Changes in rat retinal ganglion neuronotrophic factor and its mRNA during postnatal development

It is believed that chloride transport through the macula densa (MD) cells is a factor involved in the tubuloglomerular feedback (TGF) mechanism and in MD-mediated renin release. In this study isolated and perfused rabbit kidney cortical thick ascending limb (cTAL) segments containing MD plaques and attached glomeruli were loaded with chloride (CL-sensitive) 6 methoxy-1-fluorophore (sulphanate-propyl) quinolinium (SPQ). MD and cTAL intracellular chloride concentration ([Cl-]i) was determined by using image-intensified video microscopy and digital image-processing for measuring the intensity of the emitted SPQ fluorescence. With 150 mM NaCl in lumen and bath the [Cl-]i in MD and cTAL cells was 58.8 +/- 7.2 mM (n = 20) and 68.7 +/- 9.8 mM (n = 14), respectively. When the presumed luminal Na(+)-2Cl(-)-K+ co-transporter was blocked by adding 10(-4)M furosemide, the [Cl-]i was reduced in both, MD and cTAL cells from 55.5 +/- 11.9 to 28.6 +/- 10.0 mM (n = 10) and from 43.8 +/- 2.6 to 13.1 +/- 4.5 mM (n = 5), respectively. A reduction in luminal NaCl from 150 to 30 mM also decreased both, MD and cTAL [Cl-]i from 69.4 +/- 9.1 to 36.5 +/- 5.1 mM (n = 9) and from 82.9 +/- 14.5 to 49.4 +/- 8.0 mM (n = 8), respectively. Basolateral addition of the Cl(-)-channel blocker NPPB increased MD [Cl-]i from 31.1 +/- 2.0 to 100.7 +/- 17.0 mM (n = 5) and cTAL [Cl-]i from 44.4 +/- 12.9 to 89.7 +/- 11.7 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of fast axonally transported proteins during the early response of rat retinal ganglion cells to axotomy

We have found that the early response of axotomized rat retinal ganglion cells is characterized by the differential regulation of a number of fast axonally transported proteins. The abundance of 23 radiolabeled fast transported proteins was analyzed at 2 and 5 days after axotomy using two-dimensional gel electrophoresis. Corresponding changes in retinal GAP-43 mRNA were measured using northern analysis. Within 2 days of injury, > 40% of the transported proteins analyzed, including GAP-43, showed increased labeling above control levels. Approximately 13% of transported proteins decreased below control levels, whereas the remainder did not change. Five days after axotomy, only GAP-43 and another fast transported protein, C3, continued to sustain measurable increased labeling above control levels; all previously elevated proteins appeared to have been down-regulated by this time, which corresponds to the onset of cell death. These differential changes were accompanied by parallel increases in GAP-43 mRNA. These results suggest that the molecular changes within rat retinal ganglion cells are differentially regulated within two stages subsequent to damage, initial regenerative growth followed by cell death.     

Dendritic field development of retinal ganglion cells in the cat following neonatal damage to visual cortex: evidence for cell class specific interactions

A well-known feature of the mammalian retina is the inverse relation that exists in central and peripheral retina between the density of retinal ganglion cells and their dendritic field sizes. Functionally, this inverse relation is thought to represent a means by which retinal coverage is maintained, despite significant changes in ganglion cell density. While it is generally agreed that the dendritic fields of mature retinal ganglion cells reflect, in part, competitive interactions that occur during development, the issue of whether these interactions are cell class specific remains unclear. In order to examine this question, we used intracellular staining techniques and an in vitro, living retina preparation to compare the soma and dendritic field sizes of alpha and beta ganglion cells from normal retinae with those of cells located in matched areas of retinae in which the density of beta ganglion cells had been reduced selectively by neonatal removal of visual cortex areas 17, 18, and 19. Our intracellular data show that while an early, selective, reduction in beta cell density has little or no effect on the cell body and dendritic field sizes of mature alpha cells, it results in a 13% increase in the mean soma area and an 83% increase in the mean dendritic field area of surviving beta cells. This differential effect suggests that the soma and dendritic field sizes of alpha and beta ganglion cells in the mature cat retina result primarily from competitive interactions during development that are cell class specific.     

Identification of retinal ganglion cells projecting to the lateral hypothalamic area of the rat

Previous gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. We report that expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. We further demonstrate that the ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants is dependent upon a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants was found to be dependent on endogenous Wnt signaling. We conclude that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways.     

