Kenneth A. Linberg

Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment.

Retinal detachment, the separation of the neural retina from the retinal pigmented epithelium, starts a cascade of events that results in cellular changes throughout the retina. While the degeneration of the light sensitive photoreceptor outer segments is clearly an important event, there are many other cellular changes that have the potential to significantly effect the return of vision after successful reattachment. Using animal models of detachment and reattachment we have identified many cellular changes that result in significant remodeling of the retinal tissue. These changes range from the retraction of axons by rod photoreceptors to the growth of neurites into the subretinal space and vitreous by horizontal and ganglion cells. Some neurite outgrowths, as in the case of rod bipolar cells, appear to be directed towards their normal presynaptic target. Horizontal cells may produce some directed neurites as well as extensive outgrowths that have no apparent target. A subset of reactive ganglion cells all fall into the latter category. Muller cells, the radial glia of the retina, undergo numerous changes ranging from proliferation to a wholesale structural reorganization as they grow into the subretinal space (after detachment) or vitreous after reattachment. In a few cases have we been able to identify molecular changes that correlate with the structural remodeling. Similar changes to those observed in the animal models have now been observed in human tissue samples, leading us to conclude that this research may help us understand the imperfect return of vision occurring after successful reattachment surgery. The mammalian retina clearly has a vast repertoire of cellular responses to injury, understanding these may help us improve upon current therapies or devise new therapies for blinding conditions.

Retinal neurons of the California ground squirrel, Spermophilus beecheyi: A Golgi study

Although the optic nerve fibers of the cone‐dominant ground squirrel retina have been well studied physiologically, the morphological details of the retinal neurons have not. To that end, retinal neurons of the California ground squirrel have been studied in Golgi‐impregnated wholemounts.

Experimental retinal reattachment: a new perspective.

In the feline model, retinal detachment initiates a cascade of changes that include photoreceptor-cell “deconstruction,” apoptotic death of some photoreceptors, neurite outgrowth from second-and third-order neurons, remodeling of photoreceptor synaptic terminals, and Müller-cell gliosis. We have previously shown that reattachment within 24 h halts or reverses many of these presumed detrimental changes. However, in patients with retinal detachments, reattachment cannot always be performed within this 24-h window. Moreover, recovery of vision following successful reattachment surgery in the macula is often imperfect. Here, we examine the ability of relatively long-term reattachment (28 d) to stop or reverse several cellular events that occur at 3 d of detachment. In contrast to earlier studies of reattachment, which focused on the regeneration of outer segments, we focus our attention here on other cellular events such as neuronal remodeling and gliosis. Some of these changes are reversed by reattachment, but reattachment itself appears to stimulate other changes that are not associated with detachment. The implications of these events for the return of vision are unknown, but they do indicate that simply reattaching the retina does not return the retina to its pre-detachment state within 28 d.

Ultrastructure of aesthetasc innervation and external morphology of the lateral antennule setae of the spiny lobster Panulirus interruptus (Randall)

SummarySix types of setae and one type of cuticular depression were examined on the lateral antennule of the spiny lobster Panulirus interruptus using scanning electron microscopy. The organization and ultrastructure of the innervation of the most numerous setal type, the aesthetasc, were investigated using light-and transmission electron microscopy.Each aesthetasc is innervated by approximately 300 bipolar neurons whose sensory dendrites penetrate the hair and extend toward the tip, and whose axons project towards the central nervous system. The neuronal somata and two types of glia form a cluster within the antennular lumen. The inner sheath-cell somata encircle the dendritic tract distal to the sensory somata. These cells appear to extend distal processes which wrap the dendritic tract to the base of the aesthetasc. Elongate outer sheath cells are interposed between the glia-wrapped dendritic tract and the hypodermis which underlies the antennule cuticle. A continuous investment of neural lamella separates the hypodermis, the entire cluster of somata, and sensillar nerve from the antennule lumen. The organization of the neuronal somata and their association with outer and inner sheath cells in this marine species appear similar to those of crustaceans from freshwater and terrestrial habitats.

A burst of differentiation in the outer posterior retina of the eleven-week human fetus: an ultrastructural study.

Many studies on human retinal development have cited the third gestational month as a period when the posterior retina undergoes rapid differentiation and maturation, including a lining up of cone precursors. Ultrastructural data on the posterior retina during the third month are very limited, and totally lacking for the cone monolayer. We have examined two human fetal retinas between ten and 11 gestational weeks. Before the appearance of the cone monolayer, the outer neural retina consists of a homogeneous population of undifferentiated neuroblasts. Mitotic figures are still evident, even posteriorally. There is no outer plexiform layer (OPL). The interface of neural retina to retinal pigment epithelium (RPE) is largely featureless. By 11 weeks, the posterior retina has a thin OPL that separates the many rows of cells in the developing inner nuclear layer from the single tier of macular cone precursors. The RPE monolayer consists of cuboidal cells whose apical surface elaborates ridges of cytoplasm and branched processes that project into the subretinal space. The large, cuboidal cones are linked to each other and Müller cells at the outer limiting membrane. They show definitive signs of the structural polarity typical of vertebrate photoreceptors. Their apical cytoplasm contains many organelles common to the inner segment, while the basal cytoplasm has synaptic ribbons and vesicles, and receives invaginating contacts from processes in the OPL neuropil arising from differentiating second-order neurons. Lateral cone surfaces are mutually underlain by large subsurface cisterns.

