D. Howard Dickson

Fine structure of the lamprey photoreceptors and retinal pigment epithelium (Petromyzon marinus L.)

Abstract The retinal photoreceptor cells and pigment epithelium of the lamprey, Petromyzon marinus have been examined with the electron microscope. The pigment epithelial cells in this avascular retina are characterized by surface specializations in the form of apical microvilli and basal infoldings. These cells contain myriad myeloid bodies, some phagosomes and residual bodies, as well as sparse, apically positioned melanin granules. Two types of photoreceptor cells are described in the retina of this animal. The long receptors have conical outer segments, slender elongated myoid regions and their nuclei are positioned in the outer portion of the outer nuclear layer near the external limiting membrane. The pyramid-shaped synaptic terminals of these cells are found deep in the outer plexiform layer, are less electron dense than those of the short receptors and their synaptic vesicles appear to be less densely packed than in the long receptor terminals. The short receptors have slender, elongated outer segments, their nuclei are positioned deeper in the outer nuclear layer, while the spherical synaptic terminals are located in the scleral region of the outer plexiform layer. The outer segments of both cell types however, show similar cone-like characteristics at the ultrastructural level, being composed of membrane-bound stacks of double-membrane discs, some of which are in continuity with the extra-cellular space. The outer segment of both types of receptors is joined to the inner segment through a slender ciliary stalk and is surrounded by calycal processes. Autoradiographic analysis shows that protein renewal in the photoreceptor outer segments of both long and short receptors takes place in a similar diffuse manner, giving rise are to the suggestion that both photoreceptor types in the retina of Petromyzon marinus cones.

A cytochemical study of myeloid bodies in the retinal pigment epithelium of the newt Notophthalmus viridescens.

SummaryIt has been suggested (Yorke and Dickson 1984) that myeloid bodies (MBs) in the retinal pigment epithelium (RPE) of the newt, Notophthalmus viridescens, may represent areas of endoplasmic reticulum where lipids, such as 11-cis retinal derived from phagocytized outer segment tips, accumulate prior to esterification. Experiments in which an artificial ester substrate was added during in-vitro incubations have shown that esterase activity is represented in all areas of the newt RPE endoplasmic reticulum, including sites adjacent to all MBs. In related tests in which the localization of enzyme activity was restricted to areas of the cell where there had been accumulations of naturally-occurring (endogenous) esters, the products of ester hydrolysis were restricted to profiles of endoplasmic reticulum associated with lipid droplets, and with the interior of about 20% of those MBs that appeared completely circular in sections. This enzyme activity was not associated with other MB configurations. Results from endogenous-ester hydrolysis were identical to those obtained after staining with ZIO. This ZIO-reactivity was not affected by pre-incubation with agents that blocked or protected sulphydryl groups, and ZIO-reactive sites associated with MBs did not form complexes with digitonin. These observations suggest that MBs are a site of lipid-ester formation, but that they do not represent unique intracellular areas for this activity.

Photoreceptor synaptic ribbons: Three-dimensional shape, orientation and diurnal (non) variation*

A variety of different lighting regimes have been reported to cause alterations in the length and number of synaptic ribbons within photoreceptor synaptic terminals. Whether these alterations occur in mammalian visual systems during a diurnal lighting cycle was tested using computer-assisted morphometrics. Adult guinea pigs were entrained to a 12:12/ligh:dark regime and subsequently killed at various times throughout the cycle. Representative samples from all quadrants of the eyes were processed for electron microscopy and the synaptic ribbons within the synaptic terminals of the three different types of photoreceptors in this retina (alpha and paranuclear rods, and cones) were analyzed with the aid of an image analysis computer (Zeiss IBAS) for their length and absolute number per terminal. Contrary to previously published reports on other species, the synaptic ribbons in all three terminal types exhibited no statistically significant change in either number per terminal or in length, throughout the 24 hr cycle. Computer-based three-dimensional reconstructions from serial thin sections revealed horseshoe-shaped synaptic ribbons with pleats in numerous planes conforming to the contours of the invaginating postsynaptic elements.

