Epi-polarization and incident light microscopy readily resolve an autoradiographic or heavy metal label from an obscuring background or second label.

Difficulty encountered in resolving grains of exposed photographic emulsion in autoradiographs of the densely melanized retinal pigment epithelium was solved by using epi-polarized or incident light microscopy. The apparatus used included a metallurgical illuminator specifically designed for epi-polarization microscopy or, as a less expensive but only slightly less effective alternative, a modified fluorescence illuminator. The black melanin granules absorb incident light (as they do in vivo) while the silver grains reflect it producing a "darkfield-like" representation. Brightfield and darkfield-like images can be alternated easily and quickly, or both can be viewed simultaneously. Epi-polarization microscopy has wider application in resolving a reflective label over any opaque background staining or dark second label.

Two classes of Limulus ventral photoreceptors.

Ventral photoreceptors of the horseshoe crab, Limulus polyphemus, have been important in the study of visual transduction, due to their large size and hardiness in vitro. This study shows that there are two classes of ventral photoreceptors that can be distinguished on the basis of differences in cellular and nuclear dimensions, soma and rhabdom morphology, and axon size. Large protoreceptors, which have been the subject of many physiological studies, have an extensive superficial rhabdom, a nuclear diameter of 20-24 microns, and measure 100-150 microns in length. In contrast, small photoreceptors measure 45-65 microns in length and have a nucleus 13-16 microns across. Small photoreceptors are found singly or in association with large photoreceptors. The rhabdom of isolated small photoreceptors is surrounded by a calyx originating from the soma, so that it appears to be located internally. The rhabdomeral lobe of small photoreceptors associated with large photoreceptors characteristically is divided into several segments, each of which invaginates the rhabdomeral lobe of the adjacent large photoreceptor. The entire external rhabdom of the associated small photoreceptor abuts the rhabdom of the large photoreceptor. Morphometric analysis of the ventral nerves shows that there are two size classes of photoreceptor axons, corresponding to the two classes of photoreceptors. The numbers of axons in each size class are nearly equal. Unlike the ventral eye, none of the other eyes of Limulus have been reported to have more than one morphological class of photoreceptor. Functional differences between the two classes of ventral photoreceptors are suggested by experiments, reported in the accompanying paper (Herman (1991), J. Comp. Neurol. 303:11-21), showing that the large photoreceptors exhibit light-stimulated rhabdom turnover while the small ones do not.

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

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

Regional specializations in the eye of Aplysia, a neuronal circadian oscillator.

The eye of the opisthobranch mollusc, Aplysia californica, contains a neuronal circadian oscillator system as well as a photoreceptor system. The retina contains five classes of receptors, several of which are described for the first time in this paper, and two types of neurons. The most conspicuous photoreceptor has long microvilli and is densely packed with small vesicles. The other four receptor types bear both microvilli and cilia and lack densely packed vesicles. Because of their small size, these four receptors occupy only a small fraction of the retinal area, but numerically they account for about half of the receptors. There are marked differences between the dorsal and ventral portions of the eye of Aplysia. The optic nerve head and associated bundles of axons within the retina form a boundary between two anatomically distinct regions of the eye. The microvillous photoreceptor and one of the receptors bearing both microvilli and cilia are found throughout the eye. The other three receptor types are restricted to the region ventral to the optic nerve head. One type of neuron, which has been shown in other studies to produce compound action potentials whose frequency varies with a circadian rhythm, is also found only ventral to the optic nerve head and associated axon bundles. There are also marked regional variations in cellular dimensions. The rhabdom originating from the microvillous photoreceptors is thickest in the dorsal and central retina, and the cross-sectional areas of these photoreceptors are largest dorsally. The pigmented layer is also much thicker in the dorsal retina. No other molluscan eye has been reported to have as many receptor types as Aplysia, nor has restriction of a receptor or neuronal type to a limited area been described. Regional variations in cellular dimensions have been reported previously primarily in the advanced cephalopod eyes. The significance of these unusual features is discussed in relation to both the visual properties of the eye and the circadian oscillator it contains.

