Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration.

PURPOSE: Our objective was to comprehensively assess the nature and chronology of neural remodeling in retinal degenerations triggered by light-induced retinal damage (LIRD) in adult albino rodents. Our primary hypothesis is that all complete photoreceptor degenerations devolve to extensive remodeling. An hypothesis emergent from data analysis is that the LIRD model closely mimics late-stage atrophic age relared macular degeneration (AMD). METHODS: Sprague-Dawley (SD) rats received intense light exposures of varied durations and survival times ranging from 0 to 240 days. Remodeling was visualized by computational molecular phenotyping (CMP) of a small molecule library: 4-aminobutyrate (gamma), arginine (R), aspartate (D), glutamate (E), glutamine (Q), glutathione (J), glycine (G), and taurine (tau). This library was augmented by probes for key proteins such as rod opsin, cone opsin and cellular retinal binding protein (CRALBP). Quantitative CMP was used to profile 160 eyes from 86 animals in over 6,000 sections. RESULTS: The onset of remodeling in LIRD retinas is rapid, with immediate signs of metabolic stress in photoreceptors, the retinal pigmented epithelium (RPE), the choriocapillaris, and Müller cells. In particular, anomalous elevated aspartate levels appear to be an early stress marker in photoreceptors. After the stress phase, LIRD progresses to focal photoreceptor degeneration within 14 days and extensive remodeling by 60 days. RPE and choriocapillaris losses parallel Müller cell distal seal formation, with progressive neuronal migration, microneuroma evolution, fluid channel formation, and slow neuronal death. The remaining retina in advanced light damage can be classified as survivor, light damage (LD), or decimated zones where massive Müller cell and neuronal emigration into the choroid leaves a retina depleted of neurons and Müller cells. These zones and their transitions closely resemble human geographic atrophy. Across these zones, Müller cells manifest extreme changes in the definitive Müller cell tauQE signature, as well as CRALBP and arginine signals. CONCLUSIONS: LIRD retinas manifest remodeling patterns of genetic retinal degeneration models, but involve no developmental complexities, and are ultimately more aggressive, devastating the remaining neural retina. The decimation of the neural retina via cell emigration through the perforated retina-choroid interface is a serious denouement. If focal remodeling in LIRD accurately profiles late stage atrophic age-related macular degenerations, it augurs poorly for simple molecular interventions. Indeed, the LIRD profile in the SD rat manifests more similarities to advanced human atrophic AMD than most genetically or immunologically induced murine models of AMD.

Actin-dependent motility in Cryptosporidium parvum sporozoites.

The present study investigated the role of actin polymerization and myosin motor protein activity in the gliding motility of Cryptosporidium parvum sporozoites. Short motility trails were detected using an indirect immunofluorescent assay (IFA) with a polyclonal antisporozoite antibody following incubation of sporozoites on poly-L-lysine-coated glass slides. Sporozoite motility was blocked following exposure to cytochalasin D, a myosin light-chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapin e, and the myosin ATPase inhibitor 2,3-butanedione monoxime. Sporozoites were observed to form rounded, blunt-ended shapes when exposed to these same inhibitors. Incubation of purified oocysts with these compounds did not significantly inhibit in vitro excystation or subsequent infectivity in cultured epithelial cells. Indirect IFA revealed a uniform distribution of actin protein throughout the body of the sporozoite; immunoelectron microscopy confirmed a diffuse intracellular pattern of gold particles in excysted sporozoites. Collectively, these findings show that sporozoite motility is dependent upon an intact actin-myosin motor system, and the dynamic interaction of F-actin and myosin motor proteins has a further role in maintaining the structural integrity of excysted sporozoites. Further, in vitro excystation and infectivity of C. parvum occurs in the absence of dynamic sporozoite locomotion.

Pathways of cerebral calcium accumulation in a model of focal ischemia in rats.