Lamellar to tubular conformational changes in the endoplasmic reticulum of the retinal pigment epithelium of the newt, Notophthalmus viridescens

SummaryThe retinal pigment epithelium (RPE) of the newt (Notophthalmus viridescens) was examined ultrastructurally under both in-vivo and in-vitro conditions. Five distinct conformations of smooth endoplasmic reticulum (SER), two lamellar and three tubular, were observed. The two lamellar conformations included myeloid bodies, which have previously been described (Yorke and Dickson 1984), and fenestrated SER. The latter appeared as layers of flattened or curved cisternae which were penetrated by fenestrations. Fenestrated SER became indistinguishable from the highly branched and convoluted random-tubular SER through the formation of an intermediate configuration (“tubular sheets”). The remaining tubular SER conformations appeared to arise from random-tubular SER through a progressive reduction in branching and a straightening of individual tubules. Fascicular SER was represented by the hexagonal organization of straight, unbranched tubules into bundles (fascicles). Spiral SER consisted of a similar hexagonal arrangement, but the unbranched tubules spiralled about one another. Neighbouring tubules in areas of spiral SER were also joined together by pairs of electrondense bars. Although lamellar (especially myeloid bodies) and random-tubular configurations of the SER were common features in vivo, fascicular and spiral SER were primarily conformations encountered in vitro. Conditions favouring bilayer lipid phases also appear to facilitate the formation of both myeloid bodies and fascicular SER. These conditions included increased duration of incubation, low (<20° C) incubation temperatures, and Ca2+-free incubations with EGTA. Random-tubular SER was most prevalent in media supplemented with fetal calf serum and also after warmer (30° C) incubation temperatures. We speculate that the different conformations of SER observed in the newt RPE may be due, in part, to lipid phase transitions within the membranes of this organelle. However, the specific formation of fascicular and spiral SER may also involve some additional factor, possibly a protein.

Neuro-glial relationships at the external limiting membrane of the newt retina.

Abstract The ultrastructure of the external limiting membrane of the adult newt retina and of the seleral, outer portions of the Muller cells in this region, has been examined in detail. The radial fibers of Muller expand in a conical fashion at their scleral ends to compensate for the narrowing of the photoreceptor myoids in this region. The Muller cell conical expansions are divided into two regions, a scleral mitochondria-free zone and a more vitreal mitochondrial zone. Apical Mullerian microvilli, contain fine filaments which are continuous with the filamentous mitochondria-free zone, where coated vesicles and multivesicular bodies are found. Lateral extensions of the mitochondria-free zone appear in longitudinal sections to overlap, and in tangential sections to interlock with adjacent Muller cells and so fit together like the pieces of a jig-saw puzzle. The external limiting membrane is composed of both interglial and neuroglial junctions. The most scleral component of the interglial junction consists of large gap junctions which form an extensive network between the interlocking apical processes of the Muller cells and are consistently observed in a plane perpendicular to the long axis of the Muller fibers. A second interglial junctional component, resembling a zonula adherens, is found vitreal to the interglial gap junction network. The neuroglial junctions resemble zonulae adherentes but display a frequent narrowing of the intercellular cleft and gap junctions were never observed between these cells. A second population of large interglial gap junctions are observed but these are distinguished from gap junctions of the external limiting membrane, by their proximity to the mitochondria-filled portions of the conical Muller cell expansions. Glio-glial and neuro-glial relationships are discussed with regard to the nutritional role of the Muller cell in this avascular retina.

Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium.

SummaryMyeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 μm long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.

Phospholipid composition of myeloid bodies from chick retinal pigment epithelium

The phospholipid composition of a myeloid body (MB) enriched subcellular fraction of chick retinal pigment epithelium (RPE) was determined in order to further characterize the origin and functional significance of these lamellar membrane organelles. The major MB phospholipids found were phosphatidylcholine and phosphatidylethanolamine which represented 43% and 34% of the total MB lipids respectively. Sphingomyelin and phosphatidylinositol comprised the remaining detectable phospholipids. The fatty acyl chain composition of all detected phospholipids showed that the long-chain polyunsaturated fatty acids [arachidonic (20:4 n-6), docosapentaenoic (22:5 n-3) and docosahexaenoic (22:6 n-3)] account for greater than 45% of the fatty acids in MB membranes. This high proportion of long-chain polyunsaturated fatty acids in MBs is particularly striking when compared to the long-chain fatty acid composition of the photoreceptor outer segments from this predominantly cone retina which contains less than 25% long-chain polyunsaturated fatty acids. The results from this study clearly demonstrate that MB lipids represent a significantly enriched pool of long-chain polyunsaturated fatty acids.