Phagosome movement and the diurnal pattern of phagocytosis in the tapetal retinal pigment epithelium of the opossum.

The diurnal pattern of phagocytosis and the movement of phagosomes was studied in the tapetal retinal pigment epithelium (RPE) of the opossum, Didelphis virginiana. The opossum was chosen because of its rod-dominated retina and large tapetal RPE cells (up to 100 micrometers in height), which are packed with reflective granules and contain little melanin. Thus phagosomes and their passage from apical to basal cell border were easily seen. Opossums were maintained on a 12 hr light/12 hr dark cycle and were sacrificed during the day and night. Phagosomes were consulted by light microscopy in sections extending 2 mm along the eye's vertical meridian. The diurnal pattern of rod phagocytosis was generally similar to that reported for other species, although an elevated phagosome content in two animals that in some cases there might be a smaller nighttime peak in addition to the large burst occurring after light onset. To determine the spatial distribution of phagosomes at different times, the RPE cells were divided into apical, middle, and basal thirds, and the phagosomes in each region were counted. A large number of phagosomes was observed in the basal RPE 1 to 2 hr after light onset, after which the number declined, reaching a low level late in the light period. In contrast, there were few phagosomes in the apical and mid-RPE even after light onset. This suggested that phagosomes remained in the apical and mid-RPE for only a short time. To examine its effect on phagosome movement, colchicine was injected intravitreally prior to the burst of phagocytosis. Colchicine blocked the movement of phagosomes producing a row of phagosomes along the apical margin of the RPE. These results suggest that there is a rapid, microtubule-mediated movement of phagosomes from apical to basal border.

Phagosome degradation in the tapetal retinal pigment epithelium of the opossum.

Ultrastructural and cytochemical features of phagosome degradation were examined in the tapetal retinal pigment epithelium (RPE) of the opossum. Didelphis virginiana. The tapetal RPE cells of the opossum measure as much as 100 micrometers in thickness, and the phagosomes traverse these cells so as to occupy a narrow region along the basal border. Both ultrastructural and cytochemical observations showed that degradation of phagosomes by lysosomes occurs only in this basal region. Acid phosphatase activity was present only in the basal RPE, where phagosomes appeared degraded and were observed to interact with each other and with lysosomes. Phagosomes in the apical and mid-RPE always had two membranes surrounding the discs and were acid phosphatase negative. Ultrastructural changes, which may occur in the absence of lysosomal enzymes, were examined in phagosomes that were, on the basis of several criteria, undegraded. These changes were accentuated in phagosomes trapped in the apical RPE by colchicine.

Melanosome metabolism in the retinal pigmented epithelium of the opossum.

Melanosomal metabolism, including both formation and degradation of melanosomes, was studied in the retinal pigmented epithelium (RPE) of the adult opossum. The majority of the observations were made on a transitional zone between the tapetal and non-tapetal RPE, the region where melanosome metabolism was at its highest level. Formation of melanosomes, demonstrated ultrastructurally by the presence of stage-II and -III premelanosomes, was also examined autoradiographically following the incorporation of the melanin precursor, dihydroxyphenylalanine. The autoradiographic evidence indicated that many newly formed melanosomes were rapidly incorporated into complexes. Ultrastructural observations suggested that melanosome complexes were formed by at least two methods, via the fusion of melanosomes with phagosomes derived from outer segments of photoreceptors, or by the sequestration of melanosomes by cisternae. A central finding of this study, supported by both ultrastructural and histochemical data, is that there are specialized cellular regions that vary in melanosomal formation and lysosomal activity. Stage-II premelanosomes were observed only in the basal parts of the RPE cells, whereas stage-III and -IV melanosomes were found primarily in the apical RPE. Both ultrastructural and cytochemical observations indicated that degradation of melanosomes occurs only in the basal RPE. These findings are interpreted in terms of the expression of both tapetal and nontapetal characteristics in transitional cells. Finally, this study illustrates the role of lysosomal enzymes in shaping the pattern of pigmentation, and shows that the association of lysosomal activity with melanosomes depends on the functional state of the melanosome.