Focal cerebral ischemia was produced in anesthetized rats by a minimally invasive photothrombic procedure. Rose bengal was injected into a tail vein and the right middle cerebral artery region irradiated for 5 min through the skull with the right common carotid artery temporarily occluded. This resulted in focal cerebral infarction which was restricted to the cortex as shown by autoradiography and histopathology. Edema and the uptake of 45Ca were determined 1, 3 or 24 hours after ischemia in different regions of the brain, ipsilateral and contralateral to the ischemic injury, the tracer uptake at three time points after administration. The values of 45Ca uptake and edema were the highest at the center of the infarction. Simulation of the 45Ca uptake kinetics in the 24 h post-ischemic group, enabled the determination of the contributions of different physiological pathways to cerebral calcium flux. The results indicated the breakdown of the blood-brain barrier to be primarily responsible for the increased uptake of the tracer by the ischemic cortex. A concomitant, presumably intracellular, sequestration resulted in a ca. 16-fold increase in the tissue pool of exchangeable calcium. Simulation such as proposed here would be of value in predicting the outcome of tracer accumulation in pathological situations.

Glutamate as a transmitter in the sensory pathway from prostomial lip to serotonergic Retzius neurons in the medicinal leech Hirudo.

The involvement of glutamate in putative ingestive sensory pathways affecting the excitability of serotonergic Retzius neurons (RZ) in the leech CNS was investigated with a pharmacological approach. Exposure of the prostomial lip to 150 mM NaCl and 1 mM arginine produced excitatory as well as inhibitory responses in RZ found in the reproductive segments, while only excitatory responses were elicted in standard midbody RZ. Antagonists of glutamatergic receptors of the kainate/quisqualate type effectively inhibited chemosensory dependent excitation of RZ. Antagonists of glutamatergic receptors of the N-methyl D-aspartate type were ineffective in this regard. Cephalic nerve stimulation, like chemical stimulation of the lip, produced segment-specific responses in midbody RZ. Both the polysynaptic and monosynaptic components of the excitatory response of standard midbody RZ following cephalic nerve stimulation were inhibited in the presence of the kainate/quisqualate antagonist DNQX. These data suggest a role for glutamate as a transmitter in the neural circuitry from receptors of the leech prostomial lip to serotonergic RZ.

Dextran backfill tracers combined with Lucifer yellow injections for neuroanatomic studies of the leech head ganglion.

Several neuronal tracing substances were applied to the cut ends of leech cephalic nerves and the resulting backfills into the subesophageal ganglion (sbEG) were mapped. A 12 h incubation in 3 kDa dextrans conjugated either to a fluorochrome or to biotin (subsequently tagged with peroxidase) was satisfactory. In separate experiments, possible targets of cephalic nerve afferents (R3 Retzius neurons) were injected with Lucifer Yellow (LY) to visualize their projections. Comparison of the LY-R3 Retzius neuron map with that of the dextran-backfilled D1 nerve revealed extensive overlap in the sbEG. Experiments were performed combining the two protocols, confirming this observation. Moreover, confocal microscopy placed D1 nerve processes in close proximity to R3 Retzius neuron processes, suggesting that they could make synaptic contact with one another in the sbEG. With modifications, this method could be used to identify such contacts using electron microscopy.

Ingestive sensory inputs excite serotonin effector neurones and promote serotonin depletion from the leech central nervous system and periphery.

Thermal and chemical stimuli known to promote ingestive behaviours in the medicinal leech Hirudo medicinalis were tested for their physiological effects on Retzius neurones and for their biochemical effects on serotonin levels in the central nervous system, pharynx and body wall. Retzius neurones throughout the leech nerve cord receive excitatory synaptic input during thermal or chemical stimulation of the prostomial lip. These neurones respond to the rate of change of temperature as well as to absolute temperature at the lip. Exposure of the lip to sodium chloride excites Retzius neurones, whereas exposure to arginine has little effect. Thermal stimulation of the lip elicits a more rapid but less prolonged excitation of Retzius neurones than does chemical stimulation. Stimulation of the prostomial lip is associated with afferent activities in the cephalic nerves D1, D2 and V1-2. Thermal stimulation of the prostomial lip results in depletion of serotonin from midbody ganglia, whereas chemical stimulation has no effect. Conversely, chemical stimulation of the lip results in depletion of serotonin from the body wall, whereas thermal stimulation does not. Pharyngeal serotonin content is decreased with either modality. These data distinguish two important feeding-related sensory input pathways to central serotonergic effector neurones in Hirudo medicinalis.