Distribution of S- and M-Cones in Normal and Experimentally Detached Cat Retina

The lectin peanut agglutinin (PNA) and antibodies to short (S)‐ and medium to long wavelength (M/L)‐sensitive cones were utilized in order to define the relative distributions of the two spectral types of cone across the domestic cats retina. These values, in turn, were compared to those from retinas that had been experimentally detached from the retinal pigment epithelium. The pattern of cone distribution in the normal cats retina is established by the preponderance of M‐cones that constitute between 80% and 90% of all cones. Their peak density of over 26,000 cells/mm2 resides at the area centralis. Though M‐cone density decreases smoothly to the ora serrata where they have densities as low as 2,200 cells/mm2, the density decrease along the nasotemporal axis is slower,creating a horizontal region of higher cone density. S‐cones constitute between 10% and 20% of all cones, the number being quite variable even between individual animals of similar age. The highest S‐cone densities are found in three distinct locations: at the superior far periphery near the ora serrata, immediately at the area centralis itself, and in a broad zone comprising the central and lower half of the inferior hemiretina. S‐cones in the cat retina do not form a regular geometrical array at any eccentricity. As for the detached cat retina, the density of labeled S‐cone outer segments (OS) decreases rapidly as early as 1 day postdetachment and continues decreasing to day 28 when the density of cones labeling with anti‐S opsin has dropped to less than 10% of normal. This response points to a profound difference between rods and cones; essentially all rods, including those without OS, continue to express their opsin even in long‐term detachments. The implications of these results for visual recovery after retinal reattachment are discussed. J. Comp. Neurol. 430:343–356, 2001.

Intercellular junctions in the early human embryonic retina

Previous studies have shown that adult retinal pigment epithelial cells and early undifferentiated neural retinal cells are connected by intercellular junctions. In this study we found intercellular junctions between pigment epithelial cells, between neural retinal cells, and between these two cell types in the 28 and 36 mm human embryo. Intercellular junctions described include the zonula adhaerens, zonula occludens, macula adhaerens, and gap junctions. The gap junctions are of particular interest because of their role in intercellular communication. It appears that all cell types in the early embryonic human retina are coupled by extensive gap junctions.

Ultrastructure of the larval firefly light organ as related to control of light emission

SummaryThe firefly larva has a pair of light organs consisting of a layer of interdigitating, light emitting cells, covered dorsally with a layer of opaque, white cells. Each light organ is ventilated by one large and several smaller tracheal branches and is innervated by a branch of the segmental nerve containing two axons. These axons branch profusely in the photocyte layer so that several nerve profiles are seen around any photocyte. Nerve terminals contain large dense-core vesicles and small light-core vesicles. Clusters of light-core vesicles surrounding irregularly shaped membrane densifications, presumably the synapses between nerve and photocyte, are common in nerve terminals. Light emitting cells in insects characteristically contain photocyte vesicles. In the larva there are both full and empty photocyte vesicles; the full vesicles contain a matrix with tubular membrane invaginations in contrast to the empty vesicles which contain amorphous membrane invaginations.

Experimental retinal detachment in the cone-dominant ground squirrel retina: Morphology and basic immunocytochemistry

The cellular responses of the cone-dominant ground squirrel retina to retinal detachment were examined and compared to those in rod-dominant species. Retinal detachments were made in California ground squirrels. The retinas were prepared for light, electron, and confocal microscopy. Tissue sections were labeled with antibodies to cone opsins, rod opsin, glial fibrillary acidic protein (GFAP), vimentin, synaptophysin, cytochrome oxidase, and calbindin D 28K. Wax sections were probed with the MIB-1 antibody to detect proliferating cells. By 10 h postdetachment many photoreceptor cells in the ground squirrel already show structural signs of apoptosis. At 1 day many photoreceptors have collapsed inner segments (IS), yet others still have short stacks of outer segment discs. At 3 days there is a marked increase in the number of dying photoreceptors. Rod and medium-/long-wavelength opsins are redistributed in the cell membrane to their synaptic terminals. At 7 days photoreceptor cell death has slowed. Some regions of the outer nuclear layer (ONL) have few photoreceptor somata. IS remnants are rare on surviving photoreceptors. At 28 days these trends are even more dramatic. Retinal pigmented epithelium (RPE) cells do not expand into the subretinal space. The outer limiting membrane (OLM) appears flat and uninterrupted. Müller cells remain remarkably unreactive; they show essentially no proliferation, only negligible hypertrophy, and there is no increase in their expression of GFAP or vimentin. Horizontal cells show no dendritic sprouting in response to detachment. The speed and extent of photoreceptor degeneration in response to detachment is greater in ground squirrel than in cat retina-only a small number of rods and cones survive at 28 days of detachment. Moreover, the almost total lack of Müller cell and RPE reactivity in the ground squirrel retina is a significant difference from results in other species.

Three-dimensional organization of nascent rod outer segment disk membranes

Significance A vertebrate photoreceptor cell depends on the elaboration of its cilium to generate its light-sensitive organelle, the outer segment (OS), which is made up of a stack of membrane disks, containing the visual receptor, opsin. How this elaboration occurs has been the subject of recent controversy. Here we used electron microscope tomography to obtain a 3D analysis of the membrane organization at the base of the OS, where new membrane disks are continually made to replace the older ones. We show that the nascent disk membrane is continuous with the ciliary plasma membrane, and appears to form by a complex reshaping of this membrane, involving an invagination, followed by outward growth, and, finally, the completion of a disk rim. The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.