Effects of temperature and bright light on myeloid bodies in the retinal pigment epithelium of the newt, Notophthalmus viridescens

SummaryMyeloid bodies (MBs) are specialized regions of endoplasmic reticulum which occur in the retinal pigment epithelium of a number of vertebrate species. In the newt, Notophthalmus viridescens, the effects of temperature and brief exposure to bright flashed-light on myeloid bodies have been studied. Morphometric analysis has shown that in animals sampled at 06.30 h, myeloid body sectional area remained unchanged in animals maintained in the cold (1°C), compared with control animals at 15°C, whereas phagosome area was significantly increased. At higher temperatures (30° C), myeloid body area was observed to decline from control values, while phagosome area was substantially increased. During the first 2 h of the light phase of a normal (15° C) 12:12 LD lighting cycle, myeloid-body sectional area dropped significantly from values recorded in the latter part of the dark phase. This reduction of MB area at the normal time of “lights-on” was greatly reduced when animals experienced an extended period of darkness. When animals experiencd a bright flashed-light at the normal time of “lights-on”, followed by a period of extended darkness, reduction in MB area was less pronounced when compared to cycled control animals. These results are discussed in the context of the hypothesis (Yorke and Dickson 1984) that MBs represent a temporary storage site for lipids entering the pigment epithelium after phagocytosis of shed outer segment tips, prior to their permanent storage in lipid droplets. These results are consistent with the proposal that myeloid bodies are removed from the cytoplasm of the newt pigment epithelium by metabolic processes which are active over time, but accelerated by increased temperatures or the presence of light.

Day‐Night Differences in the Number and Structure of Synaptic Ribbons in Chick Pineal

The objective of this study was to evaluate the effect of lighting (day‐night) changes on pinealocyte synaptic ribbon shape (conformation) and numbers. Three‐dimensional reconstruction analysis of pinealocyte basal processes revealed that 30% (6/20) of all ribbons from dark‐adapted animals were either curved or split. Synaptic ribbons from light‐adapted animals did not show this variant morphology; all were linear structures. An analysis of each section from each series, containing curved or split ribbons, revealed that 44% (16/36) of all ribbon profiles would yield inflated counts if used in random morphometric sampling protocols. Therefore, split and curved (variant) ribbon morphologies could result in an overestimation of synaptic ribbon populations of approximately 13% (0.44 × 30%) of dark‐adapted samples. In spite of this potential sampling error, the fourfold increase in the number of synaptic ribbons observed during the dark phase of a light‐dark cycle remains highly significant (P < 0.0001).

Posthatch day/night differences in synaptic ribbon populations of the chick pineal.

Pineal synaptic ribbon (SR) populations of the early posthatch white leghorn chick were counted to determine if they demonstrate a rhythm that is in accordance with the light/dark cycle. SRs were counted between day 7 and day 10 and on day 14 of posthatch development, with samples at midlight, middark (14L:10D), and constant darkness. SR populations did not exhibit significant changes on days 7 and 8 under cycled lighting conditions nor on days 9 and 10 under constant darkness. A second experiment demonstrated that the dark:light ratio of SR populations of day 14 chicks, under cycled lighting, was 3.4:1.0, indicating SR rhythmicity by that stage of development. In that a preliminary experiment had demonstrated a 4.2:1.0 darklight ratio in SR populations in a predominantly day‐10 population of chicks, we believe that SR rhythmicity begins on, or near, day 10 of posthatch development. To determine if the invasion of sympathetic fibers from the superior cervical ganglion (SCG) correlates with the initiation of SR light/dark population differences, we employed tyrosine hydroxylase immunofluorescence to reveal the distribution of catecholaminergic fibers in chick pineal follicles Follicular innervation doubled over the day 7 to day 14 period, during which time light/dark differences in SR populations were established. There is a correlation, in time, between the invasion of the pineal by the sympathetic fibers and the initiation of SR light/dark differences. The circadian rhythm of pineal N‐acetyltransferase (NAT) activity, the rate‐limiting enzyme in the melatonin pathway, is established earlier (day 2) than the light/dark differences in SR populations (day 10). It is possible that SR rhythmicity is influenced by the ingrowth of the pineal sympathetic innervation, and that SRs respond to an extrapineal oscillator rather than the independent oscillators of the chick pineal responsible for the rhythm of NAT activity and melatonin synthesis.