Axonal regeneration of retinal ganglion cells

We have developed retinal culture system of adult mammals to investigate neural regeneration from adult retinal ganglion cells (RGC). In this culture system, neurites were regenerated from RGCs of adult retinal explants. Investigation of neurotrophic effects on the neural regeneration showed that some interleukins and neurotrophins enhanced neurite regeneration from adult rat RGCs. We also found that the adult human retina had the ability of neural regeneration and that neurotrophins enhanced this ability. A novel neurotrophic factor secreted by adult rat hepatocytes also enhanced neurite regeneration not only in adult mice but also in aged RGCs. This result indicated the novel hepatocyte secreted factor is an activator which enhances neural regeneration of the aged retina. We concluded that even adult aged RGCs had the ability of axonal regeneration after injury and that neurotrophic factors might enhanced these abilities. Therefore neurotrophic factors might have practicable applications in drug treatments for intractable disease of the neural retina and optic nerve. Future progress of neuroscience is expected to rescue the retina from various diseases, and to render possible the transplantation of the retina and optic nerve.     

Functional properties of retinal ganglion cells during optic nerve regeneration in the goldfish

Perfringolysin O (theta-toxin) is a cholesterol-binding and pore-forming toxin that shares with other thiol-activated cytolysins a highly conserved sequence, ECTGLAWEWWR (residues 430-440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of the toxin, we evaluated the contribution of each Trp to the toxin conformation during its interaction with liposomal membranes. Circular dichroism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp438, and Trp439 make large contributions to near-UV CD spectra. Quenching the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 and probably Trp436, but not Trp439, contributes to toxin insertion into the liposomal membrane. Near-UV CD spectra of the membrane-associated mutant toxins indicated that both Trp438 and Trp439 are required for the CD peak shift from 292 to 300 nm, a signal related to theta-toxin oligomerization and/or pore formation, suggesting a conformational change around Trp438 and Trp439 in these processes.     

Delay of ganglion cell death by tetrodotoxin during retinal development in chick embryos

Acid-soluble collagen from rat skin was modified by active oxygen in vitro, and properties of the modified collagen as a substratum for fibroblasts were studied. When collagen was treated with ascorbate-copper ion systems, cross-linking and a little degradation occurred rapidly. The cells attached but spread poorly on the modified collagen gel as compared with on the untreated collagen gel. On the other hand, when collagen was treated with H2O2-copper ion systems, only degradation of collagen molecule occurred rapidly. This treatment did not affect the attachment and spreading of the cells on the collagen gel, but when the incubation was continued for a long time, the cells migrated actively and gathered. Thymidine incorporation by the cells was suppressed on both modified collagen gels as compared with that on untreated collagen gel, and the extent of the suppression on the H2O2-copper-treated collagen was larger than that on the ascorbate-copper-treated collagen. These results indicate that the active oxygen-induced cross-linking and degradation significantly alter properties of collagen as a substratum for fibroblasts.     

Two phases of rod photoreceptor differentiation during rat retinal development

We have conducted a comprehensive analysis of the relative timing of the terminal mitosis and the onset of rhodopsin expression in rod precursors in the rat retina in vivo. This analysis demonstrated that there are two distinct phases of rod development during retinal histogenesis. For the majority of rod precursors, those born on or after embryonic day 19 (E19), the onset of rhodopsin expression was strongly correlated temporally with cell cycle withdrawal. For these precursors, the lag between the terminal mitosis and rhodopsin expression was measured to be 5.5-6.5 d on average. By contrast, for rod precursors born before E19, the lag was measured to be significantly longer, averaging from 8.5 to 12.5 d. In addition, these early-born rod precursors seemed to initiate rhodopsin expression in a manner that was not correlated temporally with the terminal mitosis. In these cells, onset of rhodopsin expression appeared approximately synchronous with later-born cells, suggesting a synchronous recruitment to the rod cell fate induced by environmental signals. To examine this possibility, experiments in which the early-born precursors were exposed to a late environment were conducted, using a reaggregate culture system. In these experiments, the early-born precursors appeared remarkably uninfluenced by the late environment with respect to both rod determination and the kinetics of rhodopsin expression. These results support the idea that intrinsically distinct populations of rod precursors constitute the two phases of rod development and that the behavior exhibited by the early-born precursors is intrinsically programmed.     

Regenerative capacity of retinal ganglion cells in mammals

Retinal ganglion cells (RGCs) and their projections in the optic nerve offer a convenient model to study survival and regeneration of mammalian central nervous system (CNS) nerve cells following injury. Possible factors affecting the death of RGCs following axotomy and various approaches to rescue the axotomized RGCs are discussed. In addition, two main strategies currently used to enhance axonal regeneration of damaged RGCs are described. The first focuses on overcoming the unfavorable extrinsic CNS environment and the second concentrates on upregulating the intrinsic growth potential of RGCs. Thus, the failure or success of RGC axonal regrowth after injury depends on the complicated interplay between the extrinsic and intrinsic factors.     

Threshold setting by the surround of cat retinal ganglion cells

1. The slope of curves relating the log increment threshold to log background luminance in cat retinal ganglion cells is affected by the area and duration of the test stimulus, as it is in human pyschophysical experiments. 2. Using large area, long duration stimuli the slopes average 0-82 and approach close to 1 (Weber's Law) in the steepest cases. Small stimuli gave an average of 0-53 for on-centre units using brief stimuli, and 0-56 for off-centre units, using long stimuli. Slopes under 0-5 (square root law) were not found over an extended range of luminances. 3. On individual units the slope was generally greater for larger and longer test stimulus, but no unit showed the full extent of change from slope of 0-5 to slope of 1. 4. The above differences hold for objective measures of quantum/spike ratio, as well as for thresholds either judged by ear or assessed by calculation. 5. The steeper slope of the curves for large area, long duration test stimuli compared with small, long duration stimuli, is associated with the increased effectiveness of antagonism from the surround at high backgrounds. This change may be less pronounced in off-centre units, one of which (probably transient Y-type) showed no difference of slope, and gave parallel area-threshold curves at widely separated background luminances, confirming the importance of differential surround effectiveness in changing the slope of the curves. 6. In on-centre units, the increased relative effectiveness of the surround is associated with the part of the raised background light that falls on the receptive field centre. 7. It is suggested that the variable surround functions as a zero-offset control that sets the threshold excitation required for generating impulses, and that this is separate from gain-setting adaptive mechanisms. This may be how ganglion cells maintain high incremental sensitivity in spite of a strong maintained excitatory drive that would otherwise cause compressive response non-linearities.     

Potassium-evoked directionally selective responses from rabbit retinal ganglion cells

Previous studies have shown that directionally selective (DS) retinal ganglion cells cannot only discriminate the direction of a moving object but they can also discriminate the sequence of two flashes of light at neighboring locations in the visual field: that is, the cells elicit a DS response to both real and apparent motion. This study examines whether a DS response can be elicited in DS ganglion cells by simply stimulating two neighboring areas of the retina with high external K+. Extracellular recordings were made from ON-OFF DS ganglion cells in superfused rabbit retinas, and the responses of these cells to focal applications of 100 mM KCl to the vitreal surface of the retina were measured. All cells produced a burst of spikes (typically lasting 50-200 ms) when a short pulse (10-50 ms duration) of KCl was ejected from the tip of a micropipette that was placed within the cell's receptive field. When KCl was ejected successively from the tips of two micropipettes that were aligned along the preferred-null axis of a cell, sequence-dependent responses were observed. The response to the second micropipette was suppressed when mimicking motion in the cell's null direction, whereas an enhancement during apparent motion in the opposite direction frequently occurred. Sequence discrimination in these cells was eliminated by the GABA antagonist picrotoxin and by the Ca(2+)-channel blocker omega-conotoxin MVIIC, two drugs that are known to abolish directional selectivity in these ganglion cells. The spatiotemporal properties of the K(+)-evoked sequence-dependent responses are described and compared with previous findings on apparent motion responses of ON-OFF DS ganglion cells.     

Calretinin in the mouse superior colliculus originates from retinal ganglion cells

We investigated the origin of the calretinin-immunoreactive fibers in the mouse superior colliculus. The dense plexus of calretinin-positive fibers in the superficial layers of the colliculus was completely eliminated after eye enucleation. Retrograde tracing combined with immunohistochemistry revealed many calretinin-positive small-to-medium retinal ganglion cells projecting to the colliculus. These results indicate that calretinin-containing ganglion cells are the source of this calcium-binding protein in the superficial layers of the superior colliculus.     

Increased levels of TrkA in the regenerating retinal ganglion cells of fish

Retinal ganglion cells of the fish have the spontaneous capacity to regenerate after nerve crush, a phenomenon known to be facilitated by nerve growth factor (NGF). We have studied the high-affinity NGF receptor TrkA, during the regeneration of the tench (Tinca tinca L.) optic nerve, using immunocytochemical techniques. TrkA-like immunoreactivity increased during the regeneration of the retinal ganglion cells. The increase is followed by a change in the subcellular distribution from perinuclear in control cells to cytoplasmic and perinuclear in regenerating ones. This increase was observed when antibodies against the extracellular domain of TrkA were used; no changes in TrkA-like immunoreactivity were observed with antibodies against the intracellular domain of TrkA. We thus conclude that modulation of TrkA is involved in the regeneration of fish retinal ganglion cells.     

Morphology of P and M retinal ganglion cells of the bush baby

P/midget ganglion cells mediate red-green color opponency in anthropoids. It has been proposed that these cells evolved as a specialization to subserve color vision in primates. If that is correct, they must have evolved about the same time as the long-wavelength ('red') and medium-wavelength ('green') pigment genes diverged, thirty million years ago. Strepsirhines are another group of primates that diverged from the ancestor of the anthropoids at least 55 million years ago. If P/midget ganglion cells evolved to subserve color vision, they should be absent in strepsirhines. We tested this hypothesis in a nocturnal strepsirhine, the greater bush baby Otolemur. The retinal ganglion cells were labeled with the lipophilic tracer Dil and the results show that bush babies have P/midget and M/parasol cells similar to those found in the peripheral retinas of anthropoids. A number of studies have shown that the P and M pathways of bush babies share many similarities with those of anthropoids, and our results show that the same is true for their retinal ganglion cells. These results support the hypothesis that the P system evolved prior to the emergence of red-green color opponency.     

Early axon outgrowth of retinal ganglion cells in the fetal rhesus macaque

Employing retinal explants and retrograde transport techniques, we studied the formation of the arcuate fascicles by examining the growth of the central retina, the emergence of the adult fiber layer pattern, and the projections of retinal ganglion cells in the central and peripheral retina. Sixty days prior to foveal pit formation, the distance from the incipient fovea to the optic disk was equal to the adult, even though the retinal area was only 8% of the adult. Arcuate fibers, at this age, were observed to avoid the incipient fovea, with no fascicles and few axons projecting over this region. A small population of 15.2% of the ganglion cells located within 2 mm of the incipient fovea possessed an axon with an aberrant trajectory that wound around and projected 50 to several hundred microns away from the optic disk, compared to only 3% at other retinal locations. The incidence of disorder decreased with increasing fetal age, establishing mature values in late fetal periods. These findings suggest that the area of the central retina does not increase after embryonic day 60 and that guidance factors are present that allow outgrowing ganglion cell axons to distinguish and avoid that portion of the retina that will become the fovea.     

Tritiated uridine uptake in the retinal ganglion cell layer of the cat during normal development and after visual cortex ablation

The short-term metabolic response of immature retinal ganglion cells to destruction of their target cells in the dorsal lateral geniculate nucleus (dLGN) was assessed in newborn cats. Retrograde degeneration of virtually all dLGN cells was induced by ablation of the 13 contiguous areas of visual cortex on the day of birth. The metabolic response of retinal ganglion cells to this loss of target cells in dLGN was determined by exposing the ganglion cell layer to tritiated uridine, a precursor of RNA. Control measurements were made from unoperated littermates. Following sectioning and processing of the retinae from both groups of kittens for autoradiography, silver grain densities overlying the cellular profiles in the ganglion cell layer were calculated. These calculations revealed levels of uridine incorporation at Postnatal Day 4 in both groups of kittens significantly higher than at either Postnatal Day 2 or 7, but no significant differences between the two groups on any day examined. These results show that the level of RNA synthesis in retinal ganglion cells increases temporarily during the first postnatal week and that this synthesis is unaffected by the death of target cells in the dLGN. The temporary increase may be related to the establishment of synaptic connections on retinal ganglion cells by their afferent bipolar and amacrine neurons in the inner nuclear layer.     

Structural basis for ON-and OFF-center responses in retinal ganglion cells

The inner plexiform layer of the mammalian retina has a bisublaminar organization determined by restricted branching of the terminals of cone bipolar cells and dendrites of class I (large) and class II (small) ganglion cells. Comparison of dendritic field diameters and receptive fiedl center sizes of large ganglion cells suggests that neural circuitry in sublamina a conveys "OFF"-center properties and connections in sublamina b "ON"-center properties to retinal ganglion cells.