondary endocytosis of fused coated vesicles to form MVB. Morphological evidence for photoreceptor membrane resynthesis has not been found yet in crayfish but some has been obtained in other crustaceans.

Localization of the violet and yellow receptor cells in the crayfish retinula.

Cellular identification of color receptors in crayfish compound eyes has been made by selective adaptation at 450 nm and 570 nm, wavelengths near the lambda(max)'s of the two retinular cell classes previously demonstrated. By utilizing earlier evidence, the concentration of lysosome-related bodies (LRB) was used to measure relative light adaptation and thus wavelength sensitivity in 665 retinular cells from six eyes. The observed particle distributions demonstrate the following. Both violet and yellow receptors occur ordinarily in each retinula. Of the seven regular retinular cells two (R(3) and R(4) using Eguchi's numbering [1965]) have mean sensitivities significantly greater to violet and less to yellow than the other five. The latter apparently comprise "pure" yellow receptors (R(1) and R(7)) and mixed yellow and violet receptors (R(2), R(5), and R(6)). Explanations of such ambiguity requiring two visual pigments in single retinular cells or intercellular coupling of adjacent neuroreceptors are apparently precluded by previous evidence. Present data imply alternatively some positional variability in the violet pair's location in individual retinulas. Thus R(3) and R(4) are predominantly the violet receptors but in some retinulas R(2) and R(3) or R(4) and R(5) (or rarely some other cell pairs) may be. The retinal distribution of such variations has yet to be determined. In agreement with intracellular recordings the blue and yellow cells here identified belong to both the vertical and horizontal e-vector sensitive channels.

Dichroism of photosensitive pigment in rhabdoms of the crayfish Orconectes.

Microspectrophotometric measurements of isolated crayfish rhabdoms illuminated transversely show that their photosensitive absorption exhibits a dichroic ratio of 2 in situ. The major absorption axis matches the axial direction of the closely parallel microvilli comprising the receptor organelle. Since these microvilli are regularly oriented transversely in about 24 layers, with the axes of the microvilli at 90 degrees in alternate layers, transverse illumination of a properly oriented rhabdom displays alternate dichroic and isotropic bands. Because all the microvilli from any one cell share the same orientation, the layers of microvilli constitute two sets of orthogonal polarization analyzers when illuminated along the normal visual axis. Furthermore, since the dichroic ratio is 2 and transverse absorption in isotropic bands is the same as that in the minor absorbing axis of dichroic bands, the simplest explanation of the analyzer action is that the absorbing dipoles of the chromophores, as in rod and cone outer segments, lie parallel to the membrane surface but are otherwise randomly oriented. The rhabdom's functional dichroism thus arises from its specific fine structural geometry.

Mechanism of polarized light perception.

As background for a report on our current selective adaptation experiments in decapod crustaceans, the various facts and hypotheses generally relevant to intraretinal sensitivity to polarized light in arthropods as well as cephalopods have been marshaled. On the basis of this review, the following working hypotheses have been made. 1) One ommatidium in the compound eye is the functional unit in image perception but contains in its component retinular cells subunits which can work independently in detecting other visual parameters, such as polarization. 2) Single retinular cells do respond differentially to light polarized in various planes. 3) Light sensitivity, including e-vector detection, is localized in the rhab domeres, which comprise closely packed arrays of microvilli protruding axially from retinular cells; the dichroism of the photopigment molecules, which are contained within the microvilli, provides the molecular basis of e-vector detection. 4) The visual pigment molecules have their major dichroic axis aligned predominantly parallel to the long axis of the microvillus containing them; typically all microvilli in a single rhab domere are closely parallel to one another, thus comprising at the cellular level a unit dichroic analyzer with maximum optical density to photons vibrating in the direction parallel to these microvillous protrusions. 5) In most decapod crustaceans, in cephalopods, and in some insects the microvilli in all rhabdomeres of a retinula are oriented in only two directions, perpendicular. to each other. Therefore, e-vector perception must depend at the retinular level on a two channel system consisting of a pair of dichroic analyzers with their major transmitting axes fixed at a 90 degrees angle determined by the two directions of microvillus orientation. Our new results on selective adaptation in the eye of Cardisoma provide direct experimental evidence for such a two-channel analyzer in which the pair of functional units have their maximum sensitivity to polarization in the same retinal directions as the rhab dom microvilli observed in electron micrographs. In turn, these directions correspond with the vertical and horizontal axes of the animal's normal spatial orientation. In e-vector detection the seven retinular cells of a single decapod ommatidium thus form two operational subgroups of four and three cells, respectively (39). The correspondence of the electrophysiological evidence for a dual polarization analyzer with the perpendicular directions shown by the microvilli in a single rhabdom strengthens the idea that one ommatidium is enough for detecting e-vector orientation. On this evidence we may conclude that the model developed above for a two-channel polarization analyzer effectively accounts for the relevant spectrophotometric, fine-structural, electrophysiological, and behavioral data currently available for a considerable number of arthropods and cephalopods.