Acid phosphatase localization in endocytosed horizontal cell gap junctions.

Gap junction (GJ) endocytosis appears to be part of a cycle of GJ renewal in horizontal cells of the teleost fish retina. At least three stages of GJ endocytosis in these neurons have been identified using conventional electron microscopy (EM): invagination of GJ membranes (GJ blebs); free GJ vesicles; and GJ vesicle fusion with mature lysosomes (Vaughan & Lasater, 1990a). In the present study, EM-level acid phosphatase (AP) histochemistry of white bass retina was used to determine at what stage enzymatic degradation of endocytosed GJs begins. Electron-dense AP reaction product was observed within the trans face of the Golgi apparatus, mature lysosomes, and occasional, internal GJ vesicles. In contrast, GJ blebs, peripheral GJ vesicles, and most internal GJ vesicles lacked AP reaction product. These results support the idea that at least some of the GJ vesicles observed within these retinal neurons arise from endocytosis, are on a degradative pathway, and can be termed GJ "endosomes." Furthermore, GJ vesicles appear to be initially free of AP, but some later acquire it (presumably from transport vesicles bearing degradative enzymes). It is still unclear whether our previous report of GJ vesicle fusion with mature lysosomes is a subsequent step in GJ degradation or part of a different degradative pathway altogether.

Glial and neuronal markers in bass retinal horizontal and Müller cells.

Retinal horizontal cells (HCs) are second-order neurons that integrate information from photoreceptors over large retinal areas, mediating the lateral spread of visual signals in the distal retina. The 'glial' vs. 'neuronal' nature of the HC has been widely debated. For example, carbonic anhydrase (CA), glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP) are considered 'glial' markers, yet both CA and GFAP have been previously reported in HCs of the teleost retina in species-specific patterns. In contrast, the neurofilament triplet (NFT) proteins are considered 'neuronal' markers; these proteins have been immunolocalized to a mammalian HC, but are absent from teleost HCs. We have studied these cytochemical characteristics in HCs from the white bass, by immunolabeling both cryosections of intact retina and freshly isolated, identified cells attached to coverslips. We found that both HCs (neurons) and Müller cells (MCs; glia) immunolabeled with antisera to CA. Both type 1 (external) HCs and MCs immunolabeled with an antibody to vimentin. Only MCs immunolabeled with antisera to GS and GFAP. Neither HC perikarya (and their major dendrites) nor MCs immunolabeled with an antibody to the 160-kDa subunit of NFT protein. Thus, bass HCs and MCs share the presence of CA and vimentin epitopes and absence of the NFT 160-kDa epitope. Moreover, retinal cell isolation, by itself, does not affect cell-type specific immunolabeling patterns in identified cells, except for what may be lost with the finer processes of the various cells. Isolated cell studies can aid in interpreting immunolabeling patterns observed in the intact retina, especially in retinal layers where several cell types may be present.

Renewal of electrotonic synapses in teleost retinal horizontal cells.

In teleost retinas, the somata of same-type cone horizontal cells are electrically coupled via extensive gap junctions, as are the axon terminals of same-type cells. This coupling persists throughout the animal's life and is modulated by dopamine and conditions of light- vs. dark-adaptation. Gap junction particle density in goldfish horizontal cell somata has also been shown to change under these conditions, indicating that these junctions are dynamic. We have used electron microscopy to examine gap junctions in bass horizontal cells with a fixation method that facilitates detection of gap junctions. Annular gap junction profiles were observed in the somatic cytoplasm of all cone horizontal cell types in both light- and dark-adapted animals. Serial sections showed that most profiles represented gap junction vesicles free within the cytoplasm; the remainder represented vesicles still attached to extensive plasma membrane gap junctions by a thin cytoplasmic neck, suggestive of an intermediate stage in endocytosis. Observations of gap junction vesicles containing fragments of gap junctional membrane and/or fused with lysosomal bodies further supported this hypothesis. Because gap junctions persist between the horizontal cells, we propose that gap junction endocytosis and lysosomal degradation are balanced by addition of new junctions. While endocytosis has been widely demonstrated to serve in programmed removal of gap junctions (without subsequent replacement), from both nonneuronal cells and developing neurons, this study indicates that it can also function in the renewal of electrical synapses in the adult teleost retina, where gap junction elimination is not the goal.

Distribution of F-actin in bipolar and horizontal cells of bass retinas.

F-actin distribution was studied in bipolar and horizontal interneurons of white bass retina. Cryosections of intact retina and isolated cells in culture were labeled with the F-actin specific probe fluorescent phalloidin. In intact retina, labeling was heaviest in the photoreceptor-pigment epithelium region, outer limiting membrane, horizontal cell somata, and plexiform layers. In isolated bipolar and horizontal cells, labeling patterns specific to each cell type were obtained, as were patterns in distinct categories of neurites extending from cells maintained in culture for several days. Label was especially heavy where contacting horizontal cells apposed and were coupled via extensive gap junctions both in the intact retina and in culture. This observation led to the hypothesis that this F-actin domain might modulate junctional conductance. However, cytochalasin D-induced disruption of the F-actin cytoskeletons of coupled, cultured horizontal cells had no effect on normal coupling or on dopamine-induced uncoupling. The F-actin domain may instead mediate gap junction turnover.

Glial fibrillary acidic protein (GFAP) immunoreactivity in rabbit retina: effect of fixation.

The binding of monoclonal and polyclonal antibodies to glial fibrillary acidic protein (GFAP) antigenic sites in the rabbit retina was shown to be sensitive to aldehyde fixation. In chemically unfixed retina, the polyclonal anti-GFAP labeled Müller cells, astrocytes, and unidentified profiles in the outer plexiform layer; the monoclonal anti-GFAP labeled Müller cell endfeet and astrocytes only. The outer plexiform layer label with the polyclonal antibody was lost after fixation for 1 hr in 1% paraformaldehyde; elsewhere, the label was reduced. Fixation also reduced labeling by the monoclonal antibody. Such fixation sensitivity may underlie the different patterns reported for retinal GFAP immunoreactivity in the literature.

Evidence that microtubules do not mediate opsin vesicle transport in photoreceptors.

The organization of the rod photoreceptor cytoskeleton suggests that microtubules (MTs) and F actin are important in outer segment (OS) membrane renewal. We studied the role of the cytoskeleton in this process by first quantifying OS membrane assembly in rods from explanted Xenopus eyecups with a video assay for disc morphogenesis and then determining if the rate of assembly was reduced after drug disassembly of either MTs or F actin. Membrane assembly was quantified by continuously labeling newly forming rod OS membranes with Lucifer Yellow VS (LY) and following the tagged membranes' distal displacement along the OS. LY band displacement displayed a linear increase over 16 h in culture. These cells possessed a longitudinally oriented network of ellipsoid MTs between the sites of OS protein synthesis and OS membrane assembly. Incubation of eyecups in nocodazole, colchicine, vinblastine, or podophyllotoxin disassembled the ellipsoid MTs. Despite their absence, photoreceptors maintained a normal rate of OS assembly. In contrast, photoreceptors displayed a reduced distal displacement of LY-labeled membranes in eyecups treated with cytochalasin D, showing that our technique can detect drug-induced changes in basal rod outer segment assembly. The reduction noted in the cytochalasin-treated cells was due to the abnormal lateral displacement of newly added OS disc membranes that occurs with this drug (Williams, D. S., K. A. Linberg, D. K. Vaughan, R. N. Fariss, and S. K. Fisher. 1988. J. Comp. Neurol. 272:161-176). Together, our results indicate that the vectorial transport of OS membrane constituents through the ellipsoid and their assembly into OS disc membranes are not dependent on elliposid MT integrity.

Cytochalasin D disrupts outer segment disc morphogenesis in situ in rabbit retina.

Exposure of rabbit retina to cytochalasin D (CD) via a single intraocular injection results in basal rod outer segment (ROS) and cone OS (COS) discs with abnormally large diameters; the overgrown OS membranes extend along either the cell outer or inner segment. Twenty-four hours after the injection, basal ROS and COS discs appear to have recovered their normal diameter, indicating the reversibility of this drug's effect. These data support both the evagination hypothesis for disc morphogenesis and the hypothesis that f-actin's role at the ROS base is to regulate the initiation of membrane evagination and disc diameter.

An immunocytochemical study of cat retinal Müller cells in culture.

Müller cells, the specialized radial glia found in vertebrate retinas, were enzymatically dissociated from adult cat retinas and grown in culture. The cells were processed for immunofluorescence microscopy at times ranging from 4 hr to 3 months in culture. Labeling with antibodies specific to glial fibrillary acidic protein, cellular retinaldehyde binding protein, glutamine synthetase, carbonic anhydrase C and alpha crystallin, all proteins known to be found in Müller cells, was detected in the cultured cells. Immunoblot analysis of the cultured cells showed single protein bands corresponding to the appropriate molecular weights of the antigens.

Disruption of microfilament organization and deregulation of disk membrane morphogenesis by cytochalasin D in rod and cone photoreceptors.

Morphogenesis of photoreceptor outer segment disks appears to occur by an evagination of the ciliary plasma membrane (Steinberg et al., J Comp Neurol 190:501-519, '80). We tested if polymerized actin (F-actin) was necessary for the regulation of this postulated process by incubating Xenopus eyecups with 5 or 25 microM cytochalasin D for 6-28 hours. During the second hour, the incubation medium contained 3H-leucine. Both concentrations of cytochalasin resulted in: 1) dissolution of the rhodamine-phalloidin labeling pattern of photoreceptors, and 2) collapse of the calycal processes (which are normally filled with actin filaments) and disappearance of the inner segment microfilaments. In addition, the few most basal rod and cone outer segment disks appeared several times their normal diameter. These oversized disks had incorporated 3H-leucine and extended along the margin of the outer or inner segment. The nature of the overgrown disks is consistent only with a morphogenetic process involving evaginations of the ciliary plasma membrane. Deregulation by cytochalasin D was manifest by excessive growth of a few nascent disks rather than normal growth of many. Therefore, the normal network of actin filaments is apparently not necessary for continued evagination of the membrane, but it does seem to be an essential part of the mechanism that initiates the evagination of the ciliary plasma membrane and/or the mechanism that controls how far nascent disks grow.

The distribution of F-actin in cells isolated from vertebrate retinas.

Filamentous actin has been localized in isolated retinal neurons and glia using fluorescent phallotoxin. Photoreceptors, bipolar cells, horizontal cells, amacrine cells and Müller (glial) cells were isolated by gentle enzymatic digestion of frog, lizard, rabbit, rat and cat retinas. The cells were then fixed in paraformaldehyde and stained with rhodamine-phalloidin. The patterns of fluorescence recorded were specific for each cell type. All interneurons had spots of bright fluorescence along dendrites and (or) axon terminals, probably corresponding to synaptic sites. Horizontal cells and Müller cells had a continuous subplasmalemmal layer of fluorescence throughout; this layer was also present in bipolar cells, but only in the region of cytoplasm at the base of the dendrites. Müller cells also had bright fluorescence in their apical microvilli and terminal web and associated with the zonulae adhaerentes junctions between Müller cells and photoreceptors. All photoreceptors exhibited fluorescence in their synaptic terminals, in a ring just sclerad to the nucleus (corresponding to zonulae adhaerentes junctions formed with Müller cells), and in cables running longitudinally in the inner segment. Frog photoreceptors also had processes alongside their outer segments. Rods from mammals and Xenopus had distinct spots of fluorescence at the outer segment base in a region that suggests involvement with morphogenesis of new outer-segment disc